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Spinocerebellar ataxia type 3 (SCA3) is a cureless neurodegenerative disease recognized as the most prevalent form of dominantly inherited ataxia worldwide. The main hallmark of SCA3 is the expansion of a polyglutamine tract located in the C-terminal of Ataxin-3 (or ATXN3) protein, that triggers the mis-localization and toxic aggregation of ATXN3 in neuronal cells. The propensity of wild type and polyglutamine-expanded ATXN3 proteins to aggregate has been extensively studied over the last decades. In vitro studies with mass spectrometry techniques revealed a time-dependent aggregation of polyglutamine-expanded ATXN3 that occurs in several steps, leading to fibrils formation, a high status of aggregation. For in vivo experiments though, the techniques commonly used to demonstrate aggregation of polyglutamine proteins, such as filter trap assays, SDS-PAGE and SDS-AGE, are unable to unequivocally show all the stages of aggregation of wild type and polyglutamine-expanded ATXN3 proteins. Here we describe a systematic and detailed analysis of different known techniques to detect the various forms of both wild type and pathologic ATXN3 aggregates, and we discuss the power and limitation of each strategy., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Quinet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cardiovascular disease has reached a mortality rate of 470,000 patients each year. Myocardial infarction accounts for 49.2% of these deaths, and the cTnI protein is a crucial target in diagnosing myocardial infarction. A peptide-based bioreceptor design using a computational approach is a good candidate to be developed for a rapid, effective, and selective detection method for cTnI although it is still lacking in study. Hence, to address the scientific gap, we develop a new candidate peptide for the cTnI biosensor by bioinformatics method and present new computational approaches. The sequential point mutations were made to the selected peptide to increase its stability and affinity for cTnI. Next, molecular docking was performed to select the mutated peptide, and one of the best results was subjected to the molecular dynamics simulation. Finally, the results showed that the best peptide showed the lowest affinity and good stability among other mutated peptide designs for interacting with the cTnI protein. In addition, the peptide has been tested to have a higher specificity towards cTnI than its major isomer, sTnI, through molecular docking and molecular dynamics simulation. Therefore, the peptide is considered a good potential bioreceptor for diagnosing myocardial infarction diseases., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Jauhar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

P53 is a transcription factor that controls a variety of genes, primarily involved in cell cycle and other processes related to cell survival and death. We have isolated peptides targeting P53 (protein and domains) using the "phage display" technique. Interestingly, adding ZnCl2 at 5-10 mM in panning solutions helped to recover more plaque-forming units at least at round one of the screening. Subtractive docking analyses were designed by using a pool of common redundant peptides known as parasites. This rationale helped us differentiate between possibly specific and non-specific bindings. We found notable differences in docking characteristics between different sets of peptides either related to different targets or related to zinc-conditions. The set of zinc-related peptides shows advantageous docking profiles: sharper binding for some positions and distinct exclusive bound residues, including the relevant R248 and R273. Zinc would have modulating/helping role in the targeting of protein P53 by phage displayed peptides in addition to an enhancement action on bacterial infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ben Abid et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The nascent polypeptide-associate complex (NAC) is a heterodimeric chaperone complex that binds near the ribosome exit tunnel and is the first point of chaperone contact for newly synthesized proteins. Deletion of the NAC induces embryonic lethality in many multi-cellular organisms. Previous work has shown that the deletion of the NAC rescues cells from prion-induced cytotoxicity. This counterintuitive result led us to hypothesize that NAC disruption would improve viability in cells expressing human misfolding proteins. Here, we show that NAC disruption improves viability in cells expressing expanded polyglutamine and also leads to delayed and reduced aggregation of expanded polyglutamine and changes in polyglutamine aggregate morphology. Moreover, we show that NAC disruption leads to changes in de novo yeast prion induction. These results indicate that the NAC plays a critical role in aggregate organization as a potential therapeutic target in neurodegenerative disorders., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dublin-Ryan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Polyamines (PAs) including putrescine (PUT), spermidine (SPD) and spermine (SPM) are small, versatile molecules with two or more positively charged amino groups. Despite their importance for almost all forms of life, their specific roles in molecular and cellular biology remain partly unknown. The molecular structures of PAs suggest two presumable biological functions: (i) as potential buffer systems and (ii) as interactants with poly-negatively charged molecules like nucleic acids. The present report focuses on the question, whether the molecular structures of PAs are essential for such functions, or whether other simple molecules like small peptides with closely spaced positively charged side chains might be suitable as well. Consequently, we created titration curves for PUT, SPD, and SPM, as well as for oligolysines like tri-, tetra-, and penta-lysine. None of the molecules provided substantial buffering capacity at physiological intracellular pH values. Apparently, the most important mechanism for intracellular pH homeostasis in neurons is not a buffer system but is provided by the actions of the sodium-hydrogen and the bicarbonate-chloride antiporters. In a similar approach we investigated the interaction with DNA by following the extinction at 260 nm when titrating DNA with the above molecules. Again, PUT and tri-lysine were not able to interact with herring sperm DNA, while SPD and SPM were. Obviously, the presence of several positively charged groups on its own is not sufficient for the interaction with nucleic acids. Instead, the precise spacing of these groups is necessary for biological activity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rieck et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Recent research suggests that endothelial activation plays a role in coronavirus disease 2019 (COVID-19) pathogenesis by promoting a pro-inflammatory state. However, the mechanism by which the endothelium is activated in COVID-19 remains unclear., Objective: To investigate the mechanism by which COVID-19 activates the pulmonary endothelium and drives pro-inflammatory phenotypes., Hypothesis: The "inflammatory load or burden" (cytokine storm) of the systemic circulation activates endothelial NADPH oxidase 2 (NOX2) which leads to the production of reactive oxygen species (ROS) by the pulmonary endothelium. Endothelial ROS subsequently activates pro-inflammatory pathways., Methods: The inflammatory burden of COVID-19 on the endothelial network, was recreated in vitro, by exposing human pulmonary microvascular endothelial cells (HPMVEC) to media supplemented with serum from COVID-19 affected individuals (sera were acquired from patients with COVID-19 infection that eventually died. Sera was isolated from blood collected at admission to the Intensive Care Unit of the Hospital of the University of Pennsylvania). Endothelial activation, inflammation and cell death were assessed in HPMVEC treated with serum either from patients with COVID-19 or from healthy individuals. Activation was monitored by measuring NOX2 activation (Rac1 translocation) and ROS production; inflammation (or appearance of a pro-inflammatory phenotype) was monitored by measuring the induction of moieties such as intercellular adhesion molecule (ICAM-1), P-selectin and the NLRP3 inflammasome; cell death was measured via SYTOX™ Green assays., Results: Endothelial activation (i.e., NOX2 activation and subsequent ROS production) and cell death were significantly higher in the COVID-19 model than in healthy samples. When HPMVEC were pre-treated with the novel peptide PIP-2, which blocks NOX2 activation (via inhibition of Ca2+-independent phospholipase A2, aiPLA2), significant abrogation of ROS was observed. Endothelial inflammation and cell death were also significantly blunted., Conclusions: The endothelium is activated during COVID-19 via cytokine storm-driven NOX2-ROS activation, which causes a pro-inflammatory phenotype. The concept of endothelial NOX2-ROS production as a unifying pathophysiological axis in COVID-19 raises the possibility of using PIP-2 to maintain vascular health., Competing Interests: A.B.F. is a shareholder in Peroxitech LLC, a company that is developing PIP-2 as a therapeutic agent. O.P., S.C. and A.B.F. have filed a U.S. Provisional Patent Application titled “Protective Agent Against Endothelial Dysfunction”, application No. No. 63/469,255 filed May 26, 2023. No related or modified products are applicable or filed. This patent application does not affect our adherence to PLOS ONE policies on sharing data, data acquisition parameters, and materials., (Copyright: © 2024 Paul et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Canonical α/β T-cell receptors (TCRs) bind to human leukocyte antigen (HLA) displaying antigenic peptides to elicit T cell-mediated cytotoxicity. TCR-engineered T-cell immunotherapies targeting cancer-specific peptide-HLA complexes (pHLA) are generating exciting clinical responses, but owing to HLA restriction they are only able to target a subset of antigen-positive patients. More recently, evidence has been published indicating that naturally occurring α/β TCRs can target cell surface proteins other than pHLA, which would address the challenges of HLA restriction. In this proof-of-concept study, we sought to identify and engineer so-called HLA-independent TCRs (HiTs) against the tumor-associated antigen mesothelin., Methods: Using phage display, we identified a HiT that bound well to mesothelin, which when expressed in primary T cells, caused activation and cytotoxicity. We subsequently engineered this HiT to modulate the T-cell response to varying levels of mesothelin on the cell surface., Results: The isolated HiT shows cytotoxic activity and demonstrates killing of both mesothelin-expressing cell lines and patient-derived xenograft models. Additionally, we demonstrated that HiT-transduced T cells do not require CD4 or CD8 co-receptors and, unlike a TCR fusion construct, are not inhibited by soluble mesothelin. Finally, we showed that HiT-transduced T cells are highly efficacious in vivo, completely eradicating xenografted human solid tumors., Conclusion: HiTs can be isolated from fully human TCR-displaying phage libraries against cell surface-expressed antigens. HiTs are able to fully activate primary T cells both in vivo and in vitro. HiTs may enable the efficacy seen with pHLA-targeting TCRs in solid tumors to be translated to cell surface antigens., Competing Interests: This study was funded by Adaptimmune. Adaptimmune contributed to study design, data analysis, decision to publish, and preparation of the manuscript. Writing and editorial support was funded by Adaptimmune. All authors are employees of Adaptimmune or were at the time of the study. Authors may hold stock in Adaptimmune. This does not alter our adherence to PLOS ONE policies on sharing data and materials, (Copyright: © 2024 Hiscox et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The surgical sterilization of cats and dogs has been used to prevent their unwanted breeding for decades. However, this is an expensive and invasive procedure, and often impractical in wider contexts, for example the control of feral populations. A sterilization agent that could be administered in a single injection, would not only eliminate the risks imposed by surgery but also be a much more cost-effective solution to this worldwide problem. In this study, we sought to develop a targeting peptide that would selectively bind to Leydig cells of the testes. Subsequently, after covalently attaching a cell ablation agent, Auristatin, to this peptide we aimed to apply this conjugated product (LH2Auristatin) to adult male mice in vivo, both alone and together with a previously developed Sertoli cell targeting peptide (FSH2Menadione). The application of LH2Auristatin alone resulted in an increase in sperm DNA damage, reduced mean testes weights and mean seminiferous tubule size, along with extensive germ cell apoptosis and a reduction in litter sizes. Together with FSH2Menadione there was also an increase in embryo resorptions. These promising results were observed in around a third of all treated animals. Given this variability, we discuss how these reagents might be modified in order to increase target cell ablation and improve their efficacy as sterilization agents., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Fraser et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The methionine addiction of cancer cells is known as the Hoffman effect. While non-cancer cells in culture can utilize homocysteine in place of methionine for cellular growth, most cancer cells require exogenous methionine for proliferation. It has been suggested that a biochemical basis of this effect is the increased utilization of methionine for S-adenosylmethionine, the major methyl donor for a variety of cellular methyltransferases. Recent studies have pointed to the role of S-adenosylmethionine-dependent protein arginine methyltransferases (PRMTs) in cell proliferation and cancer. To further understand the biochemical basis of the methionine addiction of cancer cells, we compared protein arginine methylation in two previously described isogenic cell lines, a methionine-addicted 143B human osteosarcoma cell line and its less methionine-dependent revertant. Previous work showed that the revertant cells were significantly less malignant than the parental cells. In the present study, we utilized antibodies to detect the asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) products of PRMTs in polypeptides from cellular extracts and purified histone preparations of these cell lines fractionated by SDS-PAGE. Importantly, we observed little to no differences in the banding patterns of ADMA- and SDMA-containing species between the osteosarcoma parental and revertant cell lines. Furthermore, enzymatic activity assays using S-adenosyl-ʟ-[methyl-3H] methionine, recombinantly purified PRMT enzymes, cell lysates, and specific PRMT inhibitors revealed no major differences in radiolabeled polypeptides on SDS-PAGE gels. Taken together, these results suggest that changes in protein arginine methylation may not be major contributors to the Hoffman effect and that other consequences of methionine addiction may be more important in the metastasis and malignancy of osteosarcoma and potentially other cancers., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Y.A., Y.K., and R.M.H. are or were unsalaried associates of AntiCancer, Inc. All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright: © 2023 Holtz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The peptide domain extending from residues 49 to 57 of the HIV-1 Tat protein (TAT) has been widely shown to facilitate cell entry of and blood-brain barrier (BBB) permeability to covalently bound macromolecules; therefore, TAT-linked therapeutic peptides trafficked through peripheral routes have been used to treat brain diseases in preclinical and clinical studies. Although the mechanisms underlying cell entry by similar peptides have been established to be temperature-dependent and cell-type specific and to involve receptor-mediated endocytosis, how these peptides cross the BBB remains unclear. Here, using an in vitro model, we studied the permeability of TAT, which was covalently bound to the fluorescent probe fluorescein isothiocyanate (FITC), and evaluated whether it crossed the "in vitro BBB", a monolayer of brain endothelial cells, and whether the mechanisms were similar to those involved in TAT entry into cells. Our results show that although TAT crossed the monolayer of brain endothelial cells in a temperature-dependent manner, in contrast to the reported mechanism of cell entry, it did not require receptor-mediated endocytosis. Furthermore, we revisited the hypothesis that TAT facilitates brain delivery of covalently bound macromolecules by causing BBB disruption. Our results demonstrated that the dose of TAT commonly used in preclinical and clinical studies did not exert an effect on BBB permeability in vitro or in vivo; however, an extremely high TAT concentration caused BBB disruption in vitro. In conclusion, the BBB permeability to TAT is temperature-dependent, but at treatment-level concentrations, it does not involve receptor-mediated endocytosis or BBB disruption., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Micro-Exon Genes are a widespread class of genes known for their high variability, widespread in the genome of parasitic trematodes such as Schistosoma mansoni. In this study, we present a strategy that allowed us to solve the structures of three alternatively spliced isoforms from the Schistoma mansoni MEG 2.1 family for the first time. All isoforms are hydrophobic, intrinsically disordered, and recalcitrant to be expressed in high yield in heterologous hosts. We resorted to the chemical synthesis of shorter pieces, before reconstructing the entire sequence. Here, we show that isoform 1 partially folds in a-helix in the presence of trifluoroethanol while isoform 2 features two rigid elbows, that maintain the peptide as disordered, preventing any structuring. Finally, isoform 3 is dominated by the signal peptide, which folds into a-helix. We demonstrated that combining biophysical techniques, like circular dichroism and nuclear magnetic resonance at natural abundance, with in silico molecular dynamics simulation for isoform 1 only, was the key to solve the structure of MEG 2.1. Our results provide a crucial piece to the puzzle of this elusive and highly variable class of proteins., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Nedvedova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Long noncoding RNAs (lncRNAs) encompass short open reading frames (sORFs) that can be translated into small peptides. Here, we investigated the encoding potential of lncRNA LINC00665 in osteosarcoma (OS) cells. Bioinformatic analyses were utilized to predict the lncRNAs with encoding potential in human U2OS cells. Protein expression was assessed by an immunoblotting or immunofluorescence method. Cell viability was assessed by cell counting Kit-8 (CCK-8). Cell proliferation was detected by 5-ethynyl-2'-deoxyuridine (EdU) assay. Cell migration was gauged by transwell assay. The downstream effectors of the short peptide were verified using qualitative proteome analysis after immunoprecipitation (IP) experiments. The effect of the short peptide on protein interactions were confirmed by Co-Immunoprecipitation (CoIP) assays. We found that lncRNA LINC00665 encoded an 18-amino acid (aa)-long short peptide (named LINC00665_18aa). LINC00665_18aa suppressed the viability, proliferation, and migration of human MNNG-HOS and U2OS OS cells in vitro and diminished tumor growth in vivo. Mechanistically, LINC00665_18aa impaired the transcriptional activity, nuclear localization, and phosphorylation of cAMP response element-binding protein 1 (CREB1). Moreover, LINC00665_18aa weakened the interaction between CREB1 and ribosomal protein S6 kinase A3 (RPS6KA3, RSK2). Additionally, increased expression of CREB1 reversed the inhibitory effects of LINC00665_18aa on OS cell proliferation and migration. Our findings show that the short peptide LINC00665_18aa exerts a tumor-inhibitory function in OS, providing a new basis for cancer therapeutics through the functions of the short peptides encoded by lncRNAs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Pan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mammalian protein arginine methyltransferase 7 (PRMT7) has been shown to target substrates with motifs containing two arginine residues separated by one other residue (RXR motifs). In particular, the repression domain of human histone H2B (29-RKRSR-33) has been a key substrate in determining PRMT7 activity. We show that incubating human PRMT7 and [3H]-AdoMet with full-length Xenopus laevis histone H2B, containing the substitutions K30R and R31K (RKRSR to RRKSR), results in greatly reduced methylation activity. Using synthetic peptides, we have now focused on the enzymology behind this specificity. We show for the human and Xenopus peptide sequences 23-37 the difference in activity results from changes in the Vmax rather than the apparent binding affinity of the enzyme for the substrates. We then characterized six additional peptides containing a single arginine or a pair of arginine residues flanked by glycine and lysine residues. We have corroborated previous findings that peptides with an RXR motif have much higher activity than peptides that contain only one Arg residue. We show that these peptides have similar apparent km values but significant differences in their Vmax values. Finally, we have examined the effect of ionic strength on these peptides. We found the inclusion of salt had little effect on the Vmax value but a considerable increase in the apparent km value, suggesting that the inhibitory effect of ionic strength on PRMT7 activity occurs largely by decreasing apparent substrate-enzyme binding affinity. In summary, we find that even subtle substitutions in the RXR recognition motif can dramatically affect PRMT7 catalysis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Bondoc et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Leptospirosis is a public health concern with lethality around 15% of the total cases. The current vaccines against Leptospira infection based on bacterins have several limitations, which require urgent development of new ones. In this context, groundbreaking approaches such as peptide-vaccines could be used to come around with promising results. Our goal was to identify conserved and immunogenic epitopes from the lipoprotein LruC that could interact with Major Histocompatibility Complex (MHC) I and II. LruC is a conserved lipoprotein expressed during leptospirosis that is considered among vaccine candidates and can be used as source for development of peptide-based vaccines. We searched for peptides that would be recognized by antibodies from either serum of hamsters previously immunized with low-LPS bacterin vaccines or from serum of patients diagnosed with leptospirosis. Immuno properties of seven peptides from LruC protein were evaluated in silico and by Dot Blot assay, and validate by ELISA. Preliminary results pointed one promising peptide that was recognized by the sera. In conclusion, the immunoinformatic approach helps the search and screening of peptides, while the Dot Blot assay, a simple and effective tool, helps to test and validate them. Thus, these prospective techniques together were validated to identify and validate potential peptides for further investigation as peptide-based vaccines or diagnostic methods., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Mariano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

E. coli Nissle (EcN) is a non-pathogenic probiotic bacterium of the Enterobacteriaceae family that has been used for over a century to promote general gut health. Despite the history of safe usage of EcN, concerns have been raised regarding the presence of the pks gene cluster, encoding the genotoxin colibactin, due to its association with colorectal cancer. Here, we sought to determine the effect of pks island removal on the in vitro and in vivo robustness and activity of EcN and EcN-derived strains. A deletion of the pks island (Δpks) was constructed in wild type and engineered strains of EcN using lambda red recombineering. Mass spectrometric measurement of N-myristoyl-D-asparagine, released during colibactin maturation, confirmed that the pks deletion abrogated colibactin production. Growth curves were comparable between Δpks strains and their isogenic parents, and wild type EcN displayed no competitive advantage to the Δpks strain in mixed culture. Deletion of pks also had no effect on the activity of strains engineered to degrade phenylalanine (SYNB1618 and SYNB1934) or oxalate (SYNB8802). Furthermore, 1:1 mixed dosing of wild type and Δpks EcN in preclinical mouse and nonhuman primate models demonstrated no competitive disadvantage for the Δpks strain with regards to transit time or colonization. Importantly, there was no significant difference on in vivo strain performance between the clinical-stage strain SYNB1934 and its isogenic Δpks variant with regards to recovery of the quantitative strain-specific biomarkers d5- trans-cinnamic acid, and d5-hippuric acid. Taken together, these data support that the pks island is dispensable for Synthetic Biotic fitness and activity in vivo and that its removal from engineered strains of EcN will not have a deleterious effect on strain efficacy., Competing Interests: At the time of submission of this letter, all authors are full time employees at Synlogic, Inc and have received funding for this study from Synlogic Inc in form of salary. Also, authors may hold stock in Synlogic Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Kalantari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Much is known about environmental influences on metabolism and systemic insulin levels. Less is known about how those influences are translated into molecular mechanisms regulating insulin production. To better understand the molecular mechanisms we generated marked cells homozygous for a null mutation in the Drosophila TGF-β signal transducer dSmad2 in unmated adult females. We then conducted side-by-side single cell comparisons of the pixel intensity of two Drosophila insulin-like peptides (dILP2 and dILP5) in dSmad2- mutant and wild type insulin producing cells (IPCs). The analysis revealed multiple features of dSmad2 regulation of dILPs. In addition, we discovered that dILP5 is expressed and regulated by dSmad2 in circadian pacemaker cells (CPCs). Outcomes of regulation by dSmad2 differ between dILP2 and dILP5 within IPCs and differ for dILP5 between IPCs and CPCs. Modes of dSmad2 regulation differ between dILP2 and dILP5. dSmad2 antagonism of dILP2 in IPCs is robust but dSmad2 regulation of dILP5 in IPCs and CPCs toggles between antagonism and agonism depending upon dSmad2 dosage. Companion studies of dILP2 and dILP5 in the IPCs of dCORL mutant (fussel in Flybase and SKOR in mammals) and upd2 mutant unmated adult females showed no significant difference from wild type. Taken together, the data suggest that dSmad2 regulates dILP2 and dILP5 via distinct mechanisms in IPCs (antagonist) and CPCs (agonist) and in unmated adult females that dSmad2 acts independently of dCORL and upd2., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Goldsmith, Newfeld. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Peptide Lv is a small endogenous secretory peptide that is expressed in various tissues and conserved across different species. Patients with diabetic retinopathy, an ocular disease with pathological angiogenesis, have upregulated peptide Lv in their retinas. The pro-angiogenic activity of peptide Lv is in part through promoting vascular endothelial cell (EC) proliferation, migration, and sprouting, but its molecular mechanism is not completely understood. This study aimed to decipher how peptide Lv promotes EC-dependent angiogenesis by using patch-clamp electrophysiological recordings, Western immunoblotting, quantitative PCR, and cell proliferation assays in cultured ECs. Endothelial cells treated with peptide Lv became significantly hyperpolarized, an essential step for EC activation. Treatment with peptide Lv augmented the expression and current densities of the intermediate-conductance calcium-dependent potassium (KCa3.1) channels that contribute to EC hyperpolarization but did not augment other potassium channels. Blocking KCa3.1 attenuated peptide Lv-elicited EC proliferation. These results indicate that peptide Lv-stimulated increases of functional KCa3.1 in ECs contributes to EC activation and EC-dependent angiogenesis., Competing Interests: The authors have declared that no competing interests exist.

The actinorhizal plant Datisca glomerata (Datiscaceae, Cucurbitales) establishes a root nodule symbiosis with actinobacteria from the earliest branching symbiotic Frankia clade. A subfamily of a gene family encoding nodule-specific defensin-like cysteine-rich peptides is highly expressed in D. glomerata nodules. Phylogenetic analysis of the defensin domain showed that these defensin-like peptides share a common evolutionary origin with nodule-specific defensins from actinorhizal Fagales and with nodule-specific cysteine-rich peptides (NCRs) from legumes. In this study, the family member with the highest expression levels, DgDef1, was characterized. Promoter-GUS studies on transgenic hairy roots showed expression in the early stage of differentiation of infected cells, and transient expression in the nodule apex. DgDef1 contains an N-terminal signal peptide and a C-terminal acidic domain which are likely involved in subcellular targeting and do not affect peptide activity. In vitro studies with E. coli and Sinorhizobium meliloti 1021 showed that the defensin domain of DgDef1 has a cytotoxic effect, leading to membrane disruption with 50% lethality for S. meliloti 1021 at 20.8 μM. Analysis of the S. meliloti 1021 transcriptome showed that, at sublethal concentrations, DgDef1 induced the expression of terminal quinol oxidases, which are associated with the oxidative stress response and are also expressed during symbiosis. Overall, the changes induced by DgDef1 are reminiscent of those of some legume NCRs, suggesting that nodule-specific defensin-like peptides were part of the original root nodule toolkit and were subsequently lost in most symbiotic legumes, while being maintained in the actinorhizal lineages., Competing Interests: The authors have declared that no competing interests exist.

Hepatocellular carcinoma (HCC) is one of the world's most risky diseases due to the lack of clear and cost-effective therapeutic targets. Currently, the toxicity of conventional chemotherapeutic medications and the development of multidrug resistance is driving research into targeted therapies. The nano-biomedical field's potential for developing an effective therapeutic nano-sized drug delivery system is viewed as a significant pharmaceutical trend for the encapsulation and release of numerous anticancer therapies. In this regard, current research is centered on the creation of biodegradable chitosan nanoparticles (CSNPs) for the selective and sustained release of bee venom into liver cancer cells. Furthermore, surface modification with polyethylene glycol (PEG) and GE11 peptide-conjugated bee venom-CSNPs allows for the targeting of EGFR-overexpressed liver cancer cells. A series of in vitro and in vivo cellular analyses were used to investigate the antitumor effects and mechanisms of targeted bee venom-CSNPs. Targeted bee venom-CSNPs, in particular, were found to have higher cytotoxicity against HepG2 cells than SMMC-7721 cells, as well as stronger cellular uptake and a substantial reduction in cell migration, leading to improved cancer suppression. It also promotes cancer cell death in EGFR overexpressed HepG2 cells by boosting reactive oxygen species, activating mitochondria-dependent pathways, inhibiting EGFR-stimulated MEK/ERK pathway, and elevating p38-MAPK in comparison to native bee venom. In hepatocellular carcinoma (HCC)-induced mice, it has anti-cancer properties against tumor tissue. It also improved liver function and architecture without causing any noticeable toxic side effects, as well as inhibiting tumor growth by activating the apoptotic pathway. The design of this cancer-targeted nanoparticle establishes GE11-bee venom-CSNPs as a potential chemotherapeutic treatment for EGFR over-expressed malignancies. Finally, our work elucidates the molecular mechanism underlying the anticancer selectivity of targeted bee venom-CSNPs and outlines therapeutic strategies to target liver cancer., Competing Interests: The authors have declared that no competing interests exist.

Micropeptides, encoded from small open reading frames of 300 nucleotides or less, are hidden throughout mammalian genomes, though few functional studies of micropeptides in the brain are published. Here, we describe a micropeptide known as the Plasticity-Associated Neural Transcript Short (Pants), located in the 22q11.2 region of the human genome, the microdeletion of which conveys a high risk for schizophrenia. Our data show that Pants is upregulated in early adulthood in the mossy fiber circuit of the hippocampus, where it exerts a powerful negative effect on long-term potentiation (LTP). Further, we find that Pants is secreted from neurons, where it associates with synapses but is rapidly degraded with stimulation. Pants dynamically interacts with Rtn4/Nogo-A, a well-studied regulator of adult plasticity. Pants interaction with Nogo-A augments its influence over postsynaptic AMPA receptor clustering, thus gating plasticity at adult synapses. This work shows that neural micropeptides can act as architectural modules that increase the functional diversity of the known proteome., Competing Interests: The authors have declared that no competing interests exist.

The growing demand for natural pigments in the industrial sector is a significant driving force in the development of production processes. The production of natural blue pigments, which have wide industrial applications, using microbial systems has been gaining significant attention. In this study, we used Aspergillus oryzae as a platform cell factory to produce the blue pigment indigoidine (InK), by genetic manipulation of its non-ribosomal peptide synthetase system to overexpress the indigoidine synthetase gene (AoinK). Phenotypic analysis showed that InK production from the engineered strain was growth associated, owing to the constitutive control of gene expression. Furthermore, the initial pH, temperature, and glutamine and MgSO4 concentrations were key factors affecting InK production by the engineered strain. The pigment secretion was enhanced by addition of 1% Tween 80 solution to the culture medium. The maximum titer of total InK was 1409.22 ± 95.33 mg/L, and the maximum productivity was 265.09 ± 14.74 mg/L·d. Moreover, the recombinant InK produced by the engineered strain exhibited antioxidant activity. These results indicate that A. oryzae has the potential to be used as a fungal platform for overproduction of extracellular non-ribosomal peptide pigments., Competing Interests: The authors have declared that no competing interests exist.

The appearance of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the lack of effective antiviral therapeutics for coronavirus disease 2019 (COVID-19), a highly infectious disease caused by the virus, demands the search for alternative therapies. Most antiviral drugs known are passive defenders which must enter the cell to execute their function and suffer from concerns such as permeability and effectiveness, therefore in this current study, we aim to identify peptide inactivators that can act without entering the cells. SARS-CoV-2 spike protein is an essential protein that plays a major role in binding to the host receptor angiotensin-converting enzyme 2 and mediates the viral cell membrane fusion process. SARS vaccines and treatments have also been developed with the spike protein as a target. The virtual screening experiment revealed antiviral peptides which were found to be non-allergen, non-toxic and possess good water solubility. U-1, GST-removed-HR2 and HR2-18 exhibit binding energies of -47.8 kcal/mol, -43.01 kcal/mol, and -40.46 kcal/mol, respectively. The complexes between these peptides and spike protein were stabilized through hydrogen bonds as well as hydrophobic interactions. The stability of the top-ranked peptide with the drug-receptor is evidenced by 50-ns molecular dynamics (MD) simulations. The binding of U-1 induces conformational changes in the spike protein with alterations in its geometric properties such as increased flexibility, decreased compactness, the increased surface area exposed to solvent molecules, and an increase in the number of total hydrogen bonds leading to its probable inactivation. Thus, the identified antiviral peptides can be used as anti-SARS-CoV-2 candidates, inactivating the virus's spike proteins and preventing it from infecting host cells., Competing Interests: The authors have declared that no competing interests exist.

Background: Established MRI and emerging X-ray contrast agents for non-invasive imaging of articular cartilage rely on non-selective electrostatic interactions with negatively charged proteoglycans. These contrast agents have limited prognostic utility in diseases such as osteoarthritis (OA) due to the characteristic high turnover of proteoglycans. To overcome this limitation, we developed a radiocontrast agent that targets the type II collagen macromolecule in cartilage and used it to monitor disease progression in a murine model of OA., Methods: To confer radiopacity to cartilage contrast agents, the naturally occurring tyrosine derivative 3,5-diiodo-L-tyrosine (DIT) was introduced into a selective peptide for type II collagen. Synthetic DIT peptide derivatives were synthesised by Fmoc-based solid-phase peptide synthesis and binding to ex vivo mouse tibial cartilage evaluated by high-resolution micro-CT. Di-Iodotyrosinated Peptide Imaging of Cartilage (DIPIC) was performed ex vivo and in vivo 4, 8 and 12 weeks in mice after induction of OA by destabilisation of the medial meniscus (DMM). Finally, human osteochondral plugs were imaged ex vivo using DIPIC., Results: Fifteen DIT peptides were synthesised and tested, yielding seven leads with varying cartilage binding strengths. DIPIC visualised ex vivo murine articular cartilage comparably to the ex vivo contrast agent phosphotungstic acid. Intra-articular injection of contrast agent followed by in vivo DIPIC enabled delineation of damaged murine articular cartilage. Finally, the translational potential of the contrast agent was confirmed by visualisation of ex vivo human cartilage explants., Conclusion: DIPIC has reduction and refinement implications in OA animal research and potential clinical translation to imaging human disease., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: MMF and NHL are named inventors on patents for radiopaque compounds containing DIT (WO2018020262A1, EP3490614A1). All other authors declare that they have no competing interests.

The invertebrate LFRFamide (LFRFa) and short neuropeptide F (sNPF), consisting of 6 to 10 amino acids, are orthologs for bilaterian NPF/Y, which consist of 36 to 40 amino acids. Recently, a molluscan G protein-coupled receptor (GPCR) for NPF was characterized in Pacific abalone (Haliotis discus hannai). To address the functional evolutionary route of the invertebrate LFRFa and NPF signaling system, in this study, we identified cDNAs encoding LFRFa precursors and the sNPF receptor (Hdh-sNPFR) in Pacific abalone. Four LFRFa mature peptides with 6 or 7 amino acids were predicted: GSLFRFa, GGLFRFa, GTLFRFa, and GSTLFRFa. Hdh-sNPFR was identified as a classical rhodopsin-like GPCR and classified into a molluscan sNPFR group. In HEK293 cells, Hdh-sNPFR was mainly localized in the cell membranes and internalized in the cytoplasm following treatment with LFRFa peptides. Reporter assays demonstrated that LFRFa peptides inhibit forskolin-stimulated cAMP accumulation in Hdh-sNPFR-expressing HEK293 cells. LFRFa precursor and Hdh-sNPFR transcripts were more strongly expressed in the cerebral and pleural-pedal ganglia of Pacific abalone than in the peripheral tissues such as the ovary, gills, intestine, and hepatopancreas. The levels of LFRFa transcripts in the ovary, intestine, and hepatopancreas were significantly higher in mature female abalone than in immature females. Injection of LFRFa induced the egg release and spawning behavior of mature abalone, but suppressed food intake. These results suggest that LFRFa peptides are endogenous ligands for Hdh-sNPFR involved in food intake and reproduction through a Gαi-protein dependent signaling pathway., Competing Interests: The authors have declared that no competing interests exist.

Aminoglycoside antibiotics interfere with the selection of cognate tRNAs during translation, resulting in the synthesis of aberrant proteins that are the ultimate cause of cell death. However, the toxic potential of aberrant proteins and how they avoid degradation by the cell's protein quality control (QC) machinery are not understood. Here we report that levels of the heat shock (HS) transcription factor σ32 increased sharply following exposure of Escherichia coli to the aminoglycoside kanamycin (Kan), suggesting that at least some of the aberrant proteins synthesized in these cells were recognized as substrates by DnaK, a molecular chaperone that regulates the HS response, the major protein QC pathway in bacteria. To further investigate aberrant protein toxic potential and interaction with cell QC factors, we studied an acutely toxic 48-residue polypeptide (ARF48) that is encoded by an alternate reading frame in a plant cDNA. As occurred in cells exposed to Kan, σ32 levels were strongly elevated following ARF48 expression, suggesting that ARF48 was recognized as a substrate by DnaK. Paradoxically, an internal 10-residue region that was tightly bound by DnaK in vitro also was required for the ARF48 toxic effect. Despite the increased levels of σ32, levels of several HS proteins were unchanged following ARF48 expression, suggesting that the HS response had been aborted. Nucleoids were condensed and cell permeability increased rapidly following ARF48 expression, together suggesting that ARF48 disrupts DNA-membrane interactions that could be required for efficient gene expression. Our results are consistent with earlier studies showing that aberrant proteins induced by aminoglycoside antibiotics disrupt cell membrane integrity. Insights into the mechanism for this effect could be gained by further study of the ARF48 model system., Competing Interests: The authors have declared that no competing interests exist.

Huntington's disease (HD) is caused by an expansion of the CAG trinucleotide repeat domain in the huntingtin gene that results in expression of a mutant huntingtin protein (mHTT) containing an expanded polyglutamine tract in the amino terminus. A number of therapeutic approaches that aim to reduce mHTT expression either locally in the CNS or systemically are in clinical development. We have previously described sensitive and selective assays that measure human HTT proteins either in a polyglutamine-independent (detecting both mutant expanded and non-expanded proteins) or in a polyglutamine length-dependent manner (detecting the disease-causing polyglutamine repeats) on the electrochemiluminescence Meso Scale Discovery detection platform. These original assays relied upon polyclonal antibodies. To ensure an accessible and sustainable resource for the HD field, we developed similar assays employing monoclonal antibodies. We demonstrate that these assays have equivalent sensitivity compared to our previous assays through the evaluation of cellular and animal model systems, as well as HD patient biosamples. We also demonstrate cross-site validation of these assays, allowing direct comparison of studies performed in geographically distinct laboratories., Competing Interests: The authors have declared that no competing interests exist.

The immune response to citrullinated peptides in the mucosa has been suggested to play an important role in the transition from pre-onset rheumatoid arthritis (RA) to clinically evident RA. Although there are reports indicating the presence of anti-citrullinated peptide antibodies in the saliva, few studies have reported citrullinated peptide detection in human saliva. This study aimed to identify citrullinated peptides in human saliva and discuss their clinical significance. Saliva samples were collected from 11 patients with RA and from 20 healthy individuals. Citrullinated peptides were detected using an anti-modified citrulline (AMC) antibody. Saliva from the healthy individuals was subjected to two-dimensional protein electrophoresis to isolate citrullinated peptides, which were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and mass spectrometry by peptide mass fingerprinting. The results were corroborated by immunoprecipitation (IP)-western blotting. The signal intensities of the bands precipitated with anti-cytokeratin 13 (CK13) and AMC antibodies were quantified. The signal intensity ratio of the band produced by the AMC antibody was divided by that of the band produced by the anti-CK13 antibody to calculate the citrullinated CK13 (Cit-CK13) ratio. A citrullinated peptide band corresponding to a molecular weight of approximately 50 kDa was detected in the saliva of healthy individuals, and identified as CK13 via mass spectrometry and IP-western blotting. No significant difference was observed between the salivary Cit-CK13 ratios of patients with RA and healthy participants (p = 0.605). This is the first study to show that Cit-CK13 is present in human saliva, and that there is no significant difference between the Cit-CK13 ratios of patients with RA and healthy individuals, suggesting that salivary Cit-CK13 content and RA development may not be associated. The physiological and pathological roles of Cit-CK13 in the oral cavity, and its responsiveness to mucosal immunity, remain unknown and will be the subject of further investigation., Competing Interests: The authors have declared that no competing interests exist.

Opioids targeting mu;μ (MOP) receptors produce analgesia in the peri-operative period and palliative care. They also produce side effects including respiratory depression, tolerance/dependence and addiction. The N/OFQ opioid receptor (NOP) also produces analgesia but is devoid of the major MOP side effects. Evidence exists for MOP-NOP interaction and mixed MOP-NOP ligands produce analgesia with reduced side effects. We have generated a HEKMOP/NOP human expression system and used bivalent MOP-NOP and fluorescent ligands to (i) probe for receptor interaction and (ii) consequences of that interaction. We used HEKMOP/NOP cells and two bivalent ligands; Dermorphin-N/OFQ (MOP agonist-NOP agonist; DeNO) and Dermorphin-UFP101 (MOP agonist-NOP antagonist; De101). We have determined receptor binding profiles, GTPγ[35S] binding, cAMP formation and ERK1/2 activation. We have also probed MOP and NOP receptor interactions in HEK cells and hippocampal neurones using the novel MOP fluorescent ligand, DermorphinATTO488 and the NOP fluorescent ligand N/OFQATTO594. In HEKMOP/NOP MOP ligands displaced NOP binding and NOP ligands displaced MOP binding. Using fluorescent probes in HEKMOP/NOP cells we demonstrated MOP-NOP probe overlap and a FRET signal indicating co-localisation. MOP-NOP were also co-localised in hippocampal tissue. In GTPγ[35S] and cAMP assays NOP stimulation shifted the response to MOP rightwards. At ERK1/2 the response to bivalent ligands generally peaked later. We provide evidence for MOP-NOP interaction in recombinant and native tissue. NOP activation reduces responsiveness of MOP activation; this was shown with conventional and bivalent ligands., Competing Interests: The authors have declared that no competing interests exist.

The androgen receptor (AR) plays a central role in prostate cancer. Development of castration resistant prostate cancer (CRPC) requires androgen-independent activation of AR, which involves its large N-terminal domain (NTD) and entails extensive epigenetic changes depending in part on histone lysine demethylases (KDMs) that interact with AR. The AR-NTD is rich in low-complexity sequences, including a polyQ repeat. Longer polyQ sequences were reported to decrease transcriptional activity and to protect against prostate cancer, although they can lead to muscular atrophy. However, the molecular mechanisms underlying these observations are unclear. Using NMR spectroscopy, here we identify weak interactions between the AR-NTD and the KDM4A catalytic domain, and between the AR ligand-binding domain and a central KDM4A region that also contains low-complexity sequences. We also show that the AR-NTD can undergo liquid-liquid phase separation in vitro, with longer polyQ sequences phase separating more readily. Moreover, longer polyQ sequences hinder nuclear localization in the absence of hormone and increase the propensity for formation of AR-containing puncta in the nucleus of cells treated with dihydrotestosterone. These results lead us to hypothesize that polyQ-dependent liquid-liquid phase separation may provide a mechanism to decrease the transcriptional activity of AR, potentially opening new opportunities to design effective therapies against CRPC and muscular atrophy., Competing Interests: The authors have declared that no competing interests exist.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is an essential post-translational modification in all eukaryotes that occurs at the endoplasmic reticulum (ER) and serves to deliver GPI-anchored proteins (GPI-APs) to the cell surface where they play a wide variety of vital physiological roles. This paper describes a specialized method for purification and structural analysis of the GPI glycan of individual GPI-APs in yeast. The protocol involves the expression of a specific GPI-AP tagged with GFP, enzymatic release from the cellular membrane fraction, immunopurification, separation by electrophoresis and analysis of the peptides bearing GPI glycans by mass spectrometry after trypsin digestion. We used specifically this protocol to address the structural remodeling that undergoes the GPI glycan of a specific GPI-AP during its transport to the cell surface. This method can be also applied to investigate the GPI-AP biosynthetic pathway and to directly confirm predicted GPI-anchoring of individual proteins., Competing Interests: The authors have declared that no competing interests exist.

Antigen polymorphisms in essential malarial antigens are a key challenge to the design and development of broadly effective malaria vaccines. The effect of polymorphisms on antibody responses is fairly well studied while much fewer studies have assessed this for T cell responses. This study investigated the effect of allelic polymorphisms in the malarial antigen apical membrane antigen 1 (AMA1) on ex vivo T cell-specific IFN-γ responses in subjects with lifelong exposure to malaria. Human leukocyte antigen (HLA) class I-restricted peptides from the 3D7 clone AMA1 were bioinformatically predicted and those with variant amino acid positions used to select corresponding allelic sequences from the 7G8, FVO, FC27 and tm284 parasite strains. A total of 91 AMA1 9-10mer peptides from the five parasite strains were identified, synthesized, grouped into 42 allele sets and used to stimulate PBMCs from seven HLA class 1-typed subjects in IFN-γ ELISpot assays. PBMCs from four of the seven subjects (57%) made positive responses to 18 peptides within 12 allele sets. Fifty percent of the 18 positive peptides were from the 3D7 parasite variant. Amino acid substitutions that were associated with IFN-γ response abrogation were more frequently found at positions 1 and 6 of the tested peptides, but substitutions did not show a clear pattern of association with response abrogation. Thus, while we show some evidence of polymorphisms affecting T cell response induction, other factors including TCR recognition of HLA-peptide complexes may also be at play., Competing Interests: The authors have declared that no competing interests exist.

Proteins make up a large percentage of the Brassica seed and are second only to the oil in economic importance with uses for both animal and human nutrition. The most abundant proteins reported in the seeds of Brassica are the seed storage proteins cruciferin and napin, belonging to the 12S globulin and 2S albumin families of proteins, respectively. To gain insight into the Brassica rapa seed proteome and to confirm the presence and relative quantity of proteins encoded by candidate seed storage genes in the mature seed, shotgun proteomics was carried out on protein extracts from seeds of B. rapa inbred line R-o-18. Following liquid chromatography tandem mass spectrometry, a total of 34016 spectra were mapped to 323 proteins, where 233 proteins were identified in 3 out of 4 biological replicates by at least 2 unique peptides. 2S albumin like napin seed storage proteins (SSPs), 11/12S globulin like cruciferin SSPs and 7S globulin like vicilin SSPs were identified in the samples, along with other notable proteins including oil body proteins, namely ten oleosins and two oil body-associated proteins. The identification of vicilin like proteins in the mature B. rapa seed represents the first account of these proteins in the Brassicaceae and analysis indicates high conservation of sequence motifs to other 7S vicilin-like allergenic proteins as well as conservation of major allergenic epitopes in the proteins. This study enriches our existing knowledge on rapeseed seed proteins and provides a robust foundation and rational basis for plant bioengineering of seed storage proteins., Competing Interests: The authors have declared that no competing interests exist.

The ability to target therapeutic agents to specific tissues is an important element in the development of new disease treatments. The transferrin receptor (TfR) is one potential target for drug delivery, as it expressed on many dividing cells and on brain endothelium, the key cellular component of the blood-brain barrier. The aim of this study was to compare a set of new and previously-described polypeptides for their ability to bind to brain endothelium, and investigate their potential for targeting therapeutic agents to the CNS. Six polypeptides were ranked for their rate of endocytosis by the human brain endothelial cell line hCMEC/D3 and the murine line bEnd.3. One linear polypeptide and two cyclic polypeptides showed high rates of uptake. These peptides were investigated to determine whether serum components, including transferrin itself affected uptake by the endothelium. One of the cyclic peptides was strongly inhibited by transferrin and the other cyclic peptide weakly inhibited. As proof of principle the linear peptide was attached to 2nm glucose coated gold-nanoparticles, and the rate of uptake of the nanoparticles measured in a hydrogel model of the blood-brain barrier. Attachment of the TfR-targeting polypeptide significantly increased the rates of endocytosis by brain endothelium and increased movement of nanoparticles across the cells., Competing Interests: Conor McQuaid was supported by a PhD studentship from Midatech Pharma plc, awarded to DM (2015-2018). This does not alter our adherence to PLOS ONE policies on sharing data and materials. The company has no patents, products in development or marketed products related to the material presented in this paper. None of the authors is employed either directly or as a consultant by Midatech Pharma plc. None of the authors has ever applied for employment with the company.’

Last decade has witnessed three major pandemics caused by SARS-CoV, SARS-CoV-2 and MERS-CoV that belong to Coronavirus family. Currently, there are no effective therapies available for corona virus infections. Since the three viruses belong to the same family and share many common features, we can theoretically design a drug that can be effective on all the three of them. In this study, using computational approach, we designed a peptide (Peptide 7) that can bind to the Receptor Binding Domain (RBD) of SARS-CoV, SARS-CoV-2 and MERS-CoV thereby preventing the entry of the viruses into the host cell. The peptide inhibitor was designed as a consensus peptide from three different peptides that might individually bind to the RBD of the three viruses. Docking studies and molecular dynamic simulations using Peptide 7 has shown that it binds with higher affinity than the native receptors of the RBD and forms a stable complex thereby preventing further viral-receptor interaction and inhibiting their cellular entry. This effective binding is observed for the three RBDs, despite the Peptide 7 interactions being slightly different. Hence; this peptide inhibitor can be used as a potential candidate for the development of peptide based anti-viral therapy against Corona viruses., Competing Interests: The authors have declared no competing interest exist.

T-cell receptor mimic (TCRm) antibodies have expanded the repertoire of antigens targetable by monoclonal antibodies, to include peptides derived from intracellular proteins that are presented by major histocompatibility complex class I (MHC-I) molecules on the cell surface. We have previously used this approach to target p53, which represents a valuable target for cancer immunotherapy because of the high frequency of its deregulation by mutation or other mechanisms. The T1-116C TCRm antibody targets the wild type p5365-73 peptide (RMPEAAPPV) presented by HLA-A*0201 (HLA-A2) and exhibited in vivo efficacy against triple receptor negative breast cancer xenografts. Here we report a comprehensive mutational analysis of the p53 RMPEAAPPV peptide to assess the T1-116C epitope and its peptide specificity. Antibody binding absolutely required the N-terminal arginine residue, while amino acids in the center of the peptide contributed little to specificity. Data mining the immune epitope database with the T1-116C binding consensus and validation of peptide recognition using the T2 stabilization assay identified additional tumor antigens targeted by T1-116C, including WT1, gp100, Tyrosinase and NY-ESO-1. Most peptides recognized by T1-116C were conserved in mice and human HLA-A2 transgenic mice showed no toxicity when treated with T1-116C in vivo. We conclude that comprehensive validation of TCRm antibody target specificity is critical for assessing their safety profile., Competing Interests: The authors have declared that no competing interests exist.

Glioma is a lethal malignant brain cancer, and many reports have shown that abnormalities in the behavior of water and ion channels play an important role in regulating tumor proliferation, migration, apoptosis, and differentiation. Recently, new studies have suggested that some long noncoding RNAs containing small open reading frames can encode small peptides and form oligomers for water or ion regulation. However, because the peptides are difficult to identify, their functional mechanisms are far from being clearly understood. In this study, we used bioinformatics methods to identify and evaluate lncRNAs, which may encode small transmembrane peptides in gliomas. Combining ab initio homology modeling, molecular dynamics simulations, and free energy calculations, we constructed a predictive model and predicted the oligomer channel activity of peptides by identifying the lncRNA ORFs. We found that one key hub lncRNA, namely, DLEU1, which contains two smORFs (ORF1 and ORF8), encodes small peptides that form pentameric channels. The mechanics of water and ion (Na+ and Cl-) transport through this pentameric channel were simulated. The potential mean force of the H2O molecules along the two ORF-encoded peptide channels indicated that the energy barrier was different between ORF1 and ORF8. The ORF1-encoded peptide pentamer acted as a self-assembled water channel but not as an ion channel, and the ORF8 permeated neither ions nor water. This work provides new methods and theoretical support for further elucidation of the function of lncRNA-encoded small peptides and their role in cancer. Additionally, this study provides a theoretical basis for drug development., Competing Interests: The authors have declared that no competing interests exist.

As a new alternative to antibody-drug conjugates, we generated "ligand-targeting" peptide-drug conjugates (PDCs), which utilize receptor-mediated endocytosis for targeted intracellular drug delivery. The PDC makes a complex with an extracellular ligand and then binds to the receptor on the cell surface to stimulate intracellular uptake via the endocytic pathway. A helix-loop-helix (HLH) peptide was designed as the drug carrier and randomized to give a conformationally constrained peptide library. The phage-displayed library was screened against vascular endothelial growth factor (VEGF) to yield the binding peptide M49, which exhibited strong binding affinity (KD = 0.87 nM). The confocal fluorescence microscopy revealed that peptide M49 formed a ternary complex with VEGF and its receptor, which was then internalized into human umbilical vein endothelial cells (HUVECs) via VEGF receptor-mediated endocytosis. The backbone-cyclized peptide M49K was conjugated with a drug, monomethyl auristatin E, to afford a PDC, which inhibited VEGF-induced HUVEC proliferation. HLH peptides and their PDCs have great potential as a new modality for targeted molecular therapy., Competing Interests: Co-author ZY is employed by Interprotein Corporation. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Split fluorescent proteins have wide applicability as biosensors for protein-protein interactions, genetically encoded tags for protein detection and localization, as well as fusion partners in super-resolution microscopy. We have here established and validated a novel platform for functional analysis of leave-one-out split fluorescent proteins (LOO-FPs) in high throughput and with rapid turnover. We have screened more than 12,000 variants of the beta-strand split fragment using high-density peptide microarrays for binding and functional complementation in Green Fluorescent Protein. We studied the effect of peptide length and the effect of different linkers to the solid support. We further mapped the effect of all possible amino acid substitutions on each position as well as in the context of some single and double amino acid substitutions. As all peptides were tested in 12 duplicates, the analysis rests on a firm statistical basis allowing for confirmation of the robustness and precision of the method. Based on experiments in solution, we conclude that under the given conditions, the signal intensity on the peptide microarray faithfully reflects the binding affinity between the split fragments. With this, we are able to identify a peptide with 9-fold higher affinity than the starting peptide., Competing Interests: The authors have declared that no competing interests exist.

Gonadotropic hormones play important regulatory roles in reproduction. Relaxin-like gonad-stimulating peptide (RGP) is a gonadotropin-like hormone in starfish. However, a receptor for RGP remains to be identified. Here, we describe the identification of an authentic receptor for RGP (RGPR) in the starfish, Patiria pectinifera. A binding assay using radioiodinated P. pectinifera RGP (PpeRGP) revealed that RGPR was expressed in ovarian follicle cells. A RGPR candidate was identified by homology-searching of transcriptome data of P. pectinifera follicle cells. Based on the contig sequences, a putative 947-amino acid PpeRGPR was cloned from follicle cells. Like the vertebrate relaxin family peptide receptors (RXFP 1 and 2), PpeRGPR was a G protein-coupled receptor that harbored a low-density lipoprotein-receptor class A motif and leucine-rich repeat sequences in the extracellular domain of the N-terminal region. Sf9 cells transfected with Gαq16-fused PpeRGPR activated calcium ion mobilization in response to PpeRGP, but not to RGP of another starfish Asterias amurensis, in a dose-dependent fashion. These results confirmed the species-specific reactivity of RGP and the cognate receptor. Thus, the present study provides evidence that PpeRGPR is a specific receptor for PpeRGP. To the best of our knowledge, this is the first report on the identification of a receptor for echinoderm RGP., Competing Interests: The authors have declared that they have no competing interests.

Tup1-Cyc8 (also known as Tup1-Ssn6) is a general transcriptional corepressor. D-Mannitol (mannitol) and D-sorbitol (sorbitol) are the major polyols in nature. Budding yeast Saccharomyces cerevisiae is unable to assimilate mannitol or sorbitol, but acquires the ability to assimilate mannitol due to a spontaneous mutation in TUP1 or CYC8. In this study, we found that spontaneous mutation of TUP1 or CYC8 also permitted assimilation of sorbitol. Some spontaneous nonsense mutations of CYC8 produced a truncated Cyc8 with a C-terminal polyglutamine. The effects were guanidine hydrochloride-sensitive and were dependent on Hsp104, but were complemented by introduction of CYC8, ruling out involvement of a prion. Assimilation of mannitol and sorbitol conferred by other mutations of TUP1 or CYC8 was guanidine hydrochloride-tolerant. It is physiologically reasonable that S. cerevisiae carries this mechanism to acquire the ability to assimilate major polyols in nature., Competing Interests: The authors have declared that no competing interests exist.

Laccases, oxidative copper-enzymes found in fungi and bacteria were used as the basis in the design of nona- and tetrapeptides. Laccases are known to be excellent catalysts for the degradation of phenolic xenobiotic waste. However, since solvent extraction of laccases is environmentally-unfriendly and yields obtained are low, they are less preferred compared to synthetic catalysts. The histidine rich peptides were designed based on the active site of laccase extracted from Trametes versicolor through RCSB Protein Data Bank, LOMETS and PyMol software. The peptides were synthesized using Fmoc-solid phase peptide synthesis (SPPS) with 30-40% yield. These peptides were purified and characterized using LC-MS (purities >75%), FTIR and NMR spectroscopy. Synthesized copper(II)-peptides were crystallized and then analyzed spectroscopically. Their structures were elucidated using 1D and 2D NMR. Standards (o,m,p-cresol, 2,4-dichlorophenol) catalysed using laccase from Trametes versicolor (0.66 U/mg) were screened under different temperatures and stirring rate conditions. After optimizing the degradation of the standards with the best reaction conditions reported herein, medications with phenolic and aromatic structures such as ibuprofen, paracetamol (acetaminophen), salbutamol, erythromycin and insulin were screened using laccase (positive control), apo-peptides and copper-peptides. Their activities evaluated using GC-MS, were compared with those of peptide and copper-peptide catalysts. The tetrapeptide was found to have the higher degradation activity towards salbutamol (96.8%) compared with laccase at 42.8%. Ibuprofen (35.1%), salbutamol (52.9%) and erythromycin (49.7%) were reported to have the highest degradation activities using Cu-tetrapeptide as catalyst when compared with the other medications. Consequently, o-cresol (84%) was oxidized by Tp-Cu while the apo-peptides failed to oxidize the cresols. Copper(II)-peptides were observed to have higher catalytic activity compared to their parent peptides and the enzyme laccase for xenobiotic degradation., Competing Interests: The authors have declared that no competing interests exist.

Two commercial proteases (subtilisin-typed FNA from Bacillus amyloliquefaciens, and chymotrypsin-like NPP from Nocardiopsis prasina), porcine pepsin, porcine pancreatin having protease activity and their combinations were studied in vitro by LC-MS for their ability to digest soy protein isolate (SPI) under conditions close to those found in the stomach (pH 3.7) and small intestine (pH 6.5). The total number of peptides generated, and their size distribution were obtained under each set of the digestion conditions. These peptides were grouped according to their C-terminal amino acid (AA) residue (P1) and mass, based on which two concepts were proposed, i.e., Normalized Peptide Bond Cleavage Frequency (NPBCF) and Protease Substrate Broadness Index (PSBI). At pH 3.7, FNA+pepsin increased PSBI vs. pepsin alone by 2.7 and 4.9 percentage points (p.p.) at a SPI:protease ratio of 20:1 and 100:1, respectively. At pH 6.5, FNA+pancreatin improved PSBI by 9.1 and 10.2 p.p. at SPI:protease 20:1 and 100:1, respectively, vs. pancreatin alone. NPP generated 38% more peptides than FNA when administered with pancreatin at SPI:protease 200:1:1 and pH 6.5, but FNA alone (28.9) or FNA+pancreatin (29.1) gave a higher PSBI than pancreatin (22.2), NPP (20.3) and NPP+pancreatin (22.0). At pH 3.7 FNA generated 59% and 39% of peptides of pepsin at SPI:protease of 20:1 and 100:1, respectively, and both groups of peptides had similar size distribution. At pH 6.5 more small sized peptides were generated by FNA or FNA+pancreatin than pancreatin and NPP alone or pancreatin+NPP. In conclusion, FNA showed complementary effects with pepsin and pancreatin in terms of PSBI and generated more small sized peptides compared to NPP., Competing Interests: E. I. du Pont de Nemours and Company provided support in the form of R&D budget for all authors. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The protease FNA and soy protein isolate Supro 760 described in the “M&M” section are commercial products provided by DuPont for this research. There are no patents, products in development or other marketed products associated with this research to be declared.

Introduction: In cancer treatment an attempt has been made to pharmacologically regulate the proteasome functions, thus the aim was to test whether 20S proteasome chymotrypsin-like (ChT-L) activity has a role in glial brain tumors. Furthermore, we analyzed the correlation between proteasome activity and IL-8, CCL2, NF-κB1 and NF-κB2 concentrations, which impact on brain tumors has already been indicated., Methods: Plasma 20S proteasome ChT-L activity was assayed using the fluorogenic peptide substrate Suc-Leu-Leu-Val-Tyr-AMC in the presence of SDS. IL-8, CCL2, NF-κB1 and NF-κB2 concentration was analyzed with the use of ELISA method. Immunohistochemistry for IDH1-R132H was done on 5-microns-thick formalin-fixed, paraffin-embedded tumor sections with the use of antibody specific for the mutant IDH1-R132H protein. Labelled streptavidin biotin kit was used as a detection system., Results: Brain tumor patients had statistically higher 20S proteasome ChT-L activity (0.649 U/mg) compared to non-tumoral individuals (0.430 U/mg). IDH1 wild-type patients had statistically higher 20S proteasome ChT-L activity (1.025 U/mg) compared to IDH1 mutants (0.549 U/mg). 20S proteasome ChT-L activity in brain tumor patients who died as the consequence of a tumor (0.649) in the following 2 years was statistically higher compared to brain tumor patients who lived (0.430 U/mg). In brain tumor patients the 20S proteasome ChT-L activity positively correlated with IL-8 concentration., Conclusions: Elevated 20S proteasome ChT-L activity was related to the increased risk of death in glial brain tumor patients. A positive correlation between 20S proteasome ChT-L activity and IL-8 concentration may indicate the molecular mechanisms regulating glial tumor biology. Thus research on proteasomes may be important and should be carried out to verify if this protein complexes may represent a potential therapeutic target to limit brain tumor invasion., Competing Interests: The authors have declared that no competing interests exist.

Poly(glycine-alanine) (polyGA) is one of the polydipeptides expressed in Frontotemporal Dementia and/or Amyotrophic Lateral Sclerosis 1 caused by C9ORF72 mutations and accumulates as inclusion bodies in the brain of patients. Superficially these inclusions are similar to those formed by polyglutamine (polyQ)-expanded Huntingtin exon 1 (Httex1) in Huntington's disease. Both have been reported to form an amyloid-like structure suggesting they might aggregate via similar mechanisms and therefore recruit the same repertoire of endogenous proteins. When co-expressed in the same cell, polyGA101 and Httex1(Q97) inclusions adopted immiscible phases suggesting different endogenous proteins would be enriched. Proteomic analyses identified 822 proteins in the inclusions. Only 7 were specific to polyGA and 4 specific to Httex1(Q97). Quantitation demonstrated distinct enrichment patterns for the proteins not specific to each inclusion type (up to ~8-fold normalized to total mass). The proteasome, microtubules, TriC chaperones, and translational machinery were enriched in polyGA aggregates, whereas Dnaj chaperones, nuclear envelope and RNA splicing proteins were enriched in Httex1(Q97) aggregates. Both structures revealed a collection of folding and degradation machinery including proteins in the Httex1(Q97) aggregates that are risk factors for other neurodegenerative diseases involving protein aggregation when mutated, which suggests a convergence point in the pathomechanisms of these diseases., Competing Interests: The authors have declared that no competing interests exist.

Increased deposition of fibronectin fibrils containing EDA+fibronectin by TGFβ2 is thought to be involved in the reduction of aqueous humor outflow across the trabecular meshwork (TM) of the eye and the elevation in intraocular pressure (IOP) observed in primary open angle glaucoma (POAG). Using a fibronectin-binding peptide called FUD that can disrupt fibronectin fibrillogenesis, we examined if disrupting fibronectin fibrillogenesis would affect IOP in the TGFβ2 BALB/cJ mouse model of ocular hypertension. BALB/cJ mice that had been intravitreally injected with an adenovirus (Ad5) expressing a bioactive TGFβ2226/228 showed a significant increase in IOP after 2 weeks. When 1μM FUD was injected intracamerally into mice 2 weeks post Ad5-TGFβ2 injection, FUD significantly reduced IOP after 2 days. Neither mutated FUD (mFUD) nor PBS had any effect on IOP. Four days after FUD was injected, IOP returned to pre-FUD injection levels. In the absence of TGFβ2, intracameral injection of FUD had no effect on IOP. Western blotting of mouse anterior segments expressing TGFβ2 showed that FUD decreased fibronectin levels 2 days after intracameral injection (p<0.05) but not 7 days compared to eyes injected with PBS. mFUD injection had no significant effect on fibronectin levels at any time point. Immunofluorescence microscopy studies in human TM (HTM) cells showed that treatment with 2ng/ml TGFβ2 increased the amount of EDA+ and EDB+ fibronectin incorporated into fibrils and 2μM FUD decreased both EDA+ and EDB+ fibronectin in fibrils. An on-cell western assay validated this and showed that FUD caused a 67% reduction in deoxycholate insoluble fibronectin fibrils in the presence of TGFβ2. FUD also caused a 43% reduction in fibronectin fibrillogenesis in the absence of TGFβ2 while mFUD had no effect. These studies suggest that targeting the assembly of fibronectin fibrillogenesis may represent a way to control IOP., Competing Interests: The authors have declared that no competing interests exist.

Evidence indicate that the brain-specific protein, brevican, is proteolytically cleaved during neurodegeneration, hence positioning fragments of brevican as potential blood biomarkers of neurodegenerative diseases, such as dementia. We aimed to develop two assays capable of detecting the brevican N-terminal (N-Brev) and the ADAMTS4-generated fragment (Brev-A), cleaved at Ser401, in serum and to perform a preliminary assessment of their diagnostic potential in dementias. Monoclonal antibodies against N-Brev and Brev-A were used to develop two ELISAs detecting each epitope. A comparison of brevican fragments in serum from individuals with AD (n = 28), other dementia (OD) (n = 41), and non-dementia-related memory complaints (NDCs) (n = 48) was conducted. Anti-N-Brev and anti-Brev-A antibodies selectively recognized their targets and dilution and spike recoveries were within limits of ±20%. Intra- and inter-assay CVs were below limits of 10% and 15%, respectively. For the N-Brev biomarker, serum from patients with OD showed significantly lower levels than those with AD (p = 0.05) and NDCs (p < 0.01). The opposite pattern was evident for Brev-A: serum levels in patients with OD were significantly higher than for AD (p = 0.04) and NDCs (p = 0.01). For both N-Brev and Brev-A, levels did not differ between AD and NDCs. The ratio of N-Brev/Brev-A resulted in increased significant differences between OD and AD (p < 0.01) and between OD and NDCs (p < 0.0001). The ratio discriminated between NDCs and OD (AUC: 0.75, 95% CI: 0.65-0.85, p < 0.0001) and between OD and AD (AUC: 0.72, 95% CI: 0.59-0.85, p < 0.01). In conclusion, we developed the first assays detecting the N-terminal of brevican as well as an ADAMTS4-cleaved fragment of brevican in blood. Differential levels of N-Brev and Brev-A between AD and OD allow for these biomarkers to possibly distinguish between different forms of dementias., Competing Interests: The authors of this manuscript have read the journal’s policy and have the following competing interests: DSJ, KH and MAK are employed by Nordic Bioscience and thus received support in the form of a salary. KH and MK own stocks in Nordic Bioscience and hold patents on biomarkers not measured in the present study. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products to declare in relation to this research.

The susceptibility of newly expressed proteins to digestion by gastrointestinal proteases (e.g., pepsin) has long been regarded as one of the important endpoints in the weight-of-evidence (WOE) approach to assess the allergenic risk of genetically modified (GM) crops. The European Food Safety Authority (EFSA) has suggested that current digestion study protocols used for this assessment should be modified to more accurately reflect the diverse physiological conditions encountered in human populations and that the post-digestion analysis should include analytical methods to detect small peptide digestion products.The susceptibility of two allergens (beta-lactoglobin (β-Lg) and alpha-lactalbumin (α-La)) and two non-allergens (hemoglobin (Hb) and phosphofructokinase (PFK)) to proteolytic degradation was investigated under two pepsin digestion conditions (optimal pepsin digestion condition: pH 1.2, 10 U pepsin/μg test protein; sub-optimal pepsin digestion condition: pH 5.0, 1 U pepsin/10 mg test protein), followed by 34.5 U trypsin/mg test protein and 0.4 U chymotrypsin/mg test protein digestion in the absence or presence of bile salts. All samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in conjunction with Coomassie Blue staining and, in parallel, liquid chromatography tandem mass spectrometry (LC-MS) detection. The results provide following insights: 1) LC-MS methodology does provide the detection of small peptides; 2) Peptides are detected in both allergens and non-allergens from all digestion conditions; 3) No clear differences among the peptides detected from allergen and non-allergens; 4) The differences observed in SDS-PAGE between the optimal and sub-optimal pepsin digestion conditions are expected and align with kinetics and properties of the specific enzymes; 5) The new methodology with new digestion conditions and LC-MS detection does not provide any differentiating information for prediction whether a protein is an allergen. The classic pepsin resistance assay remains the most useful assessment of the potential exposure of an intact newly expressed protein as part of product safety assessment within a WOE approach., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interest: The authors are employees of Bayer Crop Science, a leading manufacturer of crop seeds developed through conventional breeding or biotechnology. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Inactivation of the tumor suppressor p53 resulting from the binding with a negative regulator HDM2 is among the predominant defects in human cancers. p53-mimicking peptides whose conformational and proteolytic stability is enhanced by an all-hydrocarbon staple are being recognized as promising anticancer agents for disrupting the p53-HDM2 binding and reactivating p53. Herein, we conduct a computational modeling and thermodynamic characterization of stapled p53/HDM2 complex via molecular docking, simulations, and binding free energy analysis. The binding thermodynamics analysis is done based on the end-point calculation of the effective binding energy-a sum of the direct peptide-protein interaction energy and the dehydration penalty-and on its decomposition into contributions from specific groups constituting the complex. This allows us to investigate how individual amino acids in the stapled p53 and HDM2 contribute to the binding affinity. We find that not only the epitope residues (F19, W23 and L26), but also the hydrocarbon linker of the stapled p53 impart significant contributions. Our computational approach will be useful in designing new stapled peptides in which the staple location is also optimized to improve the binding affinity., Competing Interests: The authors have declared that no competing interests exist.

Hair growth is the cyclically regulated process that is characterized by growing phase (anagen), regression phase (catagen) and resting phase (telogen). Hair follicle stem cells (HFSCs) play pivotal role in the control of hair growth cycle. It has been notified that stem cells have the distinguished metabolic signature compared to differentiated cells, such as the preference to glycolysis rather than mitochondrial respiration. Crif1 is a mitochondrial protein that regulates the synthesis and insertion of oxidative phosphorylation (OXPHOS) polypeptides to inner membrane of mitochondria. Several studies demonstrate that tissue-specific knockout of Crif1 leads to mitochondrial dysfunction. In this study, we investigated the effect of mitochondrial dysfunction in terms of Crif1 deficiency on the hair growth cycle of adult mice. We created two kinds of inducible conditional knockout (icKO) mice. In epidermal specific icKO mice (Crif1 K14icKO), hair growth cycle was significantly retarded compared to wild type mice. Similarly, HFSC specific icKO mice (Crif1 K15icKO) showed significant retardation of hair growth cycle in depilation-induced anagen model. Interestingly, flow cytometry revealed that HFSC populations were maintained in Crif1 K15icKO mice. These results suggest that mitochondrial function in HFSCs is important for the progression of hair growth cycle, but not for maintenance of HFSCs., Competing Interests: The authors have declared that no competing interests exist.

Expanded CAG nucleotide repeats are the underlying genetic cause of at least 14 incurable diseases, including Huntington's disease (HD). The toxicity associated with many CAG repeat expansions is thought to be due to the translation of the CAG repeat to create a polyQ protein, which forms toxic oligomers and aggregates. However, recent studies show that HD CAG repeats undergo a non-canonical form of translation called Repeat-associated non-AUG dependent (RAN) translation. RAN translation of the CAG sense and CUG anti-sense RNAs produces six distinct repeat peptides: polyalanine (polyAla, from both CAG and CUG repeats), polyserine (polySer), polyleucine (polyLeu), polycysteine (polyCys), and polyglutamine (polyGln). The toxic potential of individual CAG-derived RAN polypeptides is not well understood. We developed pure C. elegans protein models for each CAG RAN polypeptide using codon-varied expression constructs that preserve RAN protein sequence but eliminate repetitive CAG/CUG RNA. While all RAN polypeptides formed aggregates, only polyLeu was consistently toxic across multiple cell types. In GABAergic neurons, which exhibit significant neurodegeneration in HD patients, codon-varied (Leu)38, but not (Gln)38, caused substantial neurodegeneration and motility defects. Our studies provide the first in vivo evaluation of CAG-derived RAN polypeptides in a multicellular model organism and suggest that polyQ-independent mechanisms, such as RAN-translated polyLeu peptides, may have a significant pathological role in CAG repeat expansion disorders., Competing Interests: The authors have declared that no competing interests exist.