Month: December 2023

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.

Although the water flux for freshwater extraction stop is encouraging, the rate of absorbing water using hydrogel is significantly slower than other draw agents. To improve performance, the team envisioned running numerous desalting sponges in parallel, and further investigations need to be conducted. Overall, since the average salinity of seawater is 35 ppt, which is around 17 times more than the saline used in the test , it shows a promising application in seawater desalination on a lower NaCl concentration.There are many factors affecting the efficiency of forward osmosis, such as concentration polarization, membrane fouling, reverse solute diffusion, membrane development, and draw solution design. Concentration polarization is the most important factor among all of them. Various studies conducted regarding forward osmosis, these studies share an identical focus, which is reducing concentration polarization. The existence of concentration polarization can weaken the actual osmotic pressure difference on both sides of the membrane, which is one of the limiting factors that affect the performance of forward osmosis in water flux recovery. Pressure retarded osmosis has been defined as osmosis through asymmetric membranes. Most forward osmosis membranes used are either an asymmetric structure membrane including an active layer/a porous support layer , or symmetric structure membrane . There are two types of concentration polarizations based on the placement of the membranes: external concentration polarization and internal concentration polarization . External concentration polarization and internal concentration polarization can be further categorized into two sub-categories: dilutive and concentrative. In general applications, forward osmosis membranes are commonly placed in a way that the active layer faces feed solution,fodder growing system and the support layer faces the draw solution.

One of the exceptions is touse forward osmosis with the function of damping osmotic pressure. In this membrane orientation, when the solution is drawn from the feed solution and enters the active layer to the support layer, the feed solution can be diluted in the pores of the support layer and its surface, thus causing dilutive external concentration polarization and dilutive internal concentration polarization respectively. To be more concise, the solution that lingers in the support layer has greater osmotic pressure than that of the feed solution. When the solute from the process is transported by porous support and active layer, it can further dilute the outlier of the draw solution, causing dilutive external concentration polarization. Selecting a good draw solution is crucial for the FO process. The ideal DS should have high solubility, high osmotic pressure, and stability. Non-toxicity of the draw solution has little to no effects on the performance and structure of the FO membrane. There are three categories of DS that are generally recognized: inorganic DS, organic DS, and other DS such as nanoparticles. Currently, inorganic draw solutions are most widely used in FO technology. They usually have extremely high osmotic pressure due to the small inorganic molecular mass and high solubility, which makes them more favorable in dealing with hypersaline wastewater. However, in the reverse osmosis process, the inorganic draw solution could increase the salinity of the feed solution. The mainstream of inorganic DS is ammonium bicarbonate and sodium chloride. In 2005, McCutcheon and Elimelech et al. conducted forward osmosis experiments using ammonium bicarbonate as the draw solution and achieved ideal results; through heating, ammonia-carbon dioxide can be regenerated. Nevertheless, there can still be a certain amount of ammonia gas present in the water. As a result, in more practical applications and pilot-scale tests, ammonium bicarbonate is the most widely used draw agent.

Ammonia and carbon dioxide are evaporated in the form of gas, which is effective for recovery and re-concentration. Since the ammonium bicarbonate extraction and recovery system can make full use of low-grade waste heat and reduce energy consumption, it is especially practical for places with available waste heat, such as thermal power plants, and regions with abundant solar.As the population increases rapidly, the demand for irrigation raises correspondingly. Almost 70% of the global water is used to irrigate. At the same time, freshwater demand is raising, water reuse treatment process and drinking water treatment process became vital technologies nowadays. Under most situations, wastewater reuse and seawater reuse are a large portion of the water reuse system. However, brackish groundwater reuse became an emergent freshwater resource recently. Brackish groundwater is often located at depths of 4,000 feet or deeper under the Earth’s surface, and it has a dissolved concentration between 1,000 to 10,000 milligrams per liter . Brackish groundwater could be used for power generation, aquaculture, industry, and public drinking water supply. There are profuse brackish groundwater resources located in the United States, including Utah, New Mexico, Arizona, Virginia, Nevada, Texas, California, Idaho, and Colorado. For instance, Texas has an estimation of 2.7 billion acre-feet of brackish groundwater; In New Mexico, 75 percent of the groundwater is too saline to use without any treatment. According to what has been discussed previously, there are bountiful resources of brackish groundwater in the United States, and one of the common implements of treated brackish groundwater is direct fertigation since there are lavish nutrients in the groundwater. The combination of nanofiltration and fertilizer drawn forward osmosis is an ideal solution for brackish groundwater treatment. Since brackish groundwater has a relatively low total dissolved solid , it requires minor desalination and nutrient removal processes before direct fertigation.

To maintain a qualified number of nutrient components in brackish groundwater for direct fertigation, researchers have compared different models combining nanofiltration and forward osmosis. The first model is fertilizer drawn forward osmosis alone without nanofiltration, the results have shown that treated water samples still contain excessive nutrients for plant growth, which indicates that the water quality would not qualify for direct fertigation. The second model applies Nanofiltration as a pre-treatment. This model can remove most of the scaling and organic fouling species, enhancing the performance of fertilizer drawn forward osmosis. However, scaling became one of the major issues due to the excess amount of scaling ions . The third model applies nanofiltration as posttreatment, this system not only has the highest reduction rate of fertilizer nutrients but is also able to recycle the excess nutrients for further reuse as draw solutions. For all the models above, researchers applied an NE90 membrane with an MWCO of 220kDa. Generally, a1KDa MWCO refers to about 1.3 nm in membrane pore size, whereas 220KDa corresponds to a pore size of 3.84 nm. Out of variousfertilizers were tested, ammonium phosphate monobasic , ammonium sulfate ,chicken fodder system and mono-potassium phosphate have the highest reduction rates of nitrogen. Research has shown that ammonium sulfate contains the highest water recovery rate at 76%. Potassium dihydrogen phosphate has a second ranking water flux recovery of up to 75% while ammonium phosphate monobasic shows the lowest nutrient concentration among three of them. FDFO demonstrates its potential with fertilizer draw solution, which acts as a low-energy osmotic dilution. Researchers also proved that most fertilizers can be used as draw solutions, different combinations of various draw solutions can have numerous removal rates for a certain nutrient. . For instance, the combination of KCI and NH4H2PO4 can result in a lower concentration of N/P/K , which shows a higher nutrient removal rate than using KCI or NH4H2PO4 individually as draw solution. Moreover, different draw solutions /fertilizers have different rejection rates of nitrogen compounds. For example, Urea has a lower rejection rate compared to ionic compounds, such as nitrate and ammonium. This phenomenon indicates that Urea may have a higher nitrogen organic removal rate after ammonification. It is proven that the hybrid system of fertilizer drawn forward osmosis with nanofiltration as a post-treatment has the most effective removal rates of nutrients when it comes to brackish groundwater treatment. When nanofiltration is applied as pre-treatment, the system has a higher removal rate on scaling precursor ions and organic fouling species treating brackish groundwater. When nanofiltration is served as post-treatment, the nitrogen removal rate is the highest compared to the FO alone without NF and NF applied as a pre-treatment. The system can also recycle excess nutrients for further reuse as draw solutions when NF is applied as a post treatment. The water flux is analogously higher when this hybrid treatment process is orientated as pressure-retarded osmosis instead of normal forward osmosis mode.

Integration of nanofiltration with fertilizer drawn forward osmosis can reduce the nutrient concentration to meet the water quality standard for direct fertigation. It brings the nitrogen input in fertigation to a lower scale compared to the standard scale. This hybrid system can also adjust the input of different nutrients for varied types of crops/situations. The sources of N2O are mainly from microbial processes in soil and oxidation of NH3 in fertilizer. This research focuses on exploring the agricultural factors and providing solutions to the issue of redundant N2O emissions. At the same time, managing these controllable factors can reduce agricultural emissions by applying water treatment methods. Since California contributes 12% of the national food production, reducing N2O emissions could have a consequential effect on air quality and public health. Studies show that exposure to long-term N2O would cause ebbed lung function and asthma, especially to young ages. People that live nearby farms have a higher risk of getting respiratory diseases. Moreover, accession of N2O in water is caused by the excess nutrient runoff to the river. According to this review, N2O emissions can be reduced significantly by managing the fertigation nitrogen input appropriately. Consequently, the air quality and water quality could be improved by reducing Nitrous oxide emissions. A higher Nitrogen input fertilizer could increase the loss of certain plant species and the death of marine organisms. At the same time, low-nitrogen fertilizer would not be as nutritious as nitrogen fertilizer, it might slow down the growth rates of plants and crops. Since fertigation is commonly used in agriculture, relatively low nitrogen input could have a negative impact on the efficiency of crop production. As a result, the nitrogen amount in fertigation should be controlled to a certain amount to maintain the balance. Besides the dinitrogen and nitric oxide emission from soil denitrification, agricultural Nitrous oxide emission has the dominant contribution to the total greenhouse gas emission. According to previous studies, Nitrate oxide is 300 times more harmful than carbon dioxide towards climate change. Nitrate oxides in the atmosphere contained 270 parts per billion in 1750, and it has increased to 331 parts billion in 2018. The increasing rate of Nitrate oxide in the atmosphere breaks the record every 5 years. In the year 2021, the global temperature increased conspicuously, one of the reasons is the overt Nitrate oxides emission since the food demand is rising every year with the population growth. This review explores the relationship between agricultural factors of N2O emissions and water treatment solutions. The result of this review shows that agricultural N2Oemission is related to different factors including soil oxygen content, soil porosity, soil organic carbon content, soil temperature, PH value of soil, soil bacteria content, and Nitrogen input in soil. By adjusting these external factors, including limiting the supplement of oxygen, reducing soil water content, choosing the soil with a lower porosity , increasing the soil PH values, increasing the soil organic carbon content, etc., lowering the nitrogen input to prevent over-fertilization could be the most effective solution. As a result, the agricultural N2O emissions decrease spontaneously. Nitrous oxide has different impacts on the atmosphere. In the stratosphere, N2O depletes ozone levels by acting with halogen oxides. In the troposphere, N2O is one of the paths depleting ozone. Over 3500 measurements of N2O existences in surface water and marine troposphere, the exactitude for tropospheric, surface water, and marine measurement are 0.3%, 1.2%, and 2.2%. These numbers indicate that almost two-thirds of the worldwide flux of N2O in the atmosphere derives from sources in the northern hemisphere. Data from surface water proposes that the oceanic flux of N2O would be less than 60 Gmol/year. Deep water N2O concentration is estimated using the values of salinity of water, water temperature, water oxygen content, and the water dissolved nitrogen content. Raise of N2O concentration in water is caused by anthropogenic nitrate denitrification, resulting in tremendous depletion of marine life, especially in deep water.

Examples include cover cropping, water conservation, erosion control, integrated pest management, or organic certification. Although the level of abstraction might be conceptualized as a continuum, this simple categorization is useful for analysis. Another way of thinking about this categorization is that goals are value-driven outcomes of sustainability and strategies represent beliefs about the means to achieving those goals . Hypothesis 2 is that mental models of sustainable agriculture will reflect geographic variation and local context. Differences in farmer knowledge and the practice of agriculture reflect regional biophysical and social differences . In particular, although abstract goals of sustainability are likely to be more universal across geographies, the concrete strategies used to achieve those goals may reflect geographical variation in terms of challenges and opportunities for realizing the goals . For example, achieving the goal of environmental responsibility in the Napa Valley requires water management and cover-crop strategies for reducing soil erosion by surface water runoff on steep hillsides. In Lodi, strategies for wind-born soil erosion control are more relevant across the gentle valley floor topography of the region. Hypothesis 3 is that farmers who subscribe to more central concepts in the mental model are also more likely to engage in a range of sustainability behaviors. In particular, the sophistication of a farmer’s definition of sustainability should be correlated with their participation in extension programs and adoption of sustainability practices. The extension programs in California viticulture explicitly train farmers in the idea of sustainability and also identify specific sustainability practices. Thus, participation, practices,dutch bucket hydroponic and mental models represent a set of coevolving and synergistic processes.We constructed a mental model based on responses to surveys of wine grape growers in all three study regions.

Using content analysis of farmers’ self-reported definitions of sustainability , we classified 56 concepts into 19 abstract goals of sustainability and 37 more concrete strategies. We operationalized the mental model as a network where the concepts are nodes and valued ties represent the number of times two concepts co-occur together in a single definition of sustainable agriculture . We first identified an overall mental model by taking the union of the regional concept networks. A union network is defined as the combination of nodes and ties from two or more networks . The union network provides a comprehensive picture of farmer thinking about sustainable agriculture. The overall mental model from the union network is visualized in Fig. 1. Nodes are scaled by a measure of centrality we call “prominence,” which indicates a concept’s importance in the mental model. Prominent concepts are widely recognized among farmers as legitimate dimensions of sustainability and they are cognitively associated with many other central concepts. Technically, prominence combines the frequency that a concept appears in the network with its centrality . Because prominent concepts are linked to many other concepts, they are effective cognitive entry points for leveraging farmer thinking about sustainability. Ties are unscaled. Nodes are shaded by classification, with yellow-colored nodes representing goals of sustainable agriculture and aqua-colored nodes representing strategies. Table S1 lists all of the concepts, examples of coded text for each concept, classification as goal or strategy , and three measures of centrality: prominence, occurrence probability, and eigenvector centrality. We chose the examples of coded text that best illustrate the core ideas of the concept.Our mental model analysis identified key concepts that are factored into a practitioner’s decision-making process. The goals of economic viability, environmental responsibility, continuation into the future, and crop value are powerful drivers of decision making, with relevance across different social-ecological contexts. The hierarchical structure of the overall mental model suggests that although practitioners focus on achieving a common set of broad goals, the strategies they associate with realizing them are numerous and diverse.

Key strategies include practice adoption, stewardship of resources, reduction or elimination of agrochemicals, and water conservation and quality enhancement. Because of their association with many other goals and strategies, central concepts are potential cognitive entry points for leveraging practitioner thinking about sustainability.Sustainability is notoriously difficult to define for the reason that it is a relative concept , which varies widely across space, time, and scale . Furthermore, diverse stakeholders often have divergent and even conflicting values and goals . Practitioners must grapple with the questions of what is to be sustained, for how long, for whose benefit, at what cost, over what geographical area, and measured by what criteria . We argue that definitions of sustainability that are grounded in practitioners’ viewpoints will have greater relevance to real-world contexts and therefore be more useful for guiding actions . Empirically measuring mental models of sustainability is crucial to know whether the normative ideas about sustainability discussed within academic, policy, and public circles are relevant to on-the-ground decisions. Our study of mental models provided two main insights into practitioners’ definitions of sustainability. First, mental models of sustainability are organized hierarchically along a continuum of abstractness from general goals of sustainability to concrete strategies for achieving those goals. At least among wine grape growers, the overall mental model is sophisticated and reflects many of the concepts discussed in the academic literature and among policymakers . Definitions that focus on central goals are likely to prompt practitioner thinking about their linked strategies, and are more likely to resonate with a greater number and diversity of practitioners. To the extent these goals and strategies are grounded in more general environmental values and norms, the network approach used here emphasizes the interdependent and relational aspects of sustainability thinking. Second, more central abstract concepts are universal across geography, with only anecdotal evidence that strategies are customized to specific social-ecological contexts.

This may be a feature of our study system because sustainability extension programs are advanced within California viticulture and wine grape-growing regions that have more similarities than differences. Mental models from social-ecological systems with more stark differences may show larger differences in how goals are linked to strategies. More research is needed to confirm or disconfirm the hypothesis that concrete strategies are more sensitive to geographic and other contextual variation.Managing knowledge systems to link knowledge and action is a core goal in sustainability science . Knowledge systems include the institutional arrangements, organizations, and social networks that facilitate the transmission of knowledge among decision makers. Our results suggest that knowledge about sustainability, participation in extension programs, and practice adoption are mutually reinforcing processes. In agriculture, local extension programs and partnerships have played a crucial role in managing knowledge systems . In the case of California viticulture specifically, there is a substantial body of literature demonstrating that these programs have had a positive influence on farmer adoption of sustainability practices . The positive association we found between farmer sustainability cognition, participation in extension activities, and practice adoption indicates that knowledge systems do help expand practitioner understanding of social-ecological systems and influence their management behaviors. Extension programs can accelerate the development of knowledge and understanding about sustainability by clarifying the linkages among central sustainability goals and the associated strategies and practices for achieving them. An important component of this learning process may be the explicit use of the concept of sustainability,dutch buckets system as it can serve as a heuristic for guiding practitioner decision making with a framework for balancing economic, ecological, and social costs and benefits. Thinking in terms of sustainability does track with behavior, and knowledge systems have the ability to support this process by providing opportunities for learning.Sustainability and climate-focused initiatives announced by the United States federal government, states, and private sector entities could have meaningful impacts on land use sectors by affecting trends in land use and management as well as shifting commodity markets. Recent policy announcements include potential land-based greenhouse-gas mitigation strategies associated with ambitious new climate targets as part of rejoining the Paris Agreement , as well as a recent presidential executive order protecting 30% of U.S. lands and waters by 2030. The US Department of Agriculture Innovation Initiative has established ambitious targets for the next three decades to increase agricultural productivity by 40%, reduce food waste by 50%, reduce nutrient loss to run of by 30%, reduce carbon emissions, and increase bio–fuel and biomass production.Other policies may not have a primary objective that is environmental or sustainability-focused, but could nonetheless support policies in this domain by shifting resource demands and improving environmental outcomes. Two examples of indirect policy objectives that could interact with sustainability and climate initiatives include enhancing agricultural productivity growth and promoting healthier diets. If widely adopted, U.S. government recommendations for healthier diets could alter protein consumption away from beef and pork and toward plant-based foods, which could indirectly benefit climate and sustainability goals . Furthermore, previous research suggests that agricultural productivity growth can complement climate change mitigation . However, it is unclear how these policy targets could be achieved in isolation, what role market adjustments will play, and how healthier diet transitions and agricultural productivity enhancement might interact.While there have been several recent studies examining combinations of sustainability-related U.S. policy targets , the literature modeling U.S. agriculture and forestry is currently lacking in its representation of demand-side sustainability policies, including transitions to healthier diets.

While shifting to healthier diets is critical to reducing the noncommunicable disease burden , understanding how dietary change could shift resource-intensive commodity production, land use and ecosystem services can help inform complementary sustainability and climate policy actions. U.S. food systems are characterized by high levels of grain and oil seed production to support a highly productive domestic livestock sector and domestic diets that are relatively rich in meat-based proteins and oils , as well as international demands for U.S.-sourced agricultural products. Sustainability priorities such as increasing biodiversity protection or ecosystem service provision could benefit from dietary shifts that reduce pressure on U.S. agriculture’s intensive and extensive margins. Simultaneously, increasing productivity growth in U.S. agriculture could increase incomes and increase comparative advantage for international trade, which may or may not have land sparing effects. The literature on environmental impact of human diets has converged on the multiple sustainability benefits of diets lower in animal-based foods and higher in plant-based foods . These studies have either examined the global impacts of all countries adopting more sustainable or healthier diets or the domestic impacts of changes to a single country’s dietary preferences . Rarely have studies quantified both domestic and global sustainability metrics of a single county’s dietary changes or the country-specific sustainability impacts of the rest of the world adopting healthier diets. In addition, many studies focus on quantifying the impacts of specific personal dietary preferences , rather than a healthier average national diet. Several studies in the U.S. have quantified the sustainability impacts of omnivorous healthy diets recommended by the Dietary Guidelines for Americans . However, there is significant disagreement about whether the DGA diets have lower GHG, land use, or water use than the average American diet today . A handful of these studies have reported slightly lower land use requirements , and three out of four available studies showed similar or greater GHG emissions . The majority of studies quantifying the sustainability of alternative diets and dietary shifts in the U.S. use life-cycle assessments to measure environmental impacts of food production chains . However, LCA studies are limited in being able to quantify land use and land use change and allow for regional variation . Moreover, for projecting the environmental impacts of future dietary changes, it is critical to provide estimates that represent dynamic, rather than steady-state, industry and economic conditions.Alternative approaches such as economic partial-equilibrium models represent the agricultural, forestry, and other land use sectors in detail, and are deliberately designed to estimate land-use-related impacts, a key gap in the existing literature on the sustainability of U.S. diets . The global scale of many of these models allows representation of international trade and thus evaluation of leakage effects of domestic policies. Indirect sustainability levers such as shifting dietary preferences have received substantially less attention in the land use modeling literature relative to carbon pricing , bioenergy, and traditional conservation incentives. However, recent analysis has started to move in this direction . Partial-equilibrium models of the land sectors, such as GLOBIOM, which we employ in this study, are designed to maintain empirically observed market relationships between supply, demand, and prices. These models endogenously determine the demand for certain foods, productivity of specific crops, and the productivity of the livestock sector.

In order to elucidate the biochemical mechanism of BZS1 function, we performed a SILIAIP-MS analysis of the BZS1 protein complex. We transformed Arabidopsis with a construct that over expresses a BZS1 protein fused with the yellow fluorescence protein at the C-terminus driven by the constitutive 35S promoter . A transgenic line that showed mild dwarf and dark-green-leaf phenotypes, resembling the bzs1-D mutant , was selected for the analysis. Pair-wised comparison was designed to seperately compare BZS1-YFP and 35S::YFP transgenic plants with non-transgenic wild type, to determine proteins associated with BZS1-YFP and YFP alone, respectively. To obtain complete 15nitrogen labeling of young seedlings, we first grew BZS1-YFP, YFP and wild-type plants hydroponically in medium containing 15N, and obtained stable isotope-labeled seeds . These 15N-labeled seeds and regular 14N seeds were grown again on corresponding 15N or 14N medium to obtain 5-day-old seedlings for further analysis . For each pair of isotope-labeled sample and control, equal amount of tissues was mixed, and the protein extract was used for immuno precipitation using the GFPtrap beads. The immuno precipitated proteins were separated in SDS-PAGE, gel bands were in-gel digested, and the tryptic peptides were analyzed by mass spectrometry . Mass spectrometry analyses of the two BZS1-YFP immuno precipitation experiments identified 514 and 383 proteins, respectively, with 279 proteins identified in both repeats . A smaller number of proteins were identified in the YFP experiments . Quantitation of isotope ratios showed median ratios of 1.16 and 1.23 for the two BZS1-YFP experiments,planting gutter and 1.0 and 0.92 for the two YFP control experiments. The protein ratios of the YFP control datasets had standard deviation of 0.23 and 0.57 .

Using 2× median as cutoff, 16 proteins were enriched in BZS1-YFP compared to wild-type control in the two repeat experiments. The YFP and wild type comparison identified 2 proteins that were enriched over 2× median, presumably due to association with YFP or false discovery, suggesting a false discovery rate <0.8% . The 15 proteins enriched by BZS1-YFP were not enriched by YFP alone, and thus were considered BZS1-associated proteins . Among the BZS1-associated proteins are COP1 and HY5, two key regulators of the light signaling pathways, as well as BZS1/BBX20’s homologs STH2/BBX21 and STO/BBX24 . To verify the interaction between BZS1 and COP1 in vivo, we performed immuno precipitation of BZS1-YFP from the BZS1-YFP transgenic Arabidopsis seedlings using anti-GFP antibody, and probed the immunoblot with anti-COP1 antibody. The results showed that COP1 co-immuno precipitates with BZS1-YFP , confirming that BZS1 interacts with COP1 in plants. Consistent with BZS1’s interaction with the COP1 E3 ubiquitin ligase, the immuno precipitated BZS1-YFP can be detected by anti-ubiquitin antibody, and the level of ubiquitination was increased by treatment with proteasome inhibitor MG132 . We further confirmed the direct interaction of BZS1 and HY5 by yeast two-hybrid assays . Further, when transiently co-expressed in Nicotiana benthamiana, the BZS1-myc protein was co-immuno precipitated by the HY5-YFP protein , confirming their interaction in plant cells. Similarly, the STH2-myc protein was co-immuno precipitated by BZS1-YFP . These results confirmed the SILIA-IP-MS results that BZS1 interacts with COP1, HY5, and STH2/BBX21. To determine the functional relationship between BZS1 and HY5, we first compared previously published transcriptomic data from BZS1-overexpression plants with chromatin immuno precipitation-microarray data of HY5 direct target genes . The result showed that 56.3% of BZS1-activated genes are HY5 targets while only 13% of BZS1-repressed genes are HY5 targets . Such significant overlap betweenBZS1-activated and HY5-bound genes suggests that BZS1 interacts with HY5 to activate gene expression.

Fusing a transcription repressor domain, such as the SRDX domain, to a transcription activator has been shown to have a dominant negative effect . Over expression of the BZS1-SRDX fusion sequence driven by 35S promotor in Arabidopsis caused a long-hypocotyl phenotype and reduced anthocyanin accumulation , which were similar to the phenotypes of loss-of-function mutant hy5-215 but opposite to the phenotypes caused by BZS1 over expression, further supporting that BZS1 functions as a transcription activator together with HY5. The BZS1-SRDX plants grown in the dark did not show any obvious phenotype , consistent with HY5 and BZS1 being degraded in the dark. To further investigate whether BZS1 function requires HY5, we crossed BZS1-YFP with hy5-215. The BZS1-YFP/hy5-215 plants showed similar phenotypes of long hypocotyls and low anthocyanin accumulation as hy5-215 , demonstrating that BZS1 activity requires HY5. Interestingly, the BZS1-YFP protein accumulates at a higher level in the hy5-215 mutant than in wild-type background , suggesting that HY5 negatively regulates BZS1 accumulation while required for BZS1 function. On the other hand, the RNA levels of HY5 and HYH are higher in BZS1-YFP line but lower in BZS1-SRDX seedlings as compared with those in wild type . Immunoblot analysis also confirmed that the HY5 protein level was increased in the BZS1-YFP line and reduced in the BZS1-SRDX line . These results indicated that BZS1 and HY5 proteins not only interact directly, but also influence each other’s protein abundance. A previous study showed that HY5 is required for SL inhibition of hypocotyl elongation. The HY5 protein level is increased by SL treatment and the hypocotyl elongation of hy5 is partially insensitive to SL . Since BZS1’s function is dependent on HY5 in the light, we examined if BZS1 is also involved in SL signaling. As reported previously , treatment with 1 μM GR24, an analog of SL, dramatically inhibited the hypocotyl elongation of wild-type seedlings but had no effect on the SL insensitive mutant max2-3 . We found that the hypocotyl elongation of BZS1-SRDX seedlings was partially insensitive to GR24, similar to the hy5-215 mutant.

The GR24 treatment decreased the hypocotyl length of wild-type seedlings by about 72% compared to the untreated control, but only by about 17% for hy5-215 and 30% for the BZS1-SRDX seedlings . GR24 also increased the chlorophyll content in wild-type plants by about 24%, but had no significant effect in max2-3, hy5-215 and BZS1- SRDX seedlings . Additionally, GR24 induced HY5 accumulation in wild-type background but not in the BZS1-SRDX seedlings . These results indicated that, like HY5, BZS1 also plays an important role in SL regulation of hypocotyl elongation and chlorophyll accumulation. We then tested if SL regulates the expression of BZS1/BBX20 and its homologs. Real-time reverse transcription PCR analysis showed that GR24 increased the expression level of BZS1/BBX20 mRNA in wild type, but not in the max2-3 mutant . Interestingly, expression levels of other members of BBX IV family, including STH2/ BBX21, were not dramatically affected by GR24. Immunoblot analysis confirmed that GR24 treatment increased the levels of the BZS1-myc protein expressed from the BZS1 native promoter and the BZS1-YFP protein expressed from the constitutive 35S promoter, suggesting that SL regulates BZS1 at both transcriptional and post transcriptional levels . These results indicated that BZS1 plays a positive role in SL signaling downstream of MAX2 at the early stage of seedling development. Seedling development is crucial for establishment of life for a plant, and is thus highly responsive to a wide range of environmental and hormonal signals. The signaling pathways that transduce these signals are highly integrated at the molecular level to ensure coherent cellular responses and optimal growth according to environmental condition and endogenous physiology . This study uncovers additional mechanisms for such signal integration. Our quantitative proteomic analysis of the BZS1 complex reveals BZS1’s interaction with HY5,gutter berries as well as provides direct evidence for in planta BZS1-COP1 interaction. Genetic analyses using over expression and dominant negative loss-of-function transgenic plants demonstrate that BZS1 interacts with HY5 to activate gene expression and promote photomorphogenesis. Further, we find that BZS1 also mediates SL regulation of HY5 level and hypocotyl elongation. Together with previous finding of BZS1 function downstream of the BR pathway , our study establishes BZS1 as a key integrator of light, BR, and SL signals for regulating seedling morphogenesis. IP-MS is a powerful method for identification of interacting proteins, which has been widely used in dissecting signal transduction pathways . With increased sensitivity of modern mass spectrometers, IP-MS tends to identify not only specific interacting proteins but also large numbers of non-specific proteins. Under our experimental conditions, over 300 proteins were identified in each IP-MS analysis. Distinguishing specific from non-specific interactors is challenging without quantitative measurement. SILIA-IP-MS provides an ideal quantitative method for this purpose, as the sample and negative control can be mixed at an early step of the immuno precipitation experiment to avoid technical variations. Indeed, among the large numbers of proteins identified by mass spectrometry, only 29 showed enrichment by the BZS1-YFP fusion protein, and thus were considered BZS1-associated proteins. The interactions of BZS1 with HY5, COP1, and its homolog STH2/BBX21 were confirmed by yeast two-hybrid or coimmuno precipitation assays. Consistent with COP1-mediated ubiquitination of BZS1, our BZS1-interactome data includes ubiquitin and one proteasome activating protein PA200 .

In theory, the ratio between sample and negative control should be infinite for proteins that specifically interact with the bait protein in SILIA-IP-MS. However, due to background signals in the control samples, either from non-specific binding of proteins in immuno precipitation or interfering signals in MS1, the ratios actually distribute within a wide range. For example, Hubner et al. observed that pull-down with Aly-GFP leads to only moderate enrichment because Aly itself binds to control beads as well. In our study, only 2 of the 254 proteins identified in the YFP sample were enriched over 2× median, suggesting that even 2-fold cutoff yields low false discovery rate when two reverse-labeled replicates are used. Our genetic analyses support that BZS1 interacts with HY5 to activate gene expression and promote photomorphogenesis. First, comparison of genome-wide data shows that BZS1 tends to activate, rather than repress, HY5 direct target genes . Second, dominant inactivation of BZS1 causes similar phenotypes as the hy5-215 mutant , supporting that BZS1 and HY5 act in the same or overlapping pathway. Third, the phenotypes of BZS1-YFP plants are suppressed by hy5-215 , confirming that BZS1 functions in a HY5-dependent manner. These results together provide strong evidence for a model that BZS1 interacts with HY5 to activate HY5-bound target genes. BBX proteins contain one or two B-box zinc finger motifs in their N-terminal regions, and are organized into five subfamilies . The fourth subfamily includes eight B-box proteins containing two tandem B-boxes without CCT domain . Our study together with previous studies show that five members of the BBX subfamily IV interact with COP1 and HY5 . Thus, interaction with HY5 seems to be a common mechanism for these B-box proteins to regulate gene expression. Interestingly, BZS1/BBX20, STH2/BBX21 and LZF1/STH3/BBX22 are positive regulators in photomorphogenesis, while BBX19, STO/BBX24 and STH/BBX25 are negative regulators . Our finding of STH2/BBX21 and STO/BBX24 as interactors of BZS1/BBX20 suggests that these factors form hetero-dimers. The dominant negative effect of the BZS1-SRDX fusion indicates that BZS1/BBX20 normally functions as a transcription activator, which is consistent with previous finding that STH2/BBX21 functions as a transcription activator . It has been reported that STO/BBX24 and STH/BBX25 interact with HY5 and most likely inhibit HY5 function by forming inactive heterodimers . Our identification of STO/BBX24 as a BZS1-associated protein suggests another possibility that STO/BBX24 may form a non-functional heterodimer with BZS1/BBX20 and hence inhibit BZS1/BBX20 activity. In addition to direct interaction between BZS1 and HY5 proteins in regulating target gene expression, BZS1 and HY5 also regulate each other’s expression level. BZS1 positively regulates the RNA and protein levels of HY5 . Recent studies have shown that HY5 binds to its own promoter to regulate its own level , thus BZS1 may regulate HY5 transcription through interaction with HY5 protein. In contrast, the BZS1 protein level is increased in hy5-215, suggesting a negative regulation by HY5 at the protein level. HY5 may promote BZS1 degradation by interacting with COP1. Similarly, a previous study showed that the degradation of BBX22 is also promoted by both COP1 and HY5 , whereas BBX22 transcription is directly activated by HY5 and repressed by BBX24 . Such positive and negative regulation between interacting partners potentially contributes to the signaling dynamics during dark-to-light transition and fluctuating light intensities.Our study uncovers a major role for BZS1 in SL response.

As sessile organisms, plants are presented with numerous biotic challenges such as herbivory and pathogen attack. Plants initiate responses to these challenges by harnessing tightly regulated phytohormone networks. Salicylic acid levels increase in plants following pathogen infection and SA is critical for the development of systemic acquired resistance . There are two enzymatic pathways for the generation of SA: one via phenylalanine ammonia lyase and the other via isochorismate synthase . In tomato , Arabidopsis and Nicotiana benthamiana, most pathogen-induced SA appears to be synthesized via the ICS pathway . Plants with compromised SA synthesis or signaling have greatly diminished defenses against pathogens, as is the case with SA-deficient transgenic plants expressing a bacterial salicylate hydroxylase or ICS mutants like sid2 , and mutants in downstream targets of SA such as npr1 . SAR induction by biotic agents coincides with increases in SA levels and a systemic transcriptional reprograming that primes the plant to respond rapidly to minimize the spread or severity of further infections . This transcriptional reprograming includes the expression of pathogenesis-related genes and deployment of peroxidases and other defense factors. In addition to induction by biotic agents, SAR responses are induced by exogenous application of SA to the foliage or roots . Plant activators are chemicals that have no direct antimicrobial activity but induce disease resistance . A number of synthetic compounds have been developed that induce SAR by increasing SA accumulation and/or by acting on downstream targets of SA . For example, the plant activator, probenazole, effective against bacterial, fungal, and oomycete diseases, stimulates SAR by increasing SA levels . 1,2,3-Benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester , sold under the trade name, Actigard,grow bucket stimulates SAR in many plant species without inducing SA accumulation . Tiadinil [TDL; N–4-methyl-1,2,3-thiadiazole-5-carboxamide] is a plant activator that was registered in Japan in 2003 under the trade name, V-GET. TDL was developed for disease management in rice where it is applied to nursery-grown seedlings for transplanting to production fields . TDL is very effective for control of rice blast disease caused by Magnaporthe oryzae and appears to induce resistance in a manner similar to BTH by acting on downstream targets of SA .

The TDL metabolite,4-methyl-1,2,3-thiadiazole-5-carboxylic acid, is responsible for the SAR activation .The effect of brief episodes of root stress such as salinity and water deficit at levels that commonly occur in agriculture is well documented in plant–oomycete interactions, wherein stress events predispose plants to levels of inoculum they would normally resist . The phytohormone abscisic acid accumulates rapidly in roots and shoots as an adaptive response to these abiotic stresses, but also contributes to the increased disease proneness of the plants . Antagonism between SA and ABA is well documented in relation to plant defense responses to pathogens . Previously, ABA was found to have an antagonistic effect on SAR which was induced by 1,2-benzisothiazol-3-one1,1-dioxide and BTH in Arabidopsis and tobacco . However, it is not known if plant activators that target SA signaling impact the ABA-mediated susceptibility to root pathogens that occurs following predisposing root stress in tomato. Because of the potential for unwanted trade offs and signaling conflicts in plants exposed to different stresses, as can occur in the field, we investigated how predisposing root stress impacts chemically induced resistance in tomato. The objective of this study was to determine the effect of pretreatment of tomato seedlings with TDL and BTH on salt-induced predisposition to the foliar bacterial pathogen Pseudomonas syringae pv. tomato and to the soil borne oomycete pathogen Phytophthora capsici. TDL is of particular interest in the context of soil borne pathogens such as Phytophthora capsici because it is often applied to plants as a root dip. We also determined the impact of SA, TDL and BTH on ABA accumulation during a predisposing episode of salt stress. The results show that TDL applied to roots strongly protects the leaves from disease caused by Pst in both non-stressed and salt-stressed plants. In contrast, neither TDL nor BTH protects roots from Phytophthora capsici.

The protection induced by plant activators against Pst does not result from reduced ABA accumulation and, although overall disease is less in both non-stressed and salt-stressed plants by chemically induced SAR, plant activators do not reverse the salt-induced increment in disease severity.To determine the effect of SA on ABA accumulation during salt stress, ABA levels were measured in WT plants pre-treated with SA, TDL, or BTH. Following salt stress treatment for 18 h, roots and shoots were collected and immediately frozen in liquid N2.The tissues were lyophilized and placed at −20◦C until extraction. The lyophilized tissue was ground in liquid N2 to a fine powder with a mortar and pestle, 50–100 mg samples were collected, and each sample transferred to a micro-fuge tube. Cold 80% methanol containing butylated hydroxytoluene at 10 μg ml−1 was added to each tube, which was then vortexed. The extracts were placed on ice and agitated occasionally for 30 min. The tubes were centrifuged for 5 min at 10,000 × g, and the supernatants collected. The pellet was extracted with 0.5 ml of 80% methanol and centrifuged to collect the supernatant. This step was repeated, all three supernatants were combined, and the methanol concentration of the extract adjusted to 70%. The extracts were applied to pre-wetted Sep-pak C18 columns and eluted with 5 ml of 70% methanol. The eluate containing ABA was concentrated to near dryness at 37◦C under vacuum and the volume adjusted to 300 μl with deionized water. The samples were analyzed by competitive immuno assay with an ABA immuno assay kit according to the manufacturer’s directions. Results are expressed as nanomoles of -ABA per gram dry weight of tissue. To determine the effect of the nahG transgene on ABA levels, roots and shoots from WT and NahG plants were processed using the same procedure as above.To determine if plant activators induce resistance to Pst under different stress regimes in our experimental format, roots of hydroponically grown seedlings of cv. “New Yorker” were treated with TDL and then either not salt-stressed or exposed to 0.2 M NaCl for 18 h prior to inoculation.

In preliminary experiments, several concentrations of TDL were evaluated for phytotoxicity and for efficacy against bacterial speck disease with 10 ppm TDL selected as this concentration provided an optimal response. Concentrations higher than 10 ppm of TDL caused a slight bronzing of the roots and depressed growth of the seedlings, suggesting a mild phytotoxicity of the chemical in our experimental format at these higher levels. Inoculated salt-stressed seedlings had more severe disease symptoms and a significantly higher titer of pathogen than non-stressed, inoculated plants. Pretreatment with TDL at 10 ppm significantly reduced Pst colonization and symptom severity in “New Yorker” plants in both non-stressed and salt-treated seedlings . However, TDL did not prevent the proportional increase in Pst colonization observed in salt-stressed plants relative to the non-stressed controls.Since TDL harnesses SA-mediated defenses, we treated SA deficient NahG plants to see if TDL induces resistance under the different stress regimes in this highly susceptible background. As expected, NahG plants were more susceptible to Pst and accumulated significantly less SA following Pst infection than the WT background “New Yorker.” However, TDL provided strong protection in the NahG plants and mitigated the predisposing effect of salt-stress on bacterial speck disease.In a previous study we showed that ABA-deficient tomato mutants displayed a much reduced predisposition phenotype to salt stress . To determine if the protective effect of TDL is altered within an ABA-deficient tomato mutant,dutch bucket for tomatoes seedlings of WT and an ABA-deficient mutant within this background, sitiens, were treated in the same format and stress regimes as above. TDL significantly reduced Pst symptoms and colonization in both non-stressed and salt-treated plants of “Rheinlands Ruhm.” However, 3.6- and 5.4-fold increases in pathogen titer as a result of salt-stress were observed in both the control and TDL-treated plants, respectively, indicating that TDL did not prevent the proportional increase in Pst colonization in salt-stressed plants, similar to the results with “New Yorker” and NahG plants. In contrast, the sitiens mutant was not predisposed to Pst by salt stress and had significantly reduced symptoms and colonization by the pathogen than the background “Rheinlands Ruhm” . Nonetheless, TDL pretreatment of sitiens provided further protection against Pst .To determine if plant activators protect tomato roots and crowns against the oomycete pathogen, Phytophthora capsici, and predisposing root stress, tomato seedlings were treated with TDL or BTH , not stressed or salt-stressed as above, and then inoculated. There was no protection provided by the plant activators against disease caused by Phytophthora capsici in either the control or salt-treated plants, as reflected in symptom severity and pathogen colonization .Because elevated levels of ABA in tomato can enhance susceptibility to Pst and Phytophthora capsici, the effect of SA, TDL, and BTH on ABA levels was determined in roots and shoots. ABA concentrations in either shoots or roots at the time selected for inoculation in our treatment sequence were not altered by SA . However, a trend of increasing ABA accumulation was observed in TDL- and BTH treated “New Yorker” plants relative to the corresponding control plants . Although the increase in ABA accumulation in the plants treated with these plant activators is not statistically significant at P ≤ 0.05, it can be said that SA, TDL, and BTH do not reduce ABA content relative to untreated plants . In addition, salt stress did not further increase the levels of ABA in plants that had been pretreated with TDL or BTH, which were similar to the salt stressed controls.In a previous study, we demonstrated the predisposing effect of salt stress and a role for ABA as a determinative factor in predisposition in the tomato–Phytophthora capsici interaction .

The present study is the first report of salt-induced predisposition to the bacterial speck pathogen, Pst, in tomato. Furthermore, the results with the ABA-deficient sitiens mutant are consistent with the salt-induced susceptibility to Pst being mediated by ABA . These results conform to studies in Arabidopsis where ABA has been reported to promote susceptibility to Pst .Because SA has been shown to protect tomato against salt stress, possibly by an ABA-dependent mechanism , plant activators that operate via the SA pathway were evaluated for effect on salt-induced predisposition. Protection of tomato against bacterial speck disease by BTH is well documented , and TDL has previously been shown to reduce the severity of bacterial and fungal infections without inducing SA accumulation . Here, TDL was shown to protect against Pst in both non-stressed and salt-stressed tomato plants. TDL pretreatment strongly reduced disease and colonization by Pst in both “New Yorker” and SA-deficient NahG plants. TDL, or more likely its biologically active metabolite, SV-03, presumably allows the NahG plants to mount an SAR response to Pst infection in the absence of SA accumulation . TDL provided protection in both non-stressed and salt-stressed plants, but did not reverse the predisposing effect of salt stress. An increase in Pst colonization was observed in the salt-stressed, TDL-pretreated plants of both genotypes, with comparable percentage increases relative to the corresponding non-stressed controls in “New Yorker” and NahG plants. This indicates that TDL does not reverse the salt-stress effect on disease, per se, and likely targets stress network signaling independently of an ABA-mediated process that conditions the salt-induced susceptibility observed in this system . “Rheinlands Ruhm” also displayed salt-induced predisposition to Pst. Pretreatment with TDL significantly reduced Pst colonization in both “Rheinlands Ruhm” and sitiens . Similarly, TDL provided protection in both non-stressed and saltstressed plants, but did not reverse the predisposing effect of salt stress in “Rheinlands Ruhm” plants. The salt-induced increment in colonization by the pathogen was comparable in both the untreated and TDL-treated plants . The ABA-deficient mutant, sitiens, is considerably less susceptible to Pst than its background “Rheinlands Ruhm,” and does not exhibit salt-induced predisposition .Protection by plant activators against foliar pathogens is well established . However, relatively few studies have examined these compounds against soilborne pathogens and so TDL and BTH were evaluated for protection against root infection by Phytophthora capsici. Neither TDL nor BTH induced resistance or impacted salt-induced predisposition to Phytophthora capsici . Phytophthora capsici is an aggressive root and crown pathogen with a hemibiotrophic parasitic habit that triggers both SA- and jasmonic acid-mediated responses during infection of tomato .

The whole-plant N2 fixation potential was calculated by multiplying the total dry nodule biomass of each plant and the N2 fixation potential, which had been normalized to dry nodule biomass. To understand how plant effects were related to CNM concentration-dependent agglomeration in moist soils, the short- and long-term stabilities of CNMs were studied in soil water extracts. Briefly, control soil was weighed into separate 50 mL centrifuge tubes with 1:5 w/v Nanopure water . The centrifuge tubes were sealed securely and shaken horizontally on a shaker for 3 h . The extract was centrifuged to separate large solids, and the supernatant was decanted. The supernatant was vacuum filtered through a 0.22 μm membrane filter , and the filtrate was collected as the final soil extract and stored prior to use. A CNM stock solution was prepared by weighing dry CNM powder into the filtered soil extract, then mixing by brief sonication using a Branson 1510 bath sonicator . Aliquots of the dispersed CNM stock solution were further diluted by the filtered soil extract to yield a final lower concentration of 10 mg L−1. These two CNM concentrations were chosen for comparing the effect of lower versus higher CNM concentrations on CNM agglomeration in moist soil; both concentrations are relevant to the CNM doses used in the plant exposure experiment . The CNM suspensions were bathsonicated immediately before use in static agglomeration and sedimentation studies, hydroponic nft channel which were performed over a long time period . The changes of CNM hydrodynamic diameter and derived count rate with time were measured using dynamic light scattering in a Zetasizer NanoZS90 . DLS measurements were made every 15 s for the first 12 h, then daily from 1 to 7 d, and finally weekly until 56 d.

Meanwhile, dynamic CNM sedimentation in the soil extract was monitored by measuring the suspension absorbance at 600 nm using a UV-1800 spectrophotometer . Sedimentation patterns were inferred from the time course of normalized suspension absorbance at 600 nm . The UV-1800 spectrophotometer was zeroed using Nanopure water. The absorbance of the filtered soil extract alone was monitored over time as well, to confirm there was no interfering absorbance from the soil extract in the CNM suspensions. The zeta potential and electrophoretic mobility of the filtered soil extract and of 10 mg L−1 CNMs were also obtained using the Zetasizer NanoZS90. For either DLS, absorbance, ζ potential, or EPM, at least three replicate measurements were performed. Environmental scanning electron microscopy was performed to visualize the agglomerate morphologies of 10 and 300 mg L−1 CNMs in the soil extract, against a clean quartz sand substrate. Specimens were prepared by dispensing approximately 100 μL of the CNM suspensions onto clean quartz sand overlaying a 10 mm stainless steel conical-well Peltier stub. Imaging was by an FEI Co. XL30 field emission gun microscope , operated at 15 kV accelerating voltage, in a 3.5-torr chamber pressure with a gaseous secondary electron detector in environmental mode. Data are shown as the mean ± SE . For each CNM type, one-way analysis of variance with Tukey’s or Games-Howell post hoc multiple comparisons was used to determine significant differences between treatments . Homogeneity of variance was tested with Levene’s test. To explore dose–response relationships, correlations were performed between plant growth and end point metrics with soil CNM concentrations, using both two-tailed linear and power regression models. Correlation analyses were conducted both with and without the control data. Statistical analyses were performed using Microsoft Excel 2013, IBM SPSS Statistics 23, and SigmaPlot 12.3.P. vulgaris is characterized by a particular evolutionary history.

Recent analyses based on sequence data presented clear evidence of the Mesoamerican origin of common bean, which was most likely located in México . The expansion of this species to South America resulted in the development of two ecogeographic distinct genetic pools with partial reproductive isolation . After the formation of these genetic pools -between 500,000 and 100,000 years ago – domestication took place, independently in the Mesoamerican and the southern Andean regions of the American continent . Genome analysis of BAT93 and G19833 , P. vulgaris sequenced model genotypes, has initially revealed interesting differences, for example between their genome size and number of annotated genes . The common bean is the most important legume for human consumption. In less favored countries from Latin America and Africa, common bean are staple crops serving as the primary source of protein in the diet. Soil acidity in these tropical regions is a major constraint for crop productivity, usually resulting in a combination of nutrient deficiency and metal toxicity . In acidic soils, aluminum toxicity is the primary factor of growth restriction, resulting in the inhibition of root growth and function, as well as in the increased risk of plants to perish of drought and mineral deficiencies, thus decreasing crop production . High Al levels mainly affect roots causing an arrest of the growth of the principal and lateral roots . In Arabidopsis, the regulation of root growth is modulated by an ABC transporter‐like protein, annotated as ALUMINUM SENSITIVE PROTEIN 3 , which is localized in the tonoplast, suggesting a role in Al vacuolar sequestration . The LOW PHOSPHATE ROOT 1 ferroxidase, an ALS3– downstream protein of the phosphate-deficiency signaling pathway, is involved in root growth inhibition, by modulating iron homeostasis and ROS accumulation in root apical meristem and elongation zone . In root cells, AlT can affect multiple areas, as the plasma membrane, the cell wall and symplastic components .

Common bean is known to be highly sensitive to AlT but this sensitivity is genotype-dependent . In 2010, the evaluation of the root morphological traits related to AlT of 36 P. vulgaris genotypes revealed that Andean genotypes were more resistant to Al than Mesoamerican ones . Mendoza-Soto et al. reported that Mesoamerican common-bean plants subjected to high Al levels for short periods showed decreased root length as well as characteristic symptoms of AlT, such as ROS accumulation, callose deposition, lipoperoxidation and cell death in roots. Along other regulators, plant response to metal toxicity involves also microRNAs as part of the regulatory mechanisms. These molecules are a class of non-coding small RNAs of about 21 nucleotides in length, regulating gene expression at post-transcriptional level, guided by sequence complementarity, inducing cleavage or translational inhibition of the corresponding target transcript . The relevance of miRNA regulation in heavy metal tolerance is well documented; it has been demonstrated that heavy metal-responsive miRNAs show differential expression according to the toxicity level. Target genes of these miRNAs generally encode transcription factors that transcriptionally regulate networks relevant for the response to heavy metals. Additionally these encode transcripts for proteins that participate in metal absorption and transport, protein folding, antioxidant system, phytohormone signaling, or miRNA biogenesis and feedback regulation . High-throughput small RNA sequencing analyses have identified miRNAs that respond to AlT in roots of different plants species, however their function in response to AlT is largely unknown. Some of the target genes cleaved by AlT-responsive miRNAs encode disease resistance proteins, transcription factors or auxin signaling proteins . Our previous research indicated that P. vulgaris is no exception to this phenomenon. We identified common-bean miRNAs that respond to Al, these include conserved miRNAs that are Al-responsive in other plant species -i.e. miR319, miR390, miR393- and also miR1511 . miRNAs from the miR1511 family have been identified in non-legume plants like strawberry and poplar tree ,nft growing system although in the latter its nature as a miRNA has been discussed as it has been considered as part of a retrotransposon . Regarding legumes, miR1511 has been identified in Medicago truncatula and soybean . Also, miR1511 was identified in Mesoamerican common-bean cultivars, being more abundant in flowers and roots . However, this miRNA was not identified when analyzing the Andean G19833 reference genome . Genetic variation in MIR1511 has been reported in a comparative genotyping analysis of different Asian accession of domesticated soybean as well as its wild type progenitor Glycine soja. While sequences of mature miR1511 and miR1511* were found in G. max accessions, the sequences of annual wild G. soja showed insertion/deletion in the stem-loop region of MIR1511 that included complete or partial deletions of mature miR1511 sequence . Updated research indicates that the miR1511 target gene is not conserved in the different plants where it has been identified. In strawberry, the miR1511 targets an LTR retrotransposon gene .

Inconsistencies about the nature of miR1511 target gene also hold for legume species. For instance, different targets have been proposed for soybean ranging from genes coding for proteins involved in the regulation of nitrogen metabolism to proteins relevant in plant cell development . While in other species such as M. truncatula target genes have been searched but have not been identified. The SP1L1 transcript has been proposed as the common-bean miR1511 target , however despite several efforts from our and other groups this prediction could not be experimentally validated. These results suggested a species-specific selection of the corresponding target thus it was essential to experimentally validate the nature and possible function of the miR1511 target gene in common bean. Recent analyses led us to predict an ABC-2-type transporter-related gene, annotated as Aluminum Sensitive Protein 3 , as the target for miR1511. In this work we present its experimental validation. In addition, we genotyped MIR1511 in ecogeographically different common-bean cultivars and investigated the role of miR1511 and its corresponding target in the regulation of plant response to AlT. The comparison of MIR1511 sequence from BAT93 vs. G19833 P. vulgaris reference sequences showed a 58-bp deletion in the G19833 genotype. Such deletion comprised around 57% of pre-miR1511 sequence and included 7-bp and 10-bp of mature and star miR1511, respectively . To explore this phenomenon at a larger scale within the Phaseolus genus, we analyzed Genotyping-By-Sequencing data from 87 genotypes originated from a single genetic population , called non-admixed genotypes. These included genotypes from three Phaseolus species and different populations of wild P. vulgaris: three populations from the Mesoamerican , one from the Andean , and one from the Northern Peru–Ecuador gene pools . All the genotypes belonging to the Andean gene pool and part of the Mesoamerican genotypes displayed a truncated MIR1511, in contrast to the Northern Peru– Ecuador genotypes and the other Phaseolus species that presented a complete version of the MIR1511 in their genome. A population clustering of P. vulgaris genotypes confirmed these results and showed that in the three Mesoamerican populations only a part of the MW1 cluster presented the MIR1511 deletion . Predicted target genes for P. vulgaris miR1511 include SP1L1-like  and isopentyl-diphosphate delta-isomerase , previously reported , and a protein with unknown function and the Aluminum Sensitive Protein 3 , from our recent bio-informatic analysis. From these predicted targets, ALS3 is the only one possibly related to AlT, as reported for Arabidopsis , and showing an adequate binding-site penalty score , thus the 5’RLM-RACE assay was used to experimentally validate the ALS3 mRNA cleavage site. As shown in Figure 3a, a significant number independently cloned transcripts mapped to the predicted site of cleavage, between the nucleotides at positions 457 and 458 of the transcript, which corresponds to position 9 and 10 of the predicted miR1511 binding site, thus confirming a miR1511-induced degradation. The other two degradation events mapped to 7 nucleotides upstream and 17 nucleotides downstream of the miRNA-associated degradation site, suggesting random degradation. An additional action of miR1511 to induce translation inhibition of ALS3 mRNA in common bean, cannot be excluded. miR1511 target genes differ among plant species . In order to evaluate the specificity of the miR1511/ALS3 regulatory node in common bean, we analyzed the miR1511/ALS3 binding site sequence alignment from eight model plant species, including five legumes, which contain a precursor gene of miR1511 in their genome . Because of the deletion in MIR1511 from the G19833 genotype, we used the mature miR1511 and the corresponding ALS3 binding site sequences from the BAT93 Mesoamerican genotype, as representative of P. vulgaris. Among plant species analyzed, P. vulgaris was the only one that showed a binding-site penalty score lower than 5, corresponding to a score recommended to consider a small RNA-target binding as probably functional. For other species, the high penalty scores, ranging from 7.5 to 9, indicate a very low probability for the existence of a functional miR1511/ALS3 regulatory node .