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The current work focused on AVHSE as a research tool, but similar techniques could be further developed for the development of grafts. Grafting would require addressing many of the structural and biological limitations noted above, as well as modifications to address host immunity issues. Overall, we have demonstrated AVHSEs as a research platform with regards to photoaging effects, but expansions of this model could be utilized for clinical skin substitutes, personalized medicine, screening of chemicals/cosmetics, drug discovery, wound healing studies, and therapeutic studies. Perfluoroalkyl substances are stable, water-soluble compounds that persist in the environment and are of major concern for the public. These substances are widely used in surfactants, lubricants, paints, polishes, paper/textile coatings, food packaging, and fire retardant . The most widely studied PFAS chemicals are PFOS and PFOA. These are organic compounds in which all hydrogens on all non-functional group associated carbons have been replaced by fluorine; thus, PFOS and PFOA are extremely stable due to their numerous strong carbon-fluorine bonds100 . Due to their stability and bio-accumulative properties, PFAS are ubiquitous and are found in food and water . PFASs have come under recent scrutiny as bio-accumulating toxins; once they reach a certain concentration in the body, their hydrophobic long chain structures penetrate cellular lipid bilayers and displace and disrupt membrane structures. Concerningly,gallon pot in people aged 12 or older, PFAS chemicals including PFOS, PFOA, PFNA, and PFHxS have been detected in more than 95% participants of a ~7800 sample study in the U.S.. These chemicals have also been found to environmentally bio-accumulate with detection in several animals including mammals and aquatic species. The primary route of PFAS exposure in adults is through food and water ingestion.

There are also a few studies demonstrating toxicity of PFAS through skin exposure in human tissue engineered skin and in animals263–265. In US children , the exposure from dust ingestion and dietary ingestion is nearly equal258 . Bio-accumulation of PFAS results in multiple health detriments with exposure ranging from pre-natal time points into adulthood. Toxicity mechanisms of PFAS within the cell and on tissue development are poorly understood but implications have been made regarding disruption of viability and proliferative capacity, immune response, pro-estrogenic endocrine processes, lipid profile and fat development , cell membrane, endothelial barrier, gap junctions, and cytoskeleton . Higher PFAS concentrations in adults are associated with greater weight regain. Evidence for non-favorable lipid profiles linked to PFAS plasma concentrations has also been found including greater total cholesterol low-density-lipid , higher triglycerides, increased very low-density lipoprotein , and increased gamma glutamyl aminotransferase. Further, hormonal effects have been linked to PFAS compounds in adult subjects. Reproductive hormones such as sex-hormone binding globulin, follicle stimulating hormone, and testosterone concentrations have been found to be inversely related to PFOA and PFOS in people aged 12-30 years. Thyroid stimulating hormones and total T4 have been positively associated with PFAS while negative associations have been found with kidney function . Children have a higher burden of PFAS partially due to mouthing behaviors, their lower body size to area ratio, and possible exposure via breastfeeding . Studies with concerning outcomes concluded associations of increased PFAS exposure in utero and during childhood with adolescent/adult obesity, cholesterol, and increased beta cell dysfunction. Childhood obesity and overweight risk is in turn associated with higher adult risk of obesity and multiple chronic diseases including cardiovascular disease and diabetes. Several PFAS compounds are able to penetrate the placental barrier in pregnant women and reach fetal circulation. Although the mechanism of accumulation is not yet understood, multiple studies have shown that this transfer is preferential, that is, fetal concentration is higher than maternal concentration-possibly due to differences in fetal v. maternal blood.

Studies have associated higher maternal serum concentrations/exposure with decreased birthweight but increased infant adiposity. A growing number of studies have also linked high maternal serum concentrations of PFAS to increased pediatric/young adult adiposity and changes in lipid profiles. Hypothesized mechanisms of PFAS association with low birthweight and increased infant/childhood adiposity include lipid metabolism changes, reduced food/water intake by the mother, direct fetotoxic effect, endocrine disrupting effects, and other altered hormone levels. Much evidence points toward hormonal effects and their downstream interference of tissue development and function including body weight and adipose regulation. One direct example of hormone sensitive development effects include changes in anogenital distance which have been observed in female infants. Negative PFAS effects have also been explored with mechanisms acting on constitutive andostane receptor, pregnane-X receptor, estrogen receptor beta, and the phosphatidylinositol 3-kinase-serine/threonine protein kinase pathway. Hypothesized mechanisms for greater weight regain in adulthood include PFASs possible involvement in changing energy metabolism and homeostasis of thyroid hormones through transcriptional factor activation like peroxisome proliferator-activated receptors. PPARα and PPARγ are key regulators in fatty acid oxidation, differentiation, adipocyte proliferation and function, glucose breakdown, and lipid and lipoprotein metabolism. In mice, PFOA was shown to affect leptin and adiponectin release during differentiation of fat cells ; leptin is a regulator of energy homeostasis while adiponectin is secreted by mature adipocytes and effects insulin responsiveness. At this time, the high PFOA/PFOS exposure association with decreased birthweight is based on multiple conflicting literature. However, to shed light on conflicting conclusions, a case study that systematically reviewed 18 epidemiological and 21 animal studies regarding PFOA toxicity concluded that exposure to PFOA is in fact toxic to human reproduction and development300.

Conflicting evidence of the associations between PFAS maternal concentrations and childhood adiposity is also present, although a recent mass analysis of cohort studies has concluded an association with early life exposure to PFOA and increased risk for childhood obesity. Speculation upon the conflicting evidence of associations between PFAS maternal concentrations and childhood adiposity have been made regarding the differences in concentrations of PFAS evaluated in study populations and differences in method of subject weight measurement. In many studies PFAS associations with negative health effects were only observed in the highest fraction of serum concentration studied while in lower concentrations associations were not present. More evidence is required to fully understand PFAS associations and how they may vary with concentrations. It has been suggested that low maternal serum concentrations show positive associations with childhood adiposity and higher serum concentrations show positive, negative, or non-monotonic dose responses with childhood obesity; this evidence is supported by the fact that PFAS are endocrine disrupting chemicals 106, since EDCs have been shown to induce NMDR. Much of this association has been investigated due to concrete developmental effects PFASs have on rodent birthweight when exposed in utero. Importantly, animals have differences in PFAS metabolization and gestational duration when compared to humans and many animal effects were observed with very high levels of PFAS. Lipid profiles are associated with adult weight and prevalence of obesity. There is much conflicting evidence on whether PFAS plasma concentrations are associated with less favorable or beneficial outcomes on lipid profiles. Several studies have linked PFAS with higher total cholesterol, higher triglyceride levels,gallon nursery pot and higher low-density lipoprotein while other studies have linked higher PFAS plasma concentrations with beneficial lipid profile effects. Drawing conclusions based on adiposity effects of PFAS in animals should be carefully considered as well. Notably, mice have differences in adipocyte derived hormones; for example, resistin is secreted by adipocytes in the mouse but in humans is not expressed in adipocytes and is primarily present in monocytes and macrophages. Conflicting literature of PFAS and associations with health elucidate a need for further understanding at the cell and biological signaling level of PFAS effect on humans. These contradictions are exacerbated due to the poor understanding of the toxic mechanisms PFAS elicits. Several of these mechanisms share common modulators, the Hippo signaling pathway and the cell cytoskeleton. Hippo signaling is a crucial cellular pathway involved in organ development, growth, homeostasis, stem cell maintenance, and regeneration. Briefly, the classic Hippo cascade involves kinase regulators mammalian Ste20-like kinases 1/2 and large tumor suppressor 1/2 which act to regulate phosphorylation and degradation of Yes-associated protein and transcriptional co-activator with PDZ-binding motif. As downstream effectors of the Hippo pathway, YAP and its paralog TAZ target genes involved in cell growth, proliferation, differentiation and development. When Hippo is active, YAP/TAZ are phosphorylated leading to YAP/TAZ degradation and cytoplasmic retention. In the inactive state, YAP/TAZ accumulate in the nucleus and target proteins of TEA-domain containing family , runt-related transcription factor , and others .

Several factors can mediate activation of the Hippo pathway including the classic cascade involving Mst1/2 and Lats1/2, mechanical cues, cell polarity and adhesion mechanisms, metabolic pathways, ligand-dependent activation, and hormone/growth factor control. Further, YAP/TAZ are involved in pathway crosstalk as well. YAP and its core upstream regulators Mst1/2 and Lats1/2 have been investigated in several organs and their developmental dysregulation which may be linked to changes in body weight. Many organ systems have been studied to identify developmental problems after knockout of Mst1/2, Lats1/2, or YAP. Through these investigations, it has been shown that changes in the main regulators and effector YAP have caused lung epithelial defects, faults in kidney structure development, bone to fat ratio disruptions, over proliferation during intestinal development, improper regulation of liver development including epithelial and hepatocyte maturation changes , and pancreas mass/size changes. It is still unclear how PFAS may disrupt YAP regulation and how these mechanisms tie into body weight and organ development/size control. PFAS may regulate the Hippo pathway via mechanosensing extracellular matrix changes through focal adhesions and the actin cytoskeleton, a known Hippo pathway propagation. YAP/TAZ regulation through Rho/ROCK activation acts as a control mechanism for transcriptional control of cytoskeleton stability, PFAS is likely effecting cell function and viability through this Hippo pathway cascade as well. Adipogenic versus osteogenic polarization of mesenchymal stem cells is dependent on YAP/TAZ localization in the Hippo signaling pathway, a relationship linked through mechanical cues and cytoskeletal tension. An increase in nuclear YAP/TAZ localization corresponds to increased osteogenic stem cell differentiation while increases in cytosolic YAP/TAZ correspond to higher adipogenic differentiation. Mechanical regulation of adipogenesis upon nuclear localization has been suggested to work through transcription factor ß-catenin or SMAD proteins rather than TEAD. Due to the cytoskeletal involvement in adipose tissue, it is plausible that PFAS may dysregulate adipose through perturbation of cell cytoskeleton. PFAS chemicals have been found to act on the cell cytoskeleton through disruption of f-actin, microtubules, and gap junctions. PFAS has been shown to disrupt and fragment actin cytoskeleton and tight junctions in mice Sertoli cells and human microvascular endothelial cells. It is likely that PFAS effects cytoskeleton integrity and could change balance of osteogenic/adipogenic polarization of mesenchymal stem cells and/or adipose tissue homeostasis. During adipogenesis, cytoskeleton remodeling is a preliminary process and it has been found that the cytoskeletal components actin, tubulin, vimentin, and septin undergo localization and expression changes. Specifically, actin forms filament bundles in the cytoplasm of pre-adipocytes and short filaments in mature adipocytes with similar organization in the microtubules, and vimentin regulates lipid droplet accumulation by forming cage structures that surround lipid droplets. Septin has been found to form filaments or rings depending on timepoint within adipocyte differentiation. These findings support the cytoskeleton’s role in regulation of adipogenesis and lipid accumulation. In a study completed with rat cardiomyocytes, it was found that the adipokine, adiponectin acts on Rho/ROCK and increases RHO GTPase activity and induces cytoskeletal remodeling to further regulate glucose uptake and metabolism. Adiponectin effects weredemonstrated by its ability to increase membrane microvillar like protrustions, and increasing actin polymerization to form filamentous actin/actin stress fibers. Potentially PFAS chemicals may be directly disrupting the cytoskeleton or disrupting it indirectly through changes in the adipokine profiles that adipose tissue secretes. On the other hand, PFAS may regulate the Hippo pathway via mechanosensing extracellular matrix changes through focal adhesions and the actin cytoskeleton, a known Hippo pathway propagation. YAP/TAZ regulation through Rho/ROCK activation acts as a control mechanism for transcriptional control of cytoskeleton stability , PFAS could be effecting cell function and viability through this Hippo pathway cascade as well. The mechanical control of adipogenic differentiation of MSCs relies on both the integrity of the actin cytoskeleton itself and tension feedback from myosin II motor which directly acts on the Hippo signaling pathway.

This means that its adaptive size prevents it from negatively impacting performance. Additionally, the thousands of read operations it satisfies enables Anticipatory Spin-Up functionality.These works generally conclude that a small non-volatile memory write-cache can significantly increase performance by reducing disk traffic. With hybrid disks soon to be available, hybrid disk/non-volatile memory file systems, such as Conquest and Hermes can be evaluated for their effectiveness at increasing file system performance by leveraging on-board non-volatile memory. Previous works have also looked at reducing hard disk power consumption using non-volatile memory. FLASH CACHE proposes to place a small amount of flash directly between main memory and disk, as an additional level in the caching hierarchy to decrease power-savings as well as increase performance. Nvcache focuses completely on reducing power management, and therefore has a completely different architecture. Although Anand et al. don’t use non-volatile memory, they propose ghost hints to anticipatively spin-up a hard disk in a mobile system context while redirecting read I/O to the Internet during disk spin-up. Microsoft proposes to use hybrid disk drives to reduce hard disk power consumption, and decrease boot-time and application launch in their upcoming Microsoft Vista Operating System. They claim a hybrid disk can be spundown by up to 96% of the time with a 1GB NVCache. Unfortunatelly, neither algorithms nor workloads are described. Duty cycle is only one metric for disk drive reliability. Disk drive reliability must also factor in duty hours, temperature, workload, and altitude. Mean Time To Failures and Mean Time Between Failures are widely used metrics to express disk drive reliability. However,black plastic pots for plants these metrics must considered with care, as they are often incorrect . IDEMA has proposed a revised MTBF rating based on disk age.

Most adaptive spin-down algorithms for traditional disks mention that disk reliability decreases when using a spin-down algorithm, but don’t quantitatively describe the impact. Greenawalt modeled the effects of different fixed time-out values and its impact on power conservation and disk reliability. They use a Poisson distribution to simulate inter-arrival access patterns and consider duty cycles as detracting X hours from the MTBF rating. Other strategies to save hard disk power involve pushing power management to applications. Weissel et al. propose that energy-aware interfaces should be provided to applications. Such interfaces can be used to convey priority or state information to an operating system. For example, deferrable I/O interfaces can be used by applications to in- form the operating system that particular I/O requests may be differed.The sediments of coastal marine wetlands in California are inhabited by a variety of algal and bacterial primary producers in addition to the more conspicuous vascular plants that provide most of the physical structure of coastal salt marshes and seagrass meadows. The non-vascular plant flora includes microscopic cyanobacteria, anoxygenic phototrophic bacteria, diatoms, and euglenoids, often collectively known as “microphytobenthos” . Larger green algae, red and brown seaweeds, and the macroscopic tribophyte, Vaucheria are also residents of these ecosystems . Ecologically, sediment-associated algae and photosynthetic bacteria are key components of wetland food webs . They account for a substantial fraction of ecosystem primary productivity in California and in other regions . While understanding of the ecological roles and spatio-temporal dynamics of these organisms has improved, the diversity and natural history of the micro- and macroalgae of salt marshes and mudflats from the northeastern Pacific, including southern California, are still poorly understood. Lack of a deeper understanding of the diversity of these organisms within and between estuaries and estuarine habitat types impedes efforts to understand how spatio-temporal variation in the composition of benthic assemblages may affect ecosystem functions or how changes in assemblages may relate to anthropogenic impacts to wetlands.

To date no comprehensive floristic account of wetland algae and photosynthetic bacteria in California has been produced by the phycological community. However, some taxonomic information on these organisms exists in scattered sources. Early research on the taxonomy of wetland algae began with William Setchell, Nathaniel Gardner, and George Hollenberg. These phycologists produced lists and/or descriptions of cyanobacteria and macroalgae from salt marshes and mudflats in several publications, but the accounts principally focused on either rocky shore cyanobacteria or wetland vascular plant floras . Several decades later, Zedler published a list of cyanobacteria, diatoms, and green algae collected from Tijuana Estuary at the southern extreme of the state. She recorded 32 species of diatoms, four cyanobacterial taxa, and the green algal genera Rhizoclonium and Enteromorpha, but noted that her account was not comprehensive. Wilson and Carpelan studied benthic diatoms from Mugu Lagoon and pelagic diatoms in four lagoons in northern San Diego County respectively. Records of wetland macroalgae have been compiled for Humboldt Bay in northern California , and for Newport Bay in southern California. Stewart’s treatment of San Diego County seaweeds also notes wetland occurrences of marine macroalgae. There are formidable obstacles to producing a comprehensive flora of tidal wetland algae for any localized region. First, the phylogenetic breadth of photosynthetic organisms in tidal wetland habitats requires a diversity of specialists, employing an array of tools from electron microscopy to culturing techniques to standard phycological methods for macroalgal identification and preservation. As Sullivan and Currin note, funding for such an endeavor is likely to be difficult to acquire. Moreover, the systematics of many groups of these organisms is in flux. In particular, study of the cyanobacteria is complicated by the existence of competing bacteriological and morphological classification schemes and by widely differing approaches to using morphology to delineate species . An additional consideration is that application of names to microalgal and cyanobacterial taxa for a given locality is at least somewhat dependent on decisions made in other geographic regions or habitats since detailed taxonomic studies are haphazardly distributed in space and time.

For instance, some important cyanobacterial reference sources either treat distant geographic areas or describe primarily freshwater and terrestrial organisms . Despite these challenges, floristic and systematic work on wetland microalgae and seaweeds provides the foundation for progress in basic biodiversity research. In addition to the possibility that cryptic taxa may be discovered in the flora, algae are excellent systems for investigation of molecular versus morphologically-based phylogenies . Better knowledge of the diversity of microproducers present in coastal wetland habitats should also enable a better understanding of ecological interactions between microphytobenthos and other wetland organisms and facilitate the use of biodiversity metrics as a means of assessing ecosystem health and dynamics. In this paper the common benthic cyanobacteria, microalgae, and seaweeds associated with sediments from tidal wetlands in southern California are described and illustrated. The goal is to provide preliminary documentation of the local flora and add to the fragmentary knowledge of these organisms in the region. The paper focuses on new collections made from Mission Bay and Tijuana Estuary in San Diego County,drainage pot but also includes some records of species previously recorded from wetlands throughout southern California . Organisms included here were assigned tentative genus names based on morphological features visible by eye or by light microscopy. Supporting references pertinent to the identification of taxa, their local distribution, and their natural history, are also included. Of the various taxa treated, documentation of the cyanobacteria is most thorough, partly filling the significant gap in information on these common inhabitants of tidal wetlands in the region. Observations and photographic documentation were made on live organisms, or occasionally on organisms grown in culture. Specimens were often kept alive by transferring moist field sediment to incubation in the laboratory. Field sediment and cultures were maintained at about room temperature with illumination . Cultured organisms were grown on sterilized f/2 media prepared in artificial seawater with or without sterilized glass particles as a substrate. Organisms living on field-collected sediment were kept and observed up to about seven months following removal from the field . Photographs were taken with a digital camera through compound microscopes . Diatoms were identified to genus where possible using Round et al. . Cyanobacterial taxa were generally identified to genus using the recent taxonomic treatments in Anagnostidis and Komárek , Komárek and Anagnostidis , and Boone and Castenholz . Humm and Wicks , Desikachary , and Setchell and Gardner were also consulted for identification and nomenclatural purposes. Macroalgae attached to sediment-associated substrates or occurring loosely in wetland habitats were pressed fresh on herbarium paper and dried. Identification and current nomenclature of macroalgae follows Abbott and Hollenberg and Gabrielson et al. . Skin is one of the largest organs of the body and has functional roles in immune response, physical protection, and thermal regulation. As aging occurs, skin function and healing capacity is reduced. Skin aging is frequently divided into two related processes: intrinsic and extrinsic aging . Intrinsic aging, also referred to as chronological aging, includes genetic and hormonal changes and the progression from cell maturity to cellular senescence. Extrinsic aging, also referred to as environmental aging, represents the impact of the environment, including: photo aging associated with sun exposure , cigarette smoking, pollution, chemical exposure, trauma. Due to the different underlying mechanisms, characteristics of each type of aged skin are different. Chronologically aged skin presents as unblemished, smooth, pale, dry, lower elasticity, and has fine wrinkles while environmentally aged skin has coarse wrinkling, rough textures, pigmentation changes, and lower elasticity.

Microstructural changes in intrinsically aged skin include decreased dermal vasculature ; changes in dermal elasticity and increased collagen disorganization; build-up of advanced glycation end products and changes in glycosaminoglycan and proteoglycan concentrations/organization contributing to stiffening of dermal structure and frailty, and decreased hydration; imbalance of tissue inhibitors and matrix metalloproteinases resulting in imbalance between collagen deposition and breakdown; and flattening of the dermal epidermal junction/loss of rete ridges. Aging also contributes to variations in epidermal and dermal thickness and reduced subcutaneous fat volume. There are also many changes related to cell population in all three main skin compartments including reduced epidermal cell turnover, drop in number of active melanocytes ; decreases in dermal fibroblast concentrations, decreases in immune cells and immune function. Abnormalities of skin barrier occur during aging and often present as dryness or skin irritation. In aged skin, barrier function has been studied in the context of decreases of filaggrin, increases in pH , altered lipid presence, and changes in cornified envelope arrangement. These changes add to fragility of older skin and higher chances of infection, it remains unclear exactly how these changes take place and what mechanisms are controlling them. On the molecular scale, expression levels of soluble factors, proteins, and vitamins are both effects and contributors to aging phenotypes. Examples include upregulation of stress regulatory proteins, increases in AP-1, and declines in vitamin D production by the epidermis. These changes are largely attributed to increases in reactive oxygen species, DNA mutations , telomere shortening, increased cell senescence, and hormonal changes. Changes in skin aging have been associated with fluctuations in expression patterns of integrins including α6 and ß1 integrins.In healthy human skin, α6 and ß1 integrin expression are localized on the basal side of basal keratinocytes. Defects in integrin expression are present in human blistering skin diseases with supporting evidence in knockout mice 34 and also in aged human skin ,although further work is necessary to understand how integrin expression changes in aging. Aging in the skin has sex-related differences as well, specifically, sex is linked to faster thinning of the dermis and collagen density decline in males as opposed to females. Males undergo a decline in androgen levels while estradiol levels are constant, these changes result in a linear decline of skin thinning and collagen content in men 10. Women experience both androgen and estrogen decline linearly and an additional post-menopausal estrogen decline which is linked to lower collagen content, lower skin moisture and capacity to hold water, lessened would healing response, thinner skin, and lower skin elasticity. Detailed summary and discussion of sex-related changes in skin aging have been previously reviewed. These intrinsic mechanisms are compounded by environmental skin aging . A key example is the effects of ultraviolet irradiation , which accelerates telomere shortening and DNA damage present with intrinsic aging. Other extrinsic aging and examples of compounding UV effects are discussed in previous literature. Overall, skin aging at the molecular, cellular, and tissue levels continues to be a field of active research.

Hybrid disks present an opportunity for spin-down algorithms to further reduce power consumption while minimizing the performance and reliability impact they impose on the media itself. We now describe four spin-down algorithm and I/O subsystem enhancements.Spin-down algorithms controlling traditional disks compute the idle period as current time − last access time, where last access time is the time of the last disk access. If the idle period exceeds the current time-out, the rotating media is spun-down. I/O type, whether read or write, is ignored because any request, regardless of type, will cause the rotating media to spin-up. This is not true of hybrid disks as one of the intents for adding an NVCache to a hard disk is to extend the duration of spun-down periods by servicing I/O to and from the NVCache. Note this assumes the block I/O layer or disk driver is aware of the rotating media’s power state, and will redirect I/O while it is at rest. Our previous work describes a mechanism implemented in the block-layer to redirect I/O to and from a physically separate flash-based NVCache while the disk is spun-down. With such support, NVCache utilization is conveyed to the spin-down algorithm in the context of extended spin-down periods. A hybrid disk-unaware spin-down algorithm will still ignore I/O type because it believes any I/O will cause a spin-up. However, with the above redirection mechanism, such an assumption is false—write requests are actually unlikely to cause a spin-up. Therefore, we present Artificial Idle Periods, a spin-down algorithm modification for a hybrid disk which considers I/O type when computing a disk’s idle time, by recording idle time as time since the last read request. When a request occurs for a disk in the active mode,black plastic plant pots the time-out value is reset only on a read request. The idle period is thus artificially increased to current time − last read access time.

As a result, even if a hybrid disk is actively servicing requests, it can be spundown and remain so, provided I/O consists only of write requests. Such a modification has several implications. First, duty cycles may be consumed faster; idle periods are artificially increased so a disk will spin-down sooner and probably more frequently. Second, I/O performance may degrade with sequential write workloads as flash sequential write throughput is only a fraction of rotating media, which must still be periodically flushed to disk. Finally, the NVCachewill endure more erase-operations resulting from its increased workload, decreasing its expected lifetime.As we will show in our evaluation, even with Artificial Idle Periods, typically less than 10% of the NVCache is used per spin-down period to cache writes. Although the NVCache is checked for desired read requests, reads are still typically responsible for initiating spin-ups. NVCache cached writes are not successful at servicing read requests because the host operating system is likely to be idle and not evicting buffer cache pages quickly. As a result, most read requests will be satis- fied by the buffer cache. To ensure that read requests are satisfied by the NVCache we propose a Read-Miss Cache, an area in the NVCache that is populated with unsatisfied NVCache read requests . The hypothesis being that readmisses causing a disk to spin-up are likely to cause a disk to spin-up again. When an NVCache read-miss occurs while the rotating media is spun-down, the requested content is read from the newly spun-up disk and returned to the file system. It is stored in the Read-Miss Cache and subsequent sequential reads are also stored in the Read-Miss Cache, which we refer to as preloading. Preloading stops when a non-sequential read or write request occurs. Only the most frequently preloaded content is stored in the NVCache— preloading data into the NVCache merely updates its frequency count, including the original read-miss.

The Read-Miss Cache size is dynamic. However, a maximum size constraint can be supplied to bound its growth, represented as a percent of the total NVCache. There is also a static minimum Read-Miss Cache size set to 1%. The Read-Miss Cache grows when a read-miss occurs causing the disk to spin-up and shrinks when a write operation cannot be stored in the NVCache because there is no available room.We implemented hybrid disk functionality in the Linux kernel, mimicking a hybrid disk using flash and a traditional disk by redirecting I/O traffic at the block I/O layer. While the disk is spun-down, I/O is intercepted at the block layer and redirected to flash memory. To evenly spread out block erase cycles and reduce page-remappings, redirected writes are appended to flash in log order. Each redirected request is prepended with a metadata sector describing the original I/O: starting LBA, length, spin-down interval, etc. The redirected LBA numbers are stored in memory to speed up read requests to flash. When the flash fills up or a readmiss to it occurs, the corresponding disk is spun-up and the flash sectors are flushed to their respective locations on disk. We also built a a simulator to model a hybrid disk to allow expedient evaluation of the proposed enhancements with several week-long block-level traces. The simulator considers power relative to the given trace for different power states: read/write, seek, idle, standby, and spin-up. For the notebook drive, power state transitions to and from performance, active, and low-power idle are done internally to the drive and are workload dependent. Therefore, we make a worst case assumption and assume the drive is always in low-power idle, providing a lower bound on spin-down algorithm performance, relative to idle power. Read/write I/O power is computed using the disk’s maximum specified I/O rate, and seek power is computed with the drive’s average seek time rating for non-sequential I/O.

The spin-down algorithm implemented is the multiple experts spin-down algorithm. It is an adaptive spin-down algorithm developed by Helmbold et al.. The spin down algorithm is based on a machine learning class of algorithms known as Multiple Experts. In the dynamic spin-down algorithm, each expert has a fixed time-out value and weight associated with it. The time-out value used is the weighted average of each expert’s weight and time-out value. It is computed at the end of each idle period. After calculating the next time-out value, each expert’s weight is decreased proportional to the performance of its time-out value.To evaluate the proposed enhancements, we use several different block-level access traces, shown in Table 2. We use four desktop workloads and a personal video recorder workload. Each workload is a trace of disk requests, and every entry is described by: I/O time, sector, sector length, and read or write. The first workload, Eng, is a trace from the root disk of a Linux desktop used for software engineering tasks; the ReiserFS file system resides on the root disk. The trace was extracted by instrumenting the disk driver to record all accesses for the root disk to a memory buffer, and transfer it to userland when it became full. A corresponding userland application appended the memory buffer to a file on a separate disk. The trace, HPLAJW, is from a single-user HP-UX workstation. The next trace, PVR, is from a Windows XP machine usedas a Home Theater PC running the personal video recording application, Beyond TV. The WinPC trace is from an Windows XP desktop used mostly for web browsing, electronic mail, and Microsoft Office applications. The block level traces for both Windows systems were extracted using a filter driver. The final trace, Mac is from a Macintosh PowerBook running OS X 10.4. The trace was recorded using the Macintosh command line tool, fs usage,black plastic planting pots by filtering out file system operations and redirecting disk I/O operations for the root disk to a USB thumb drive. The physical devices we present results for are a Sandisk Ultra II Compact Flash card, a Hitachi Travelstar E7K100 2.5 in drive, and a Hitachi Deskstar 7K500 3.5 in drive. The power consumption for each state are shown in Table 1. Note that in all figures except Figure 9, we show results using the 2.5 in drive. We present results for both a a 2.5 in and 3.5 in drive in Figure 9 to motivate placing an NVCache in a 3.5 in form factor.Figure 5 shows the results of making the I/O subsystem aware of a hybrid disk’s NVCache, such that a write request occurring while the rotating media is at rest, is redirected to the NVCache.

This figure shows the percentage of time the disk can remain spun-down as a function of the NVCache size. The Eng trace benefits the most from the write cache by increasing its spin-down time from 71% to 92%, which translates into an increase of slightly more than one and half days of spin-down time for the seven day trace. This is primarily due to the periodicity of writes-while-idle which occur more frequently than any another workload. The other workloads also benefit from a write-cache by increasing their spun-down time by 4–10%. Figure 5 shows the percentage increase in spun-down time by adding Artificial Idle Periods to write-caching. This plot shows that Artificial Idle Periods significantly increase the percentage of time a disk is spun-down. The most significant benefit comes when the NVCache is less than 1MB. By adding Artificial Idle Periods to NVCache with less than 1MB, its utilization increases as I/O redirection to the NVCache occurs sooner and more frequently. With larger NVCache sizes, Artificial Idle Periods is utilized less often, and so its impact is less pronounced. However, the percentage increase by adding Artificial Idle Periods still stabilizes between 3.5% and 5% for all but the PVR workload, which stabilizes at a 27% increase in spundown time. The PVR workload benefits from artificial write period so much because its workload consists of periodic write requests without interleaving read requests. Although write-caching and Artificial Idle Periods are excellent solutions to decrease the time a disk is spent in standby mode, it is important recognize the associated reliability impact. Figure 5 shows the number of expected years to elapse before the 2.5 in disk exceeds the duty cycle rating . By enabling write-caching while the disk is spun-down, reliability increases with respect to utilized NVCache size. As the NVCache exceeds 10MB, reliability stabilizes because it becomes under utilized beyond this point. Figure 5 also shows the expected years before the disk will exceed the duty cycle rating, but with Artificial Idle Periods on. In this figure, we see that reliability decreases relative to write-caching alone. However, the expected years before exceeding the duty cycle rating is still more than two and half years for the the Mac trace, the lowest of the five workloads. Note that without write-caching or Artificial Idle periods, the Mac workload would exceed the duty cycle rating in seven months.Figure 6 shows the results for a 256MB NVCache with write-caching, Artificial Idle Periods, and a Read-Miss Cache as a function of the maximum Read-Miss Cache size. Figure 6 shows the number operations satisfied by the Read-Miss Cache while the rotating media is spundown. This figure shows that with less than half the NVCache enabled for the Read-Miss Cache, the working set of read requests to the NVCache is captured. Figure 6 shows the average Read-Miss Cache size as a function of the maximum Read-Miss Cache size. The average Read-Miss Cache size grows linearly with respect to the maximum size until 90%, after which it deteriotes quickly, confirming that usually 10% of a 256MB NVCache is used for write-caching and Artificial Idle Periods. The PVR workload is an exception as it stabilizes at 27% because of its high NVCache utilization from television content recording. Figure 6 shows the percentage increase in spun-down time by adding a Read-Miss Cache to an NVCache with write-caching and Artificial Idle Periods. Here we see that the Read-Miss Cache only increases the spun-down time percentage by at most 1.5%. Note that with only write caching and Artificial Idle Periods enabled for a 256MB NVCache, all but the PVR workload are spun-down for 90–95% of the workloads, which leaves little room for improvement. Although the Read-Miss Cache does not increase spun-down time significantly, there is still a consistent increase when the Read-Miss Cache is allowed to use the entire NVCache.

The traces of this history may remain in plain sight but reconstructing this history requires more than critical close readings of China-themed films or Chinatown tourism publicity from the period. While close reading methodologies can help us understand the ways in which popular culture structured dominant ideas of race, gender, and nation, these methods tell us little of the motives of the Chinese Americans who helped produced these films and related performances, or of the emotional or social ties that the production of these performances elicited. To understand these performances in their historical context necessitates both close readings of popular racial representations alongside the use of archival documents and oral history interviews produced by community members. This archival work can help us piece together the lives and actions of the Chinese Americans who played such an important role in producing cultural artifacts from this period. This dissertation unites archival and oral history methodologies from social history with close textual analysis from film studies to foreground the ways seemingly everyday Chinese Americans influenced the process of racial formation. In the process, this interdisciplinary methodology produces new ways of understanding Chinatown both as a representation and as an ethnic enclave. As part of this interdisciplinary methodology, this dissertation is closely grounded in a form of social history often referred to in Asian American Studies as community history. As first deployed in the developing field of Asian Americans studies in the 1970s and 1980s, Asian American community history utilizes community-engaged methods such as oral history and collection of family and community documents to create an archive on which the history of a given place-based, ethnic enclave can be written. If the primary goal of mainstream American history has long been to produce academic interventions in society’s knowledge of the past,drainage pot the goal of Asian American community history, as it was originally developed, was much more politically informed.

Developing out of the political imperatives of the Asian American Movement of the late 1960s, community history methodology sought to address broader historical silences within mainstream narratives of American history while simultaneously documenting, building, and empowering local Asian American communities. Often produced collectively, these community histories originated both out of emerging ethnic studies departments as well as out of the first local Asian American historical associations that were then coming into being. While Asian American community history developed around the same time that “public history” was emerging as an accepted academic field within American history, the audience for these early Asian American community histories was not an unidentified public audience but rather the members of the same ethnic enclaves whose history was being told. This project may be written as dissertation in the field of Ethnic Studies, but it maintains the ethos of these earlier Asian American community histories. In particular, this dissertation builds on the work of community historians at the Chinese Historical Society of Southern California and the Chinese American Museum of Los Angeles. While other archival institutions were used, the collections of these two institutions form the foundation of the dissertation. Central to this dissertation is the Southern California Chinese American Oral History Project. Produced in the late 1970s and early 1980s as a joint effort between the Chinese Historical Society of Southern California and UCLA’s Asian American Studies Center, the project interviewed 165 people about their lives in Los Angeles until 1945. The resulting collection contains four hundred hours taped interviews and 1700 pages of summary transcripts. A collective effort that involved volunteers from both UCLA’s Asian American studies center and the CHSSC, the project remains perhaps the most comprehensive archived Chinese American oral history collection of its type focusing on the pre-war period. I employ these archival and community history methodologies alongside those of cultural studies.

Grounded in the work of scholars like Stuart Hall and Edward Said, this project sees culture as intimately tied to social, economics, and political power. Power relations of a given social structure are encoded in popular representations, and subaltern groups, such as Chinese Americans in the 1930 and 1940s, use culture as a means of engaging the intersecting structures of race, class, gender, and sexuality. This project thus sees films, newspaper articles, and above all Chinatown itself, as texts that can be read to better understand the social structure in a given place and period of time. Reading these cultural texts can lead not only to a better understanding of power relations within a given historical moment, but also to a better understanding of the ways those groups contested their subaltern position within the social structure. Historians are often criticized for their over reliance on the written word as a primary source, and certainly few of the existing studies on Chinese Americans in the first half of the twentieth century have given popular cinematic representations from the 1930s and 1940s the same attention as the written word. In contrast, my dissertation utilizes visual and material culture as “texts” that can be read in a way that will supplement rather than supplant our understanding of community history. As the first dissertation on the history of Los Angeles Chinatown and its relationship to Hollywood film, this project bridges these methodologies from social history and cultural studies to demonstrate the ways in which members of the Chinese American community in Los Angeles shaped dominant ideas of race, gender, and nation. I contend that the same transformations in the urban environment that facilitated the development of film in the late nineteenth century also transformed Chinatown into a similar type of cultural apparatus. Within the field of film studies, scholars such as Tom Gunning, Ben Singer, Laruen Rabinovitz, and Vanessa Schwartz have advanced what has come to be known as the modernity thesis.This modernity thesis posits that urbanization brought about a transformation in the social act of seeing which facilitated the development of new types of visual amusements, key among which was early silent film.A handful of film studies scholars have touched on this visual transformation and its relationship to both New York and San Francisco Chinatowns.My project builds on this earlier scholarship by briefly tracing the shared symbiotic history of Chinatowns and cinema from their roots in Chicago in the 1890s and San Francisco after the 1906 Earthquake. The dissertation then moves on to demonstrate the convergence and development of these two mediums—Chinatown and film—in Los Angeles between in the 1910s and the 1940s. Repositioning Chinatown as a medium of cultural production, symbiotically tied to the development of cinema allows for a more nuanced understanding of the amount of agency Chinese Americans were able to exercise over self-representations of their own community over the course of the first half of the twentieth century. It also allows us to see the myriad ways in which Chinese American in Los Angeles challenged, rearticulated, and at times reinforced ideas of American Orientalism.

Edward Said defines the idea of Orientalism as a system of knowledge and power through which the West defines itself against the East.For Said, the Orient is more than simply an idea. Instead it is “a mode of discourse with supporting institutions, vocabulary, scholarship, imagery, doctrines, even colonial bureaucracies and colonial styles.”While Said originally advanced the idea of Orientalism in discussing Europe’s relationship to the Middle East, a growing number of scholars have examined the way Orientalism functions within the United States. Scholars such as Gordon Chang and John Kuo Wei Tchen have all discussed the roles that discourses and popular conceptions of China played historically in constructing the idea of the United States as a modern, progressive nation.At the same time, Mary Ting Yi Lui, Anthony Lee, and Kay Anderson have all discussed various aspects of Chinatown and Orientalism.These scholars have demonstrated the ways in which popular ideas about China and Chinese people defined so many aspects of the way the United States understood itself as a nation in the late nineteenth and early twentieth centuries. While recognizing the lasting permanence of American Orientalism as a foundational ideology of the United States,large pot with drainage scholars have also acknowledged an important shift that occurred around the Second World War in the way Chinese Americans were popularly perceived. During the Chinese Exclusion Act period, American Orientalism defined Chinese Americans as legally, economically, and culturally as outside the boundaries of the US nation state. Throughout the Exclusion act period, the U.S. citizen came to be defined against the Asian immigrant.As such, representations of an American citizen of Chinese descent remained in many ways a cultural impossibility. Beginning around the Second World War, the ideology of racial liberalism took hold within the United States. With racial liberalism, the United States began the process of attempting to incorporate and manage, rather than exclude, a wider range of racial and ethnic groups within the United States.For Chinese Americans this period saw the symbolic end of the Chinese Exclusion Act in 1943 and the increasing acceptance of Chinese Americans into broader society. For the first time, large numbers of Chinese Americans were able to find jobs outside of the nation’s Chinatowns. While Orientalist ideas about Asia and Asian people did not disappear, the advent of racial liberalism transformed the ways in which American Orientalism functioned. Scholars have argued that the shift toward racial liberalism in general and the increasing incorporation of Chinese Americans into the nation-state in particular was largely the result of geopolitical factors directly linked to the war itself. In this narrative, the U.S. alliance with China during the Second World War, and the broader need to combat Japanese propaganda that labeled the United States as a racist nation, necessitated a transformation in the way in which the country treated its Chinese American residents. This accepted historical narrative leaves little room for the agency of Chinese Americans in the shifting notions of race, gender, and nation, and it further demonstrates Karen Leong observation that too often studies of American Orientalism see only whites as being able to engage these Orientalist discourses.In contrast to most earlier studies, I contend that Chinese American engagement with American Orientalism, through Chinatown performance, helped lay the foundation for the eventual incorporation of Chinese Americans into the nation state under the logic of racial liberalism during World War II. During the Chinese Exclusion Act era, Chinese Americans were forced to negotiate U.S. citizenship and national belonging through the discourse of American Orientalism. During this period, the question was not whether or not Chinese Americans would be defined as an Other against the US citizen, but rather what form this image of the Asian Other would take within the popular imagination. Therefore understanding Chinese American self representations before the Second World War necessitates an acknowledgement of the discursive possibilities and limits under which Chinese Americans operated during this period. While a few Chinese Americans such as Wong Chin Foo did attempt to present cultural representations of Chinese Americans as U.S. citizens, most Chinese Americans utilized a largely different strategy to combat Orientalist depictions of Chinese immigrants as a Yellow Peril.Examining what I call, “Chinese American Orientalism,” as a challenge to Yellow Peril stereotypes, the project foreground the ways that Chinese merchants, actors, and street performers used the medium of Chinatown to advance a vision of their community that at once challenged earlier Yellow Peril depictions while still maintaining some of the underlying assumptions about Chinese people’s differences from whites. In the face of Yellow Peril representations that defined Chinatown as an underground den of violent opium dealing tongs, Chinese American Orientalism cast the nation’s Chinatowns as clean, modern commercial areas where whites could shop and eat. These representations remained Orientalist in that they constructed Chinese culture in opposition to that of the West, but this form of Chinese American Orientalism negated rather than perpetuated ideas of Chinese Americans as a threat. This Chinese American Orientalism challenged images of Chinatown as a community of violent, opium-addicted bachelors living in underground dens and presented in its place an image of Chinatown as the modern extension of an ancient Oriental culture and tradition, one that could easily be commodified and sold to white visitors to financially support the needs of an emerging Chinese American middle class.

Same with nurses due to their specialty care facilities. So no one ever looks to come here full time. And the chiefs here know that we are posting positions to be filled with [temporary staff]. And so the cycle goes.” To overcome the cycle of temporary-staff usage, enable the sustainability of change, and maintain the spirit and knowledge of the implementation through consistent staff, a separate program was developed Nursing positions had the highest number of vacancies overall and also carried the highest fill rate. An analysis was commissioned to understand the ratio of clinical-position staffing relative to workload. Time-motion studies were carried out and combined with human resources initiatives designed to place employees in“hard-to-fill” posts. The result of these efforts was a program dedicated to nurse staffing designed to work hand in hand with CCM implementation, ultimately enabling sustainability of the overarching transformation effort.For health care quality improvement efforts to be sustainable in a correctional environment, local physician and nurse champions are required . Also important is an interdisciplinary implementation team involving a variety of health care team members. In the custodial health care setting, correctional officers are key stakeholders. Hence, identifying a local custody champion to be part of the interdisciplinary team was critical. The team of interdisciplinary champions was provided sufficient release time to participate in training and development in the areas of quality improvement, the chronic care model, and clinical diabetes content. Gaining the support of the custodial personnel who are responsible for prisoner control and safety related to health care needs was essential to overcome the institutional barrier. Correctional officers, and more specifically their captains and assistant wardens of health care, were brought into the primary care team meetings to be educated and trained on the methodology and processes. Feedback was received from team members on how to improve existing processes. The CCI implementation team decided that, as cultural change agents,vertical garden hydroponic they ought to be provided time to plan, implement, and disseminate change after inculcating an understanding of what the change meant for them, their particular departments, and their coworkers.

In this custodial environment, a reliance on one’s teammates and coworkers for success and safety was paramount, much as in work environments with nuclear reactors. In the receivership experience, champions were selected based on their expressed interest in serving as a catalyst for change in their pilot site; they were also identified as excellent clinicians who were respected by their peers. Physician involvement is critical to a successful change effort and program implementation within a health care delivery setting . Among the champions, the physician leaders’ commitment to the model and the change it represents must be asserted from the beginning. In order to achieve the goal of developing workforce competencies after receivership, several local institutions’ chief medical officers were recruited as the core project team’s clinical leaders. Practicing physicians and nurse consultants were recruited as quality improvement advisors.Health care delivery system design for prisons is a significant challenge. Contrary to models of care delivery external to the correctional environment, the primary mission of the institution from the custody perspective is security; health care delivery is procedurally treated as secondary. Typically, prisons are constructed with little or no space for clinics or medical supply storage. To implement a new CCM, the fundamental delivery system design had to start with the basics of creating adequate space for exam rooms so that the interdisciplinary team could provide integrated patient care. Once the issue of clinic space was addressed, the delivery system was changed to shift from a siloed, single-provider approach to a patient-centered team model. The Chronic Disease Management Program’s pilot prison sites adopted a managed-care-based primary care model and redefined the care team’s roles and definitions from a traditional solo provider medical model to an interdisciplinary team model. Treatment of inmate-patients was thus transformed to enable a more comprehensive level of care with each visit, with the goal of increasing quality of care and reducing the need for future medical visits.

Given the state government’s bureaucratic structure and a heavily unionized workforce, it was necessary to create new job classifications in the organizational structure as permanent positions—for example, nurse executive, nurse clinical care coordinator, nurse case manager, and medical assistant. In California, state employees are unionized, and as a result managing labor relations proactively during the delivery system design was critical to minimize employee grievances, union resistance, or both. Transparency and proactive collaborative approach were the keys to minimizing resistance from the unions. Planned group visits or health education classes proved to be a strong component of the delivery-system design in the correctional system.At the time of the program’s design the California prisons did not have enterprise-wide information technology connectivity and most clinic areas had no access to computers. The pilot prison sites therefore used the Chronic Disease Electronic Management System’s Disease Registry for asthma and diabetes as a temporary solution. As additional staffers were needed to perform data-entry functions and there was limited physical clinic space, the adoption was challenging. Some pilot prisons employed a low-tech, manual, tickler-file approach of tracking inmate-patients with chronic conditions. To overcome the identified institutionalized barrier of using memorized manual processes and tools to perform work, the benefits of the new systems were discussed during learning collaborative sessions. Additionally, continuing educational unit needs were identified and promised for delivery through new automated solutions. Communications were coordinated with other departments concerning other aspects of computerization to be implemented within the facilities as some of these programs were generally well accepted by staff. Staff also saw self-management support as reducing workload, and this further contributed to their acceptance of the changes.Confinement for high-security inmates is the primary obstacle to implementing self management support. Custodial concerns and rules greatly inhibit the inmate-patients’ ability to perform aspects of self-management of routine care. For example, some prisons prohibit the use of a medical device known as a drug delivery spacer due to it being physically sharp with the potential to be converted into a weapon.

Serum glucose monitoring and insulin injections are typically performed by a licensed vocational nurse instead of by the patient. Despite the custodial constraints against self-management,vertical farm tower peer education was successfully utilized as a prominent strategy for promoting health education and compliance for inmate-patients at lower security levels. While clinical and custodial staff had institutionalized aversion to dealing with inmates on an educational level, the cultural processes by which inmates tend to mentor other inmates was strong. As described by an associate warden of health care who was involved in the pilot program, “they, particularly the older ones or the ones trying to get themselves together—like for parole or if they found God—are all about educating each other or at least other inmates who they talk to. I knew back then I wasn’t going to tell my officers to deal with making sure they got their information on how to control their disease, but I knew they’d be passing back and forth any sort of knowledge that worked for them that they got in triage or somewhere else.” The custodial supervisor in the above passage was describing the exact institutional obstacle of organizational process that was discussed at the outset of the planning process. Custodial personnel were concerned with one issue—custody concerns. Nothing else mattered. To attempt to alter this highly institutionalized method of thought and process of organizational behavior was not the point of the CCM program, nor was it even considered feasible. Utilizing a train-the-trainer approach via another highly institutionalized process—that of inmate-to-inmate communication channels—was the preferred approach for successful implementation of the model and improved treatment outcomes. In order to make best use of the inmate communication channels to enable self management and to better understand the obstacles to self-management within this setting, a rudimentary analysis was performed. Two primary factors were identified during this analysis: self-efficacy and health literacy. From discussion sessions with the mental health clinicians, it was known that self-efficacy was a limiting factor for improving health in this population. Self-efficacy is generally defined as a person’s perception that she or he has the intrinsic capability to attain a goal . The second limiting factor identified was health literacy, which, given the average educational level of seventh grade, was considered an impediment to self-management. While health literacy does not specify a certain level of understanding within a given point of time concerning one’s health, it does consider the inmate-patient’s ability to understand and follow a clinician’s general instructions. A study of diabetic patients of various ethnic backgrounds found evidence that improvements in self-efficacy were associated with improvements in diabetes care outcomes . After controlling for ethnicity and health literacy levels, researchers found that increasing self-efficacy was related to patient self monitoring. On the basis of this and related research, the implementation’s management team concluded that nothing needed to be done immediately to improve health literacy or self-efficacy. It was felt that, as treatment outcomes improved over time, these related concerns would be naturally addressed and could be revisited as the program evolved. Patient education would be enhanced at the treatment encounter, and as treatment outcomes and inmate-patient experience improved due to better coordination of care, overall health literacy would improve due to the natural inmate communication channels.

The community at large typically marginalizes the incarcerated. Due to the high rate of recidivism, parolees become part of the broader community when released and then return to the prison environment, potentially repeating this cycle several times. If not treated in the prisons, chronic conditions and communicable diseases eventually become public health problems. While under the custodial confines of the state, the incarcerated also access the specialty services and acute care from community providers when health treatment considerations warrant such visits. Hence, care coordination, case management, and discharge planning are critical functions connecting the inmate-patients with their communities. Clinical staff within CDCR performs these specialty care visits. Community resources for the newly paroled, however, were and continue to be scarce. Community-level integration efforts with CDCR were not viewed as a priority by agency staff. While such efforts could have been integrated into CCM planning, they were not—perhaps due to agency staff’s overwhelming workload. While the literature does not currently state that community-prison integration is a primary aspect of successful CCM implementation, it is here argued to be significant due to recidivism. Unfortunately, however, it was not picked up as an element of overriding concern. The prison health care reform effort in California did not address the linkage with the community through provider-network development and community partnership. However, preliminary steps were taken to establish the public-private partnership with the local public health agencies. Discharge planning for the parolees was also deemed critical as it helped ensure continuity of care and avoided burdening the emergency departments in the community. This chapter has reviewed the process by which the new structure and processes proposed by the private-sector chronic care model were implemented within the public correctional setting. The challenges to implementation were met by carefully modifying the technical details of the program to fit the institutional context of the environment and the people who operate within it. Program level of analysis was introduced as a concept helpful for understanding the nature of departmental behavior because this project required collaboration and motivation at the program level, not at the overall organizational-mission level. This is an important concept that will carry over to the next chapter, where management behavior is explored. The actions considered by managers are reviewed at the program level of analysis in order to better understand both the motivation of this employee level and how their actions can be best guided to enable program implementation success.The ability of managers to transform organizations is an often-visited topic, debated by various disciplines within both academic and practitioner settings. Successful implementation of a program is a learning opportunity for scholars of many domains because numerous associations between the variables of performance and outcomes can be drawn. The literature specific to implementations within the public sector, particularly those studies looking at leaders to manage the change, provides many examples of failure . Peeking through the fog of these tales of derailment are the few stories of hope: implementations that provide evidence of success despite the odds.

Vaccination with CNS antigens can induce autoreactive T cell responses that home to sites of injury in the CNS and can inhibit neuronal degeneration in different models of neurological disease and injury, including spinal cord and head injury, Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, glutamate toxicity, and glaucoma . It is thought that immunization activates CNS-reactive T cells that enter the CNS, secrete neurosupportive factors and shift the phenotype of resident microglia to one that is more neurosupportive. The initial studies of this therapeutic strategy immunized animals with oligodendrocyte antigens such as myelin basic protein . Since autoimmunity to oligodendrocyte antigens can lead to a multiple sclerosis -like disease in experimental animals, subsequent studies of neuroprotective vaccines have focused on vaccinating with CopaxoneH . CopaxoneH is a mixture of synthetic polypeptides composed of four amino acids in a random sequence dissolved in an aqueous solution. Frequent CopaxoneH injection induces regulatory T cell responses that have partial cross-reactivity with myelin antigens and this treatment has been approved as a therapy for relapsing-type MS. The vast majority of neuroprotective vaccine studies in animal models of neuropathological disorders have, however, administered myelin antigens or CopaxoneH in complete Freund’s adjuvant , an adjuvant that is unsuited for human use. There have been few reports of adjuvant-free CopaxoneH having beneficial effects in animal models of neuropathological disorders other than MS. Our previous studies of antigen-based vaccine therapies for inhibiting autoimmune disease have shown that the ability of a vaccine to induce protective T cell responses depends critically on which self-antigen is administered. This is because each self-antigen has a unique expression pattern and impact on T cell selftolerance induction. Accordingly,vertical tower for strawberries self-antigens have different immunogenicities and should vary in their ability to induce neuroprotective T cell responses.

Random copolymers as CopaxoneH may not be optimal immunogens for inducing neuroprotective T cell responses since only a small portion of the induced T cell response may be capable of cross-reacting with CNS antigens. Hence, further studies are needed to examine how the nature of the antigen used in neuroprotective vaccines affects the efficacy of the treatment. Current treatments for Parkinson’s disease temporarily ameliorate its symptoms but do not slow progressive loss of dopaminergic neurons. Accordingly, new approaches to slow the degeneration of the nigrostriatal dopaminergic system are urgently needed. It is thought that oxidative stress, protein nitration andactivated microglia contribute to the loss of dopaminergic function in human PD. Additionally, there is a growing appreciation that CD8+ and CD4+ T cells significantly infiltrate the SN of patients with PD . All of these potentially pathogenic factors are elicited by treatment with the neurotoxin MPTP. The MPTP mouse model of PD has therefore been extensively used to assess neuroprotective strategies. Several studies have shown that vaccination with oligodendrocyte antigens or CopaxoneH in CFA preserves dopaminergic neurons in MPTP treated mice. These studies, however, did not determine whether the vaccine-induced immune responses limited the initial nigrostriatal dopamine system damage and/or promoted long-term neurorestoration. We began our studies asking whether vaccination with tyrosine hydroxylase , a neuronal protein involved in dopamine synthesis, could protect striatal dopaminergic neurons to a greater extent than CopaxoneH in the MPTP model of PD in mice. Contrary to our expectations, we observed that immune stimulation by the CFA adjuvant, regardless of the emulsified antigen, appeared to be the major neuroprotective factor.The BCG vaccine developed against childhood tuberculosis contains live attenuated Mycobacterium bovis that is closely related to Mycobacterium tuberculosis, and has been administered safely to billions of individuals since the 1920s [29,30]. We describe the neuroprotective effects of BCG vaccination in the MPTP mouse model and discuss possible underlying mechanisms.

Our results suggest that general immune stimulation in the periphery may provide a new strategy to help slow disease progression in some neurodegenerative diseases.Studies of neuroprotective vaccines have focused on using CopaxoneH since it induces protective immune responses that cross-react with myelin antigens and because it is in clinical use for treating MS. We wanted to test whether immunization with a dopaminergic neuron antigen might have a more beneficial effect in the MPTP mouse model of PD, since this should direct vaccine induced T cells to the brain areas that were damaged by MPTP treatment and that slowly degenerate in human PD. We chose tyrosine hydroxylase as a test antigen because it is involved in dopamine synthesis and is predominantly expressed in striatal dopaminergic neurons. We isolated TH from recombinant E. coli inclusion bodies, and purified it using affinity chromatography and preparative SDS-PAGE as described in Materials and Methods. Gel analysis of the purified TH is shown in Supplement Figure S1. Since it takes 10–14 days for vaccine-induced immune responses to peak, and MPTP has a very immediate toxic effect, we immunized mice with TH or CopaxoneH in CFA 10 days before MPTP treatment. A group of control mice received only saline. The animals were sacrificed 21 days after the last MPTP treatment, which is a relatively long time point for such studies, because we wanted to test for potential neurorestorative effects of vaccination. As an initial read-out of the vaccine’s ability to preserve dopaminergic system integrity, we measured [3 H]WIN 35,428 binding to DAT in mouse striatal homogenates. We found that the mean DAT WIN binding levels were higher in striata from MPTP-treated mice that received CFA, regardless of whether they received CFA alone, TH/CFA, or CopaxoneH/ CFA, compared to that in unvaccinated MPTP-treated mice . Specifically, compared to unvaccinated MPTP-treated mice, the levels of striatal WIN binding were 43% higher in MPTP-treated mice that received CFA alone and 34% higher in mice that received CopaxoneH/CFA . The level of striatal WIN binding was 17% higher in MPTP-treated mice that received TH/CFA, but this was not statistically significant. These results argue against our initial hypothesis that a neuronal self-antigen may provide a more efficacious neuroprotective vaccine. Rather, the results suggest that peripheral immunostimulation by CFA was the major beneficial factor.It is possible that immune responses elicited by CFA vaccination limited MPTP’s direct effects or promoted the subsequent restoration of dopaminergic neuron integrity. We therefore performed a more detailed study of the effects of CFA immunization on DAT levels 4 and 21 days after the last MPTP treatment. Groups of mice were vaccinated with CFA or saline, and 10 days later were given MPTP for 5 consecutive days.

Four days after the last MPTP treatment, the mean levels of striatal WIN binding were 18% higher in the CFA treated group than in unvaccinated MPTP-treated mice, but this increase was not statistically significant . This suggests that CFA vaccination did not differentially affect the uptake, distribution or metabolism of MPTP and that CFA-induced immune responses have little or no ability to limit the acute toxicity of MPTP. We also examined similarly treated mice 21 days after the last MPTP treatment. We found that the mean levels of striatal DAT WIN binding was 29% higher in CFA vaccinated mice that received MPTP,vertical growing compared to unvaccinated MPTP-treated mice . These data again demonstrate the beneficial effects of CFA treatment in our model. Additionally, the increase in striatal WIN binding observed in vaccinated vs. unvaccinated MPTP-treated mice from 4 to 21 days post treatment , suggests that CFA treatment promoted a greater rate of neurorestoration. Indeed, 21 days after MPTP treatment, only CFA-treated mice displayed a significant increase in striatal DAT WIN binding compared to levels 4 days post-treatment, suggestive of a neurorestorative effect.CFA is unsuitable for human use, but its main immunogenic component, inactivated Mycobacterium tuberculosis, is closely related to the live attenuated Mycobacterium bovis used in the BCG vaccine against childhood tuberculosis. We hypothesized that the peripheral immune responses induced by BCG immunization may also be neuroprotective. C57Bl/6 mice were immunized with BCG and 10 days later they, and a control group of unvaccinated mice, received MPTP. Twenty one days later, their striatal WIN binding levels were measured. Mice vaccinated with BCG had significantly higher levels of WIN binding than MPTP controls . In addition, striatum from mice vaccinated with BCG also had significantly higher DA content . Thus, BCG vaccination had a significant beneficial effect on both striatal DA content and DAT ligand binding levels.Previous studies have shown that the number of microglia increases rapidly in the striatum after MPTP treatment and play an active role in MPTP-induced nigro-striatal system damage. Inflammatory type microglia are considered detrimental to neuron survival after a neuro-toxin insult and blockade of microglia activation was neuroprotective in the MPTP mouse model of PD. To examine whether BCG vaccination also affected the microglial reaction to MPTP toxicity, we treated other groups of mice with BCG or saline prior to MPTP treatment and counted the number of microglia in their midbrains three days post-MPTP treatment. We found that the number of Iba1+ microglia cells was significantly greater in animals that received MPTP compared to that in mice that received only saline , as also reported by others . In contrast, the Iba1+ cell number in SNc of BCG-vaccinated mice that received MPTP was similar to that in mice that only received saline . We also observed that the Iba1+ cells in the unvaccinated MPTP-treated mice had large cell bodies with only a few short thick processes, a morphology associated with microglia activation. In contrast, the Iba1+ cells in mice that received BCG before MPTP treatment had small cell bodies with long-fine processes similar to those in saline-treated control mice, suggesting a resting state. Thus, BCG vaccination prevented the MPTP induced increase in the number of activated microglia in the SNc, suggesting that general immune stimulation in the periphery can limit CNS microglia responses to a neuronal insult. At 21 days post-MPTP, stereological analysis revealed that the number of TH+ cells in the SNc of animals that received BCG was on average 6% greater than that in mice that received only MPTP , although this was not statistically significant .Vaccination with CNS antigens has beneficial effects in a number of different animal models of neurological disease and injury. This strategy is based on inducing CNS-reactive T cells which home to areas of damage and exert beneficial effects locally in a process termed ‘‘protective autoimmunity’’ . Early studies of neuroprotective vaccines administered myelin antigens, which raised safety concerns because of their potential for inducing a MS-like disease. Subsequent studies used CopaxoneH which has some resemblance with MBP, and in its aqueous form is approved for MS treatment. Almost all these studies, however, used CFA as an adjuvant and often did not report on the effects of CFA alone. The studies that did examine CFA often reported that these treatments had some beneficial effect, although of lower magnitude than the myelin antigen or CopaxoneH in CFA. Contrary to our initial expectations, we found that immunization with a dopaminergic neuron antigen did not provide a greater beneficial effect. Rather, CFA itself appeared to be main factor associated with higher levels of striatal WIN binding in vaccinated MPTP-treated mice. CFA treatment did not significantly alter the level of striatal DAT WIN binding 4 days after MPTP treatment, suggesting that CFA-induced immune responses cannot limit the acute toxicity of MPTP. Twenty one days post-MPTP treatment, however, the average levels of striatal DAT WIN binding in CFA treated MPTP-treated mice was significantly greater than that in unvaccinated MPTP-treated mice. The ratio of striatal WIN binding in vaccinated mice versus unvaccinated MPTP-treated mice increased from 4 to 21 days, suggesting that the CFAinduced responses promoted a greater rate of neurorestoration. CFA-treated mice, but not unvaccinated mice, had significantly higher striatal WIN binding 21 days vs. 4 days after MPTP treatment, indicating a neurorestorative effect. Based on the neuroprotective effects of CFA, we turned to testing BCG vaccination. Potential advantages of BCG vaccination include not only its established safety record over many decades of worldwide use in humans, but also that the attenuated BCG slowly replicates in the vaccinated individual, inducing immune responses over many months. Accordingly, BCG vaccination could provide a long-term source of neurosupportive immune responses.We observed that BCG vaccination significantly preserved striatal DAT WIN binding and DA content compared to that in unvaccinated MPTP-treated mice.

Much remains to be elucidated about the assembly and activity of these core clock proteins, but insight into the molecular basis of circadian period determination is growing thanks to the integration of genetic studies from model organisms and humans with biochemical, structural and cell-based studies. High-resolution structures have now been determined for most of the globular domains of core clock proteins and some of their complexes, with a growing appreciation for the important role that flexible linkers and intrinsically disordered regions play in tuning clock protein function and clock timing. We will focus here on recent advances in our understanding of the mechanisms of period control by some of the negative elements of the core feedback loop , highlighting the nanoscale structural and dynamic properties of clock proteins that influence their functional roles as repressors within the core TTFL. For a review of mutations in human CRY1 and CRY2 that influence circadian timing, please refer to.Although transcriptional regulation by CLOCK:BMAL1 and downstream transcription factors is essential for generation of robust circadian rhythms, it is becoming increasingly clear that post-transcriptional and post-translational modifications of core clock components play an important role in both the generation of circadian rhythms and determination of its intrinsic period. While many studies have identified roles for post-translational modification of CRY and PER proteins in clock timing, we will focus here on an in-depth analysis of the regulation of PER2 by CK1δ/ε,vertical growing systems as it relies on an elaborate integration of post-translational modifications that ultimately determine the relative abundance of PER2 needed to maintain a ~24-hour period. PER1 and PER2 serve as interaction hubs for both CK1δ/ε and cryptochromes with dedicated binding sites that maintain stable complexes with these core clock proteins throughout most of the repressive phase of the clock each day , a feature that is notably absent in PER3.

The flexibility of PER proteins likely contributes to their role as labile scaffolds for transcriptional regulators of the clock; PER1–3 are all predominantly intrinsically disordered, conferring a susceptibility to regulation by post translational modifications and promiscuity for interaction partners that is common among other intrinsically disordered proteins. Two tandem PER-ARNT-SIM domains present in the N-terminus of all three PER isoforms allow them to form homodimers and heterodimers mediated by the β-sheet surface of the PAS-B domain . Deletion of the core PAS-B motif in the Per2 Brdm1 mutant leads to a loss of circadian rhythms, demonstrating that protein-protein interactions facilitated by this region are essential for clock function. Notably, mutation of just a single residue, W419E, at the dimer interface in the mouse PER2 PAS-B domain potently disrupts formation of homodimers and was recently shown to reduce phosphorylation by CK1δ. Moreover, the PER2 earlydoors mouse possesses a point mutation in the interdomain linker connecting the PAS-A and B domains that reduces PER2 stability to shorten the circadian period. Collectively, these findings demonstrate that dimerization via the PER PAS domains is critical for protein stability and clock timing, although more work is needed to understand the exact role that PAS domains play in orchestrating clock protein complexes and PER turnover.The turnover of PER2 is primarily mediated by CK1δ/ε-dependent phosphodegrons to intimately link kinase activity with PER2 stability. Dimerization of the PER2 PAS-AB domains positions the phosphodegron site of each monomer to protrude from the same face of the PAS-AB domain homodimer. This phosphodegron largely conforms to the canonical β-TrCP recognition motif, DSGϕXS, where ϕ is a hydrophobic residue and the two conserved serines become phosphorylated to make the substrate competent for β-TrCP recognition.

CK1δ/ε phosphorylation of S478 in mouse PER2, the first of the two serines in the motif, is required for interaction with the E3 ubiquitin ligases β-TrCP1/2, leading to ubiquitination of PER2 and its proteasomal degradation. However, this PAS-B phosphodegron is unique to PER2; PER1 utilizes a different CK1δ/ε-dependent phosphodegron N-terminal to the tandem PAS domains. This N-terminal phosphodegron is also conserved in PER2 and may play an auxiliary role in its turnover, as clock timing was only modestly impacted in the PER2 S478A transgenic mouse. An interaction with the E3 ubiquitin ligase MDM2 also influences PER2 stability independently of CK1δ/ε activity, opening the door for a complex integration of signals to mediate PER2 degradation. Although other kinases such as CK1α, CK2, SIK3 and Cdk5 phosphorylate PER2, CK1δ/ε are the only kinases that stably associate with PER2 throughout the night, moving from the cytoplasm into the nucleus with the other core clock proteins. CK1δ and the related isoform CK1ε bind to the Casein Kinase-Binding Domain in PER2 via two conserved motifs that flank a serine-rich region. Notably, mutation of the first residue in a series of five consecutive serines that are phosphorylated in human PER2 markedly decreases its stability and shortens circadian period by ~4 hours to manifest as Familial Advanced Sleep Phase Syndrome. Because CK1δ/ε-dependent phosphorylation of PER2 in this region links circadian timekeeping to this human sleep disorder, the serine-rich cluster in the CKBD has been named the FASP region. Recent studies have begun to elucidate the molecular basis for CK1δ/ε activity in the FASP region of PER2 to understand how it exerts such powerful control over circadian period. Phosphorylation of the first serine in this cluster by CK1δ leads to the obligately sequential phosphorylation of downstream serines.Therefore, the human S662G FASPS allele eliminates the ability of CK1δ to prime its activity downstream, disrupting all phosphorylation in the FASP region.

There is strong evidence that FASP phosphorylation plays a critical role in stabilizing PER2 protein, as the S662G mutation in human PER2 leads to premature turnover of the protein and a dramatically shorter circadian period of ~20 hours in a transgenic mouse model, while use of a phosphomimetic mutation in human PER2 that presumably leads to constitutive priming of sequential FASP phosphorylation confers a long period of ~25 hours in vivo. Although it is not yet known how FASP phosphorylation contributes to regulation of PER2 stability, mutation of the priming serine that blocks phosphorylation of FASP downstream serines increases CK1δ/ε activity at the phosphodegron site S478 to suggest that the phospho-FASP region could antagonize CK1δ/ε activity at the phosphodegron site S478. The opposing effects of FASP and phosphodegron phosphorylation likely involves cellular phosphatases like PP1 that contribute to CK1δ/ε-dependent regulation of circadian period through PER2, although there could also be a direct mechanistic link between FASP phosphorylation and regulation of CK1δ/ε activity. In fact, the functional linkage of phosphorylation at the FASP region and phosphodegron by CK1δ/ε has been described as a phospho switch that introduces a phase-specific delay to PER2 degradation necessary for proper circadian timekeeping. Interestingly, while introduction of the analogous priming site mutation in mouse PER1 destabilized PER1 and led to a shorter circadian period, it also caused an advance in feeding rhythms not seen in the PER2 mutant, suggesting that further study of the regulation of PER turnover could help uncouple distinct functions of PER1 and PER2 in control of circadian period and clock outputs. PER proteins are also subjected to a number of other post-translational modifications aside from phosphorylation. PER2 is O-GlcNacylated within the FASP region,outdoor vertical plant stands modifying the priming serine along with two sites downstream. Both O-GlcNAc transferase and O-GlcNAcase , the enzymes responsible for adding or hydrolyzing O-GlcNAc, respectively, are expressed or activated in a circadian manner, and factors that increase O-GlcNacylation also lead to a concomitant decrease in FASP phosphorylation that reduces PER2 protein levels.

These results support a model for competition between OGlcNacylation and phosphorylation at this key regulatory region, suggesting a mechanism by which glucose metabolism could modulate the circadian clock by antagonizing phosphorylation of PER2 in the stabilizing FASP region to represent a direct link between the circadian clock and metabolism as a “nutrition switch” . Acetylation also plays an important yet enigmatic role in PER2 regulation of the clock, first observed through manipulation of SIRT1, the nicotinamide adenine dinucleotide – dependent deacetylase. Although PER2 becomes acetylated as it accumulates throughout the repressive phase of the circadian clock, the identity of the acetyltransferase that modify PER2 is currently not known, nor is it known where PER2 is acetylated throughout the protein. Nonetheless, loss of SIRT1 results in elevated levels of acetylated PER2 in mouse liver to attenuate the robustness of circadian rhythms, while overexpression of SIRT1 facilitates PER2 degradation. Because acetylation and ubiquitination both target lysine residues, it is possible that these modifications compete for the same residues to directly control PER2 stability. However, there is some evidence that regulation of PER2 by acetylation could be more complicated, as acetylation at K680 on mouse PER2, located downstream of the serine cluster in the FASP region, is hyperacetylated following inhibition of SIRT1 and leads to a decrease in FASP phosphorylation, suggesting that an interplay between acetylation and phosphorylation of the FASP region could also control CK1δ/ε activity on PER2 to regulate the balance of the phosphoswitch. The timing of eukaryotic circadian rhythms from green algae to humans is heavily influenced by CK1δ/ε and its related orthologs. Like other Ser/Thr kinases, CK1δ/ε has a typical two-lobed structure , but little is known about the molecular mechanisms by which activity of the CK1 family is regulated. The activation loop is one key feature that distinguishes the CK1 family from other Ser/Thr kinases. Unlike many other kinases, the kinase domain of CK1 family members is not regulated by activation loop phosphorylation; therefore, they are considered to be constitutively active. The CK1 family acts as phosphate-directed kinases that preferentially recognize a D/E/pSxxS consensus motif, where a phosphorylated serine or a similar negative charge within the substrate templates activity at a serine located 3 or 4 residues downstream. Interestingly, at least two functionally important CK1δ/ε-dependent phosphorylation sites on PER2, the phosphodegron and the FASP priming site, do not conform to this consensus motif and likely serve as slow, rate-limiting steps for PER2 regulation. Therefore, understanding the molecular basis for kinase activity and substrate selectivity by CK1δ/ε has the potential to yield important insights into circadian timekeeping. In particular, a better understanding of the molecular mechanisms underpinning CK1δ/ε-dependent phosphorylation of PER will provide a framework for treating circadian disorders by targeting CK1δ/ε to modulate the clock. The kinase domain of CK1δ/ε contains several highly conserved anion binding sites located around the C-terminal lobe, including two that flank either side of the substrate binding cleft. We recently showed that these anion binding sites regulate the overall kinase activity of CK1δ/ε, as well as influence the substrate specificity of the kinase at both consensus and non-consensus sites. The significance of these highly conserved anion binding sites was initially suggested by the discovery of the first period-altering allele in mammals, the CK1ε tau allele that causes a dramatically shortened circadian period of ~20 hours. The R178C substitution in the tau kinase was predicted to disrupt an anion-binding pocket near the substrate binding region to decrease CK1δ/ε activity. While the tau mutant kinase did exhibit reduced activity on some generic kinase substrates as well as the FASP region of PER2, it led to a paradoxical gain of function at the PER2 phosphodegron that decreased stability of the protein. Crystal structures of the tau kinase domain recently revealed that disruption of the anion binding pocket at S1 in the mutant is linked to an allosteric structural switch in the activation loop that encodes a preference for the PER2 PAS-B phosphodegron site S478. Allostery is a common regulatory feature of protein kinases that allows for a switch-like, ultrasensitive regulation of their biological activity. The activation loop and flanking regions distinguish CK1 from all other Ser/Thr kinases, containing residues involved in the coordination of anions at three conserved sites, S1-S3. Therefore, these sites likely play a role in the CK1 family-specific regulation of kinase activity, perhaps through binding of anionic, phosphorylated residues. Interestingly, the entire substrate binding cleft that allosterically links anion binding to substrate selectivity is 95% identical from humans to green algae, suggesting that the mechanisms discovered in mammalian CK1δ/ε may also regulate kinase activity and circadian period across other eukaryotic clocks.

KineTACs are fully recombinant bispecific antibodies built of human scaffolds that utilize cytokine-mediated internalization of its cognate receptor to enable highly selective lysosomal delivery of both cell surface and extracellular proteins. To demonstrate the utility of this platform, chemokine CXCL12 was chosen as it specifically binds the atypical chemokine receptor CXCR7, a decoy receptor that constitutively internalizes and recycles.We show that KineTACs bearing CXCL12 can efficiently utilize CXCR7 internalization for lysosomal degradation applications and are generalizable against various therapeutically relevant proteins .To demonstrate proof-of-concept that CXCL12 bearing KineTACs can degrade cell surface proteins, we first targeted programmed death ligand. Overexpression of PD-L1 on cancer cells leads to inhibition of checkpoint protein programmed death protein 1 and suppression of cytotoxic T cell activity.First, we generated knob-in-hole bispecifics14 in which the human CXCL12 chemokine was N-terminally fused to the knob Fc domain and the antibody sequence for atezolizumab , an FDA approved inhibitor of PD-L1, was fused to the hole Fc . CXCL12 bearing bispecifics are not limited by the light chain mispairing problem, which is common to bispecific IgGs with Fabs on both arms, enabling full assembly of KineTACs during mammalian expression.A His tag was introduced on the knob arm to allow purification of the formed bispecific from unwanted hole-hole homodimers that may form. Next, we confirmed that the PD-L1 targeting KineTAC retains binding to PD-L1 using biolayer interferometry. Furthermore, an isotype control of the CXCL12 KineTAC, which incorporates a Fab arm to the SARS-CoV-2 spike protein,vertical planters for vegetables retained binding to endogenous CXCR7 expressed on triple negative breast cancer cell line MDA-MB-231 . This data suggested that both anti-PD-L1 and CXCL12 arms of the KineTAC were functional in the bispecific context.

To determine whether CXCL12-Atz could degrade PD-L1, MDA-MB-231 cells were treated with varying concentrations of the KineTAC. After 24 hr treatment, levels of PD-L1 were quantified using western blotting, demonstrating that both glycosylated forms of PD-L1 were substantially degraded, with a maximal percent degradation of roughly 70% . Control antibodies, such as atezolizumab Fab or CXCL12 isotype, do not induce the degradation of PD-L1 either alone or in combination, indicating that PD-L1 degradation is dependent on the bispecific KineTAC scaffold . Finally, flow cytometry and western blotting was used to verify that the PD-L1 degradation observed is due to depletion of cell surface PD-L1. We next sought to determine whether the KineTAC platform could be generalized to degrade other therapeutically relevant cell surface proteins. First, we targeted human epidermal growth factor receptor 2 , which is frequently upregulated in cancer and linked to breast cancer invasiveness and tumor progression.To develop a KineTAC targeting HER2, we incorporated the antibody sequence for trastuzumab , an FDA approved HER2 inhibitor, into the KineTAC scaffold . Various breast cancer cell lines endogenously expressing HER2 were incubated for 24 hr with CXCL12-Tras. Substantial degradation of HER2 was observed in MCF7 and MDA-MB-175VII cells, with Dmax of 51 and 62%, respectively . Unsurprisingly, lower percent degradation was observed in SK-BR-3 cells, which over expresses HER2 relative to CXCR7, suggesting that the maximal percent degradation mediated by KineTACs could be dependent on the expression of the target protein relative to CXCR7. Next, we targeted epidermal growth factor receptor for degradation. EGFR is implicated as a driver of cancer progression, and EGFR inhibitors are approved for use in non-small cell lung, colorectal, and gastric cancers. We developed KineTACs targeting EGFR by incorporating cetuximab , an FDA approved EGFR inhibitor, into the KineTAC scaffold .

Following 24 hrtreatment with CXCL12-Ctx, EGFR levels were dramatically reduced in HeLa cells, with a Dmax of 84% observed . This result was recapitulated in various breast and lung cancer cell lines, including MDA-MB-231, A431, and NCI-H292 .Using a previously described antibody against CUB domain-containing protein 1, we observed near complete degradation of CDCP1 after 24 hr treatment of HeLa cells, with a Dmax of 93% . KineTACs also enabled the degradation of tumor-associated calcium signal transducer 2 , the over expression of which has been linked to tumor progression in a variety of tumors. In MCF7 cells, we observed a Dmax of 51% after treatment with TROP2 targeting KineTAC . We then tested whether KineTACs are active to degrade the checkpoint protein PD-1 in CD8+ T cells isolated from primary human peripheral blood mononuclear cells. T cells were then activated, causing over expression of PD-1 on the cell surface along with other activation markers . Activated T cells were then treated for 24 hr with a PD-1 targeting KineTAC, which incorporates the antibody sequence for nivolumab , an FDA approved PD-1 inhibitor . Following treatment with CXCL12-Nivo, cell surface PD-1 levels were dramatically reduced, with a Dmax of 82%, compared to nivolumab isotype control, which is known to induce slight internalization of PD-1 .Overall, these results demonstrate the generality of the KineTAC platform for degrading a variety of cell surface proteins for degradation .Next, we sought to determine which properties, such as binding and receptor signaling, of the KineTAC are critical for efficient degradation. Alongside CXCR7, CXCL12 binds the signaling receptor CXCR4, which upon agonism will cause downstream signaling followed by receptor internalization and degradation. Signaling through CXCR4 could be counter-productive if using KineTACs to target cancer drivers, as CXCR4 over expression and agonism is linked to tumor metastasis.Thus, avoiding CXCR4 signaling could be an important consideration in developing KineTACs for therapeutic purposes.

To test this, KineTACs bearing previously described antagonistic variants of CXCL12 , that retain binding to both CXCR7 and CXCR4 but prevent CXCR4 signaling, were incorporated into the KineTAC scaffold with atezolizumab.Following 24 hr treatment in MDA-MB-231 cells, all three antagonistic variants retained the ability to degrade PD-L1 to a similar degree compared to CXCL12WT . This data suggests that CXCL12 signaling through CXCR4 is not critical for degradation, allowing flexibility to include or eliminate it depending on the biology we wish to affect. Next, the dependence on binding affinity to target receptor was probed by introducing alanine mutations into key interacting residues of atezolizumab’s complementary determining regions known to interact with PD-L1 based on the known structure of the complex. We generated a library of alanine scanned mutants of atezolizumab with a range of binding affinities and corresponding kinetic parameters to PD-L1, as measured with BLI . The atezolizumab mutants were then introduced into the KineTAC scaffold with CXCL12WT and tested for their ability to degrade PD-L1 . Correlating the PD-L1 levels post treatment to the different kinetic parameters of these binders,vertical farming technology we find that degradation is correlated to the KD and the dissociation rate , but not to the association rate. Of the mutants tested, wild-type atezolizumab had the highest binding affinity and induced the greatest level of PD-L1 degradation. Therefore, over this affinity range,the levels of degradation are dependent on the binding affinity of the antibody arm to the target protein. To determine whether a pH-dependent antibody binder against the target protein would affect degradation, BMS936559, an anti-PD-L1 antibody reported to release PD-L1 in acidic conditions29, was introduced into the KineTAC scaffold. Treatment with CXCL12- BMS936559 compared to CXCL12-Atz showed that pH-dependent release of PD-L1 slightly decreases the maximal level of degradation observed . This result is not due to differences in KD as atezolizumab and BMS936559 are reported to have similar binding affinities to PD-L1.30 To investigate whether the binding epitope on the protein of interest could impact degradation, we introduced additional HER2 and EGFR targeting antibodies into the KineTAC scaffold that have been described to bind different epitopes. For HER2, pertuzumab , which is known to bind a distinct epitope from trastuzumab on HER231, was introduced into the KineTAC scaffold . Following 24 hr treatment of MCF7 cells, we find that CXCL12-Tras is superior to CXCL12-Ptz at lower concentrations, indicating that epitope can alter the dose response to KineTACs . For EGFR, we introduced five different antiEGFR binders , necitumumab , and matuzumab 32 into the KineTAC scaffold. Following 24 hr treatment of HeLa cells, we observe that some epitope binders, such as necitumumab and matuzumab, retain similar levels of EGFR degradation compared to CXCL12-Ctx, while other epitope binders, such as depatuxizumab, nimotuzumab, and panitumumab, abrogate or impair the ability to degrade EGFR .

Further, the degradation observed for each binder is not correlated to binding affinity . This data highlights the dependence of KineTAC-mediated degradation on target binding epitope.Next, we asked whether glycosylation of the KineTAC Fc domain at the N297 position would impact degradation. The N297G mutation is commonly introduced into IgGs to produce an aglycosylated form to eliminate effector function. However, glycosylation at N297 can impart greater stability and favorable pharmacokinetic properties.The glycosylation site at N297 was re-introduced to the CXCL12-Atz scaffold and the degradation efficiency between the glycosylated and aglycosylated forms compared. We find that glycosylation at N297 does not significantly impact PD-L1 degradation levels . Thus, the improved stability and pharmacokinetic properties of KineTACs can be utilized for in vivo use without major disruption to degradation efficiency. Finally, we determined whether the bispecific antibody construct used could influence levels of degradation. To this end, a Fab fusion construct in which CXCL12 is fused to the N-terminus of the atezolizumab Fab heavy chain via a flexible Avidin tag linker was co-expressed with atezolizumab Fab light chain . The CXCL12-Atz Fab fusion retained binding to PD-L1 Fc fusion as measured by BLI . After 24 hr treatment in MDA-MB- 231, the levels of PD-L1 were measured by western blotting. While the bispecific IgG construct caused significant degradation of PD-L1, the Fab fusion was unable to induce significant degradation, with a Dmax of only 20% observed . The differences in degradation between these two constructs could be due to several factors, including construct rigidity and linker length. Overall, this data highlights that the bispecific IgG is a useful KineTAC scaffold. We next sought to evaluate the mechanism of KineTAC-mediated degradation. To determine whether KineTACs catalyze degradation via the lysosome or proteasome, MDA-MB-231 cells were pre-treated with either media alone, bafilomycin , orMG132 . After 1 hr pre-treatment, cells were treated with CXCL12-Atz for 24 hrs. We observed that bafilomycin pre-treatment inhibited degradation of PD-L1, while MG132 had no effect, demonstrating that KineTACs mediate degradation via delivery of target proteins to the lysosome . Immunofluorescent microscopy revealed complete removal of EGFR from the cell surface following 24 hr CXCL12-Ctx treatment as compared to the cetuximab isotype, further highlighting that KineTACs induce robust internalization of target proteins . Furthermore, KineTAC-mediated degradation occurs in a time-dependent fashion, beginning after 6 hrs post-treatment with CXCL12-Atz, with the levels of PD-L1 continuing to decrease over time to near complete degradation at 48 hrs . We next wished to determine which of the two receptors is more important for degrading surface proteins in the context of KineTACs. To confirm that KineTAC mechanism of action occurs via CXCR7 and not CXCR4, RNA interference was used to knockdown the levels of CXCR4 in HeLa cells. After 48 hr transfection with CXCR4-targeting or control siRNA pools, cells were treated with CXCL12-Ctx for 24 hrs . Western blotting analysis revealed that EGFR degradation levels are unchanged with CXCR4 knocked down. This data highlights that CXCR4 is not necessary for efficient degradation and suggests that KineTACs operate through CXCR7-mediated internalization . Furthermore, KineTACs bearing CXCL11, a chemokine that specifically binds CXCR7 and CXCR3 but not CXCR434, are capable of degrading both PD-L1 and EGFR . This result further highlights that CXCR7 is the receptor responsible for KineTAC-mediated degradation and demonstrates the exciting opportunity for using alternative cytokines in the KineTAC scaffold to degrade target proteins. We next used quantitative mass spectrometry to determine whether proteome-wide changes occur following KineTAC treatment. Both the surface-enriched and whole cell lysates were analyzed following 48 hr CXCL12-Atz or CXCL12-Ctx treatment compared to PBS treated control in MDA-MB-231 or HeLa cells, respectively. For PD-L1 degradation, the surface-enriched sample revealed no significant changes to the proteome, with PD-L1 being the only protein down regulated in CXCL12-Atz treatment compared to control . Whole cell proteomics also revealed that no major changes are occurring . PD-L1 was not detected in the whole cell sample, likely due to low abundance of cell surface proteins relative to cytosolic proteins.

Targeted and template cores were taken by a single urologist at UCLA Clark Urology Center under local anesthesia using a MR/US fusion and biopsy tracking device .A dedicated uro-pathologist interpreted all biopsy cores . FT eligibility was assessed using three different ablative strategies to determine the extent of ablation that would be needed to eliminate the index lesion. Individual biopsy cores from each subject were assessed using database software to determine eligibility for each strategy. Men with positive biopsy cores limited to the ROI were considered eligible for all FT strategies . Men with positive cores adjacent to the ROI were considered eligible for quadrant and hemi-ablation. Those with ipsilateral but distant positive cores were considered eligible for hemi-ablation. A visual representation of different strategies can be seen in Figure 3. We evaluated eligibility using both a GS ≤ 4+3 and GS ≤ 3+4 threshold .Of the 454 men with biopsy positive ROI, 64 underwent radical prostatectomy and whole mount processing to facilitate MRI-histological correlation as previously described.7 Three-dimensional printed molds were used in cases processed after 2014. Lesion contours identified on whole mount histology were loaded onto custom software and elastically warped to match the mpMRI-defined prostate contour, allowing targets on mpMRI to be directly compared to lesions identified on whole mount histology. Eligibility for FT was then re-assessed based on evaluation of whole mount sections by a dedicated urologic pathologist . Descriptive statistics for patient characteristics were calculated for each group. Confidence intervals were calculated using a binomial assumption at a 95% threshold. KruskalWallis non-parameteric one-way analysis of variance and post-hoc tests were conducted to measure differences between continuous variables.

Pearson chi-squared tests were performed on categorical variables. Statistical significance was considered at p < 0.05 for all analyses. Statistical analyses were performed by a coauthor using Stata® software, version 13.1. Additional analyses were performed using JMP Pro,vertical agriculture version 13. 1408 men in our cohort underwent MRI/US fusion biopsy from 2010–2016. 454 men met the screening criteria in Figure 1. Of the 454 men with at least one biopsypositive ROI, 175 FT candidates were identified. Of the 914 men with a MRI suspicion score or higher, 19% were FT candidates . 57 men with small-volume GS 3+3 cancers were found . Eight men who would have otherwise qualified for FT were excluded on the basis of PSA > 20 ng/mL. Baseline patient characteristics for FT eligible and ineligible patients are shown in a supplementary table . Men were considered ineligible if the ROI contained either insignificant CaP or high-risk CaP. Eligible and ineligible men differed in age, ethnicity, free PSA and PSA density, mpMRI suspicion score, average number of positive cores, incidence of bilateral CaP, MCCL, and GS . While differences between components of inclusion criteria are expected, post-hoc tests nonetheless showed an increasing trend across all three categories with total PSA, PSA density, number of positive cores, and maximum cancer core length. Of the patients with GS ≤ 3+4, 154 were eligible for hemi-ablation or less, 140 for quadrant ablation or less, and 94 for site-specific ablation. When the inclusion criteria included those with maximum GS 4+3, 175 were eligible for hemi-ablation or less, 157 for quadrant ablation or less and 105 for site specific ablation . No man within this study is known to have undergone focal Therapy.64 men in this series underwent RP as first-line therapy with whole-mount processing of the specimen; 35/64 with 3D-printed molds. Average time from biopsy to surgery was 89.1 ± 32.5 days.

Examples of whole mount histology and 3D digital reconstruction are demonstrated in Figure 3. 25/64 patients who underwent RP would have qualified for FT on the basis of biopsy findings. 15/64 men qualified for FT on the basis of whole mount histological findings, with 16 discordant findings . Of the 13 patients who were classified as eligible for FT based on fusion biopsy and did not qualify based on whole mount , 4 were discordant due to a higher GS on whole mount, and 9 were due to the lesion crossing the midline. When examining factors associated with eligibility determined after RP, no significant difference was found for PSA density , prostate volume , or total serum PSA , although the study was not powered for analysis on whole-mount prostatectomy cases. Targeted and template biopsy, when combined, had a sensitivity, specificity, and accuracy of 80.0%, 73.5%, and 75.0%, respectively for determining eligibility for FT when compared to the whole mount gold standard. Targeted cores alone yielded a sensitivity of 73.3% , and a specificity of 47.9% , with an accuracy of 54.7% . FT has recently emerged as a potentially definitive treatment for localized CaP that aims to preserve quality of life.FT appears promising in initial studies using HIFU, cryotherapy, and FLA,but long-term oncological control has not been established. One key barrier is knowing a priori which patients will benefit from partial treatment,with some arguing for FT as an alternative to surgical intervention, and others for FT as a complement to active surveillance.While the multi-focality of CaP favors whole gland treatment, studies have emphasized the importance of the index lesion as a driver of metastatic potential.Recent studies indicate that low grade, low volume lesions behave in an indolent fashion, with limited metastatic potential.Contrasting these is a case report by Haffner and colleagues which investigated the clonal origin of lethal prostate cancer and found that its origin arose from a small, low-grade cancer focus in the primary tumor.

Nevertheless, more recent and larger studies continue to support the concept that CaP is driven by a single clone,and can be serially tracked with biomarkers and targeted biopsy.Furthermore, FT has been used successfully in treatment of other multifocal solid organ malignancies where secondary lesions have proven to be indolent.In the present study we estimated the proportion of men diagnosed by MRI/US fusion biopsy who would be eligible for FT. Eligibility criteria from a recent FDA-AUA-SUO workshop on partial gland ablation were used.6 Biopsy findings were also compared to whole mount histology in a subset of cases. We found that over a third of men with a MRI target and fusion biopsy-confirmed cancer were suitable candidates for FT; nearly a quarter met criteria for site-specific ablation. Fusion biopsy findings were generally concordant with whole mount findings, in agreement with findings shown previously.The majority of the false positives was attributed to the lesion crossing the midline. In this study, we called lesions that crossed the midline by even a few millimeters as ineligible for any method of focal therapy. In practice, many of these lesions are treatable using a site-specific ablation or ‘hockeystick’ ablation as described by Ahmed et al. Only in a minority of cases was assessment through targeted biopsy a failure due to upgrading. While eligibility criteria included GS ≤ 4+3 lesions, 89% of patients met more stringent criteria limited to GS ≤ 3+4. The increased sensitivity of MRI-targeted biopsy for detection of csCaP, widely reported for other situations, also appears valuable when evaluating for FT eligibility. In our work, over half of eligible men had csCaP localized to within a single ROI, while 40% of men had csCaP outside the index lesion. Both targeted and template biopsies were important in accurately classifying patients for FT. Overall accuracy using both methods was improved by 20% over using targeted biopsy alone . This suggests that the combined targeted and template biopsy approach is effective at ruling out focal therapy. While whole mount histology of RP specimens are generally concordant with targeted biopsy findings,the moderate agreement in eligibility assessment indicates that improved criteria need to be established. In the univariate analysis of eligible and ineligible patients, PSA density was significantly different between all three cohorts. While the RP data did not show a similar significant difference in PSA density,vertical farming aeroponics the difference suggests that PSA density merits further investigation as a eligibility criterion in a larger, powered study.While one potential source of error is the registration accuracy between MRI and US ,a larger issue is the underestimation of true tumor burden by MRI. of eligible patients had csCaP ipsilateral and adjacent to the ROI, qualifying for quadrant ablation. Le Nobin et al found that tumors required a 1 cm margin to achieve complete treatment, while Priester et al found that the average uniform margin to achieve complete treatment exceeded 1.5 cm.This supports the notion that biopsy cores should be taken from beyond the margins of the ROI when evaluating for site-specific or quadrant-based FT.

Moreover, these data suggest customization of focal therapy based on data from individual biopsy site locations around the apparent tumor margin.This also suggests that perhaps improved criteria for FT would include individual consideration of the position and size of the lesion, i.e. patient-specific planning, rather than uniform classification. Several limitations exist that preclude a more general interpretation of the findings presented. This study was hypothesis-generating and retrospective in nature, and was conducted at a single site with all biopsies performed by a single physician. Limited data were available for comparing fusion biopsy findings with whole mount histology. Nevertheless, the significant concordance between the two approaches for determining FT candidacy suggests that fusion biopsy may serve as an important aid to determine eligibility. Further, these results might be used to develop a framework for future prospective studies. The present findings suggest that more than one-third of patients with a biopsy-proven target were eligible for FT using consensus criteria; fusion biopsy with both targeting and template samples accurately characterizes the grade and extent of CaP for the purposes of determining FT eligibility Barrett’s esophagus is a risk factor for the development of esophageal adenocarcinoma .Challenges in the management of patients with BE include detecting areas of dysplasia or superficial cancer and surveillance after endoscopic treatment to evaluate for residual or recurrent disease. Dysplasia in BE may not be apparent during inspection using white light endoscopy . Therefore, current guidelines recommend endoscopic surveillance of BE with random 4-quadrant biopsy sampling every 1– 2 cm , in addition to targeted biopsy sampling of any visible abnormalities.This imperfect surveillance protocol can result in missed disease, with an estimated 25.3% of EAC procedures occurring within 1 year of a surveillance endoscopy.Dysplasia can be treated with endoscopic therapies including endoscopic mucosal resection ,radio frequency ablation ,cryotherapy,and others. However, high recurrence rates have been reported, including up to 33% recurrence of intestinal metaplasia or dysplasia at 2 years in the case of RFA.Residual disease, particularly at the GEJ, and the existence of disease buried beneath neosquamous epithelium are also sources of concern.Recently, advanced imaging techniques such as narrow band imaging and confocal laser endomicroscopy have sought to improve dysplasia detection in BE patients by allowing biopsies to be taken in a targeted rather than random fashion, even when focal abnormalities are absent on WLE inspection.Volumetric laser endomicroscopy utilizes optical coherence tomography to produce high-resolution, cross sectional surface, and subsurface images of the esophageal wall over a long continuous segment .Studies have examined the efficacy of VLE as applied to dysplasia detection in pre and post treatment surveillance as well as informing treatment selection.While the safety and feasibility of esophageal VLE imaging has been shown,the objective of this registry was to determine usage patterns of VLE in clinical practice and to estimate quantitative and qualitative performance metrics as they are applied to BE management.This is a prospective observational cohort study from August 2014 to April 2016. Patients were eligible for inclusion in this study if undergoing a clinically indicated upper endoscopy during which VLE was used for evaluation of the esophagus. Procedures were performed at 18 centers throughout the United States . Each site was eligible to enroll up to 100 subjects, with an overall registry enrollment cap of 1000 patients. Investigators were free to recruit patients with a variety of disease states at various stages of clinical management. Patients for whom the VLE device would be in conflict with the manufacturer’s Instructions For Use were excluded. This included use in anatomies where catheter deployment would generate significant risk, such as the setting of a tight stricture. The research protocol and informed consent forms were approved by each of the participating institutional review boards, and informed consent was obtained from each participant prior to enrollment.

In this case, molecular methods can be used to supplement or replace a target allele present in one population with a preferred allele present in another. The key issue is, of course, the identification of the genes and alleles that should be targeted for GE in livestock species. Likely, many genes for which naturally occurring variation creates subtle phenotypic effects exist, whereas GE of these genes with novel alleles might provide profound improvements in animal health and performance. Undoubtedly, the discovery of these genes will come from research with model organisms where the effects of the allelic forms on fitness can be extensively studied, emphasizing the importance of support for basic agricultural research.Early research efforts in livestock GE were focused on increasing the efficiency and yield of production for a diversity of species. The use of more productive GE animals [i.e., animals that produce more units of output with the same or less inputs] should be given due consideration in the context of sustainability. Making a conscious choice to use less productive animals necessitates the use of more land per unit of product or the use of more animals to produce a constant amount of animal product or both. This choice could cause conflict when open land is scarce, or other uses compete with the use of land for agriculture. The use of the best available technologies and inputs to produce greater output per unit of input offers overt sustainability advantages . Rapid growth and increased production result in a reduced number of animals being required for a fixed amount of output. Additionally, the environmental footprint per unit of animal product is reduced for more productive animals, regardless of the agricultural production system used. Bradford estimated that a small incremental increase of 2% per year in average milk production per cow globally,raspberry cultivation pot with no change in cow numbers, would result in a 60% increase in the global milk supply by the year 2020. The observation by Palmiter et al. that supplementing the mouse genome with an extra GH gene increased muscle growth greatly stimulated research into this approach for enhancing the productivity of meat animals.

However, early transgenic animal GH supplementation experiments achieved mixed results. Whereas growth enhancement was observed in experiments with both fish and mammals , in some experiments increased GH concentrations compromised the health of animals . From this research, much was learned about the GH axis, methods for transgenesis, the need for controlled gene expression, and best practices for transgenic animal stewardship. One successful outcome of this early work was a transgenic salmon whose genome contains an extra copy of the salmon GH gene . After nearly 15 years of research and development, the GH-transgenic salmon are now in an advanced stage of regulatory review by the FDA and could constitute the first transgenic animal product approved for human consumption in the United States. These fish produce the same amount and kind of circulating GH as wild-type salmon, but they produce it throughout the entire year. This modification has resulted in fish that reach market weight faster and consume less food per kilogram of product than wild-type salmon because they process their food 10 to 30% more efficiently. The more efficient utilization of protein in the diet of an animal leads to a reduction in the excretion of nitrogenous waste . Concerns over the potential environmental impact of feral transgenic fish have been studied extensively and practically addressed by eliminating the possibility of gene flow from transgenic to wild salmon by implementing a redundant biological and physical containment production system, which exclusively utilizes sterile female fish. These fish are reared in land-based facilities with multiple redundant physical containment features. Because of the controlled production environment, the fish are neither exposed to disease challenges nor are they reservoirs for disease transmission as they might be in conventional salmon net pen aquaculture. The ability to grow these salmon in land-based facilities closer to population centers dramatically reduces transportation costs compared with conventional salmon aquaculture, affecting the economics and environmental footprint of salmon production.

Finally, because these fish can be raised in inland fisheries, they represent a unique food security opportunity, potentially rejuvenating a nearly extinct US Atlantic salmon industry. To address the sustainability of pork production, a line of GE pigs was developed with the ability to digest and metabolize natural P in their feed, which non-GE pigs cannot accomplish. These pigs referred to as Enviropigs, have a genome supplemented with a gene from Escherichia coli that produces phytase exclusively in their salivary glands . This genetic modification reduces the excretion of undigested P in pig feces by 30 to 60%, which will ameliorate surface water eutrophication from swine production, as well as eliminate the environmental footprint of phytase production as a feed supplement . Clinical analysis of the health of Enviropigs using hematology,As mentioned, a focus on animal welfare positively influences productivity, and, therefore, indirectly enhances the sustainability of animal production. Numerous targets of GE aim to deliver even more direct, simultaneous improvements in animal welfare and sustainability . For example, GE could provide a humane method for sex selection in dairy and egg industries, where cows and hens provide the animal product. The development of male animals could be avoided ab initio and eliminate inefficiencies in animal production and welfare concerns associated with sex selection and castration. Gene supplementation that feminizes male embryos or eliminates the production of male sperm in sires is technically feasible; the latter approach has the desirable outcome that the animals that are produced are not themselves genetically engineered. Based on the global importance of pork, researchers have developed GE pigs to improve the sustainability of production, enhance animal welfare, and add nutritional value . For example, the expression of bovine α-lactalbumin and IGF in the mammary gland of lactating sows results in increased milk production, which directly enhances animal welfare as demonstrated by improved growth, intestinal development, and overall survival of piglets at weaning .

In a striking example of the value of GE in enhancing animal welfare, Wall et al. at the USDA-ARS engineered Jersey cattle to express the antibacterial protein lysostaphin in their milk, an accomplishment that dramatically enhanced the resistance of these cows to infection by Staphylococcus aureus, the most common and most difficult to treat cause of mastitis. This genetic improvement, could not only improve the well being of around 2 million dairy cattle per year in the United States alone, but also could decrease the economic costs of mastitis, which are currently estimated to exceed $2 billion per year in the United States .One promising aspect of GE is the potential for the development of functional foods that enhance food safety, human nutrition, and health . For example, in China the nutritional value of bovine milk has been improved by GE to express human α-lactoglobulin and human lactoferrin,low round pots proteins normally found in human milk but missing from bovine milk . Given the increasing prevalence of obesity and cardiovascular disease in developed nations, changes in product composition in conjunction with improvements in dietary practices could contribute to improvements in consumer health. The amounts and type of fats in animal products are topics of frequent public discourse, and from the perspective of sustainability, improved feed conversion efficiency increases the ratio of lean-to-fat deposition in livestock. Net benefits include reduced production costs, improved product quality, reduced excretion of nitrogenous wastes into the environment, decreased grazing pressure on fragile landscapes, and reduced pressure on world feed supplies . A decrease in the prevalence of deleterious fats and cholesterol and an increase in the prevalence of MUFA and n-3 fatty acids are consistent with dietary recommendations for cardiovascular health and an objective difficult to achieve in the absence of GE. In fact, 3 proof-of-principle studies have been published: 1) GE goats that expressed a rat stearoyl-CoA desaturase in the mammary gland and yielded milk with a reduced saturated fatty acid content and increased content of CLA, a beneficial antioxidant fatty acid ; 2) a GE pig made transgenic for a Δ12 fatty acid desaturase gene from spinach that produced the PUFA, linoleic acid and α-linolenic acid , which are essential for human nutrition ; and 3) a GE pig that was developed to express an n-3 fatty acid desaturase capable of converting n-6 fatty acids to n-3 fatty acids . Although fish provide an excellent source of dietary n-3 fatty acids, which are important for fertility, cardiovascular health, immune system health, mental health, and cancer prevention , worldwide fisheries will be challenged to sufficiently supply n-3 fatty acids to the developing world. As the most widely consumed meat, pork logically should be considered as an alternative source of n-3 fatty acids. Consistent with this strategy, the pigs developed by Lai et al. produce increased content of n-3 fatty acids from n-6 analogs, and their tissues have a reduced ratio of n-6/n-3 fatty acids. Such animals may be useful as models for human health and for providing a dietary source that could enhance the health of consumers in developed and developing countries. The first FDA approval of a GE animal product, the anticoagulant ATryn , firmly established the importance and safety of engineered mammary gland-based protein expression systems. Additional GE projects in cattle and goats have targeted the mammary gland for the expression of proteins to enhance the welfare of animals, and the safety and stability of milk products .

Bacterial diarrhea, which is responsible for more than 2 million infant deaths per year in developing countries, results from campylobacter, salmonellae, shigellae, and some strains of E. coli infections. Transgenic goats that express the human lysozyme protein, a natural antimicrobial protein in breast milk, were developed to produce milk with an enhanced shelf life that would improve the gastrointestinal health of goat kids and children . Experiments in vitro and in vivo have established that milk from these goats has antimicrobial properties, whether pasteurized or not, and that this milk inhibits the enteric bacteria E. coli when fed to piglets . Approval of this product could make a significant contribution to the alleviation of hunger and disease .Recent estimates indicate that 31.7% of children aged 2 to 19 years are overweight and 16.9% are obese . The issue of obesity is complex, with numerous interrelated causes. It stands to reason that any problem with this degree of complexity cannot be solved through programs that address a single contributing factor. Multiple government agencies have echoed this logic in a call for integrative and innovative strategies that demonstrate promise in promoting healthy lifestyle choices among children. These strategies include implementation at multiple levels, from individual to family to community to society . The need for healthier school environments, improved dietary and physical activity behaviors at home and community engagement in efforts to improve the health and well-being of our nation’s children was emphasized in a recent joint initiative released by first lady Michelle Obama, the surgeon general and the Department of Health and Human Services . To help reach these goals, President Obama issued a memorandum on Feb. 9, 2010, calling for the establishment of a federal task force on childhood obesity . In an Institute of Medicine report, key stakeholders were urged to commit to childhood obesity prevention and to strive not only to develop innovative programs but to monitor the progress and evaluate the efficacy of new and existing obesity prevention policies and programs and work to disseminate promising practices for maximum impact . The committee cited nutrition education and gardening as examples of promising, innovative practices to increase fruit and vegetable consumption through both Farm to School programs and school gardens. Other organizations, including the Centers for Disease Control and Prevention, encouraged creating a school environment that supports regular physical activity and healthy eating habits . Conceptual frameworks are key to the development, implementation and evaluation of successful health programs, as they can provide a system for linking and evaluating the multiple components that influence health behavior . Effective, sustainable programs targeting obesity prevention for the individual are needed within the context of the socio-ecological model, which succinctly describes the relationship of choices made by an individual to the other spheres of influence within the environment and society.