CD38 promotes hematopoietic stem cell dormancy.

A subpopulation of deeply quiescent, so-called dormant hematopoietic stem cells (dHSCs) resides at the top of the hematopoietic hierarchy and serves as a reserve pool for HSCs. The state of dormancy protects the HSC pool from exhaustion throughout life; however, excessive dormancy may prevent an efficient response to hematological stresses. Despite the significance of dHSCs, the mechanisms maintaining their dormancy remain elusive. Here, we identify CD38 as a novel and broadly applicable surface marker for the enrichment of murine dHSCs. We demonstrate that cyclic adenosine diphosphate ribose (cADPR), the product of CD38 cyclase activity, regulates the expression of the transcription factor c-Fos by increasing the release of Ca2+ from the endoplasmic reticulum (ER). Subsequently, we uncover that c-Fos induces the expression of the cell cycle inhibitor p57Kip2 to drive HSC dormancy. Moreover, we found that CD38 ecto-enzymatic activity at the neighboring CD38-positive cells can promote human HSC quiescence. Together, CD38/cADPR/Ca2+/c-Fos/p57Kip2 axis maintains HSC dormancy. Pharmacological manipulations of this pathway can provide new strategies to improve the success of stem cell transplantation and blood regeneration after injury or disease.

A vasopressin circuit that modulates mouse social investigation and anxiety-like behavior in a sex-specific manner.

One of the largest sex differences in brain neurochemistry is the expression of the neuropeptide arginine vasopressin (AVP) within the vertebrate brain, with males having more AVP cells in the bed nucleus of the stria terminalis (BNST) than females. Despite the long-standing implication of AVP in social and anxiety-like behaviors, the circuitry underlying AVP's control of these behaviors is still not well defined. Using optogenetic approaches, we show that inhibiting AVP BNST cells reduces social investigation in males, but not in females, whereas stimulating these cells increases social investigation in both sexes, but more so in males. These cells may facilitate male social investigation through their projections to the lateral septum (LS), an area with the highest density of sexually differentiated AVP innervation in the brain, as optogenetic stimulation of BNST AVP → LS increased social investigation and anxiety-like behavior in males but not in females; the same stimulation also caused a biphasic response of LS cells ex vivo. Blocking the vasopressin 1a receptor (V1aR) in the LS eliminated all these responses. Together, these findings establish a sexually differentiated role for BNST AVP cells in the control of social investigation and anxiety-like behavior, likely mediated by their projections to the LS.

Keratin filaments mediate the expansion of extra-embryonic membranes in the post-gastrulation mouse embryo.

Mesoderm arises at gastrulation and contributes to both the mouse embryo proper and its extra-embryonic membranes. Two-photon live imaging of embryos bearing a keratin reporter allowed recording filament nucleation and elongation in the extra-embryonic region. Upon separation of amniotic and exocoelomic cavities, keratin 8 formed apical cables co-aligned across multiple cells in the amnion, allantois, and blood islands. An influence of substrate rigidity and composition on cell behavior and keratin content was observed in mesoderm explants. Embryos lacking all keratin filaments displayed a deflated extra-embryonic cavity, a narrow thick amnion, and a short allantois. Single-cell RNA sequencing of sorted mesoderm cells and micro-dissected amnion, chorion, and allantois, provided an atlas of transcriptomes with germ layer and regional information. It defined the cytoskeleton and adhesion expression profile of mesoderm-derived keratin 8-enriched cells lining the exocoelomic cavity. Those findings indicate a novel role for keratin filaments in the expansion of extra-embryonic structures and suggest mechanisms of mesoderm adaptation to the environment.

A single-cell atlas of de novo ß-cell regeneration reveals the contribution of hybrid ß/δ-cells to diabetes recovery in zebrafish.

Regeneration-competent species possess the ability to reverse the progression of severe diseases by restoring the function of the damaged tissue. However, the cellular dynamics underlying this capability remain unexplored. Here, we have used single-cell transcriptomics to map de novo ß-cell regeneration during induction and recovery from diabetes in zebrafish. We show that the zebrafish has evolved two distinct types of somatostatin-producing δ-cells, which we term δ1- and δ2-cells. Moreover, we characterize a small population of glucose-responsive islet cells, which share the hormones and fate-determinants of both ß- and δ1-cells. The transcriptomic analysis of ß-cell regeneration reveals that ß/δ hybrid cells provide a prominent source of insulin expression during diabetes recovery. Using in vivo calcium imaging and cell tracking, we further show that the hybrid cells form de novo and acquire glucose-responsiveness in the course of regeneration. The overexpression of dkk3, a gene enriched in hybrid cells, increases their formation in the absence of ß-cell injury. Finally, interspecies comparison shows that plastic δ1-cells are partially related to PP cells in the human pancreas. Our work provides an atlas of ß-cell regeneration and indicates that the rapid formation of glucose-responsive hybrid cells contributes to the resolution of diabetes in zebrafish.

In vivo imaging of calcium dynamics in zebrafish hepatocytes.

BACKGROUND AND AIMS: Hepatocytes were the first cell type for which oscillations of cytoplasmic calcium levels in response to hormones were described. Since then, investigation of calcium dynamics in liver explants and culture has greatly increased our understanding of calcium signaling. A bottleneck, however, exists in observing calcium dynamics in a noninvasive manner because of the optical inaccessibility of the mammalian liver. Here, we aimed to take advantage of the transparency of the zebrafish larvae to image hepatocyte calcium dynamics in vivo at cellular resolution. APPROACH AND RESULTS: We developed a transgenic model expressing a calcium sensor, GCaMP6s, specifically in zebrafish hepatocytes. Using this, we provide a quantitative assessment of intracellular calcium dynamics during multiple contexts, including growth, feeding, ethanol-induced stress, and cell ablation. Specifically, we show that synchronized calcium oscillations are present in vivo , which are lost upon starvation. Starvation induces lipid accumulation in the liver. Feeding recommences calcium waves in the liver, but in a spatially restricted manner, as well as resolves starvation-induced hepatic steatosis. By using a genetically encoded scavenger for calcium, we show that dampening of calcium signaling accelerates the accumulation of starvation-related lipid droplets in the liver. Furthermore, ethanol treatment, as well as cell ablation, induces calcium flux, but with different dynamics. The former causes asynchronous calcium oscillations, whereas the latter leads to a single calcium spike. CONCLUSIONS: We demonstrate the presence of oscillations, waves, and spikes in vivo . Calcium waves are present in response to nutrition and negatively regulate starvation-induced accumulation of lipid droplets.

The semisynthesis of site-specifically modified histones and histone-based probes of chromatin-modifying enzymes.

Histone post-translational modifications (PTMs) on lysine residues, including methylation, ubiquitylation, and sumoylation, have been studied using semisynthetic histones reconstituted into nucleosomes. These studies have revealed the in vitro effects of histone PTMs on chromatin structure, gene transcription, and biochemical crosstalk. However, the dynamic and transient nature of most enzyme-chromatin interactions poses a challenge toward identifying specific enzyme-substrate interactions. To address this, we report methodology for the synthesis of two ubiquitylated activity-based probe histones, H2BK120ub(G76C) and H2BK120ub(G76Dha), that may be used to trap enzyme active-site cysteines as disulfides or in the form of thioether linkages, respectively. The general synthetic method we report for converting ubiquitylated nucleosomes into activity-based probes may also be applied to other histone sites of ubiquitylation in order to facilitate the identification of enzyme-chromatin interactions.

Betaine alleviates doxorubicin-induced nephrotoxicity by preventing oxidative insults, inflammation, and fibrosis through the modulation of Nrf2/HO-1/NLRP3 and TGF-ß expression.

Doxorubicin (Dox) is an anthracycline antibiotic used to treat various cancers and shows severe toxicity in multiple organ systems, including kidneys. Evidence shows that betaine's antioxidant and anti-inflammatory properties could prevent the onset of several disorders. Hence, the present study aims to investigate the therapeutic potential of betaine on Dox-induced nephrotoxicity (DIN). Nephrotoxicity was induced in male Sprague Dawley rats using Dox at a dose of 4 mg/kg (cumulative dose: 20 mg/kg) by the intraperitoneal route and cotreated with betaine through oral gavage (200 and 400 mg/kg) for 28 days. At the end of the experiment, biochemical, oxidative stress parameters, histopathology, and qRT-PCR were performed. DIN was indicated by elevated serum creatinine, urea, and decreased albumin levels representing kidney damage; the histopathological lesions (increased capsular space, renal tubule damage, and fibrosis) in renal tissues supported these biochemical findings. Interestingly, betaine treatment improves these alterations in Dox-treated rats. Further, betaine treatment decreases the lipid peroxidation and nitrite concentration and increases the superoxide dismutases and catalase enzyme concentration in Dox-treated rats. Fascinatingly, at the molecular level, DIN in rats shows upregulation of the Nrf2/HO-1 gene, while betaine treatment attenuated its expression along with the downregulation of inflammatory genes (NLRP3, TLR-4, TNF-α, and IL-6) and fibrosis-related genes (TGF-ß and Acta2) expression in Dox-treated rats. These results showed that betaine has reno-protective properties by reducing inflammatory and fibrotic mediators and enhancing antioxidant capacity in the renal tissue of rats treated with Dox. We believe betaine can be exploited as a dietary supplement to attenuate DIN.

Betaine ameliorates doxorubicin-induced cardiomyopathy by inhibiting oxidative stress, inflammation, and fibrosis through the modulation of AMPK/Nrf2/TGF-ß expression.

Doxorubicin (DOX) is a broad-spectrum antibiotic with potent anti-cancer activity. Nevertheless, despite having effective anti-neoplasm activity, its use has been clinically restricted due to its life-threatening side effects, such as cardiotoxicity. It is evident that betaine has anti-oxidant, and anti-inflammatory activity and has several beneficial effects, such as decreasing the amyloid-ß generation, reducing obesity, improving steatosis and fibrosis, and activating AMP-activated protein kinase (AMPK). However, whether betaine could mitigate DOX-induced cardiomyopathy is still unexplored. Cardiomyopathy was induced in male Sprague Dawley rats using DOX (4 mg/kg dose with a cumulative dose of 20 mg/kg, i.p.). Further, betaine (200 and 400 mg/kg) was co-treated with DOX through oral gavage for 28 days. After the completion of the study, several biochemical, oxidative stress parameters, histopathology, western blotting, and qRT-PCR were performed. Betaine treatment significantly reduced CK-MB, LDH, SGOT, and triglyceride levels, which are associated with cardiotoxicity. DOX-induced increased oxidative stress was also mitigated by betaine intervention as the SOD, catalase, MDA, and nitrite levels were restored. The histopathological investigation also confirmed the cardioprotective effect of betaine against DOX-induced cardiomyopathy as the tissue injury was reversed. Further, molecular analysis revealed that betaine suppressed the DOX-induced increased expression of phospho-p53, phospho-p38 MAPK, NF-kB p65, and PINK 1 with an upregulation of AMPK and downregulation of Nrf2 expression. Interestingly, qRT-PCR experiments show that betaine treatment alleviates the DOX-induced increase in inflammatory (TNF-α, NLRP3, and IL-6) and fibrosis (TGF-ß and Acta2) related gene expression, halting the cardiac injury. Interestingly, betaine also improves the mRNA expression of Nrf2, thus modulating the expression of antioxidant proteins and preventing oxidative damage. Here, we provide the first evidence that betaine treatment prevents DOX-induced cardiomyopathy by inhibiting oxidative stress, inflammation, and fibrosis by regulating AMPK/Nrf2/TGF-ß expression. We believe that betaine can be utilized as a potential novel therapeutic strategy for preventing DOX-induced cardiotoxicity.

An assessment of "Organization Culture" in District Program Management Units established under National Health Mission in a western state of India.

Background: State programme management unit and District Programme Management Units (DPMU) are established under National Health Mission with an expectation to strengthen the management structures at the state and district levels, respectively. This is a major initiative to bring change in organisation structure and an effort to embrace change in working culture. The study was undertaken in a western Indian state to assess and compare the organisation culture in DPMUs. Methods: Districts of the state were divided into three strata based on the geographical and demographic features. One district from each stratum was randomly selected for the study. In-depth interview of nine key officials in each sampled DPMU was conducted with the use of standardised questionnaire-openness, confrontation, trust, authenticity, proactiveness, autonomy, collaboration and experimentation (OCTAPACE), developed by Pareek. Results: Values of OCTAPACE were diversely distributed with significant difference in distribution of scores between these domains (p < 0.05). Pro-action and Trust scores were relatively higher and consistent in the study units. Values of experimentation were lower and inconsistent. There was no significant difference in the distribution of scores between three sampled DPMUs for openness, confrontation, trust, authenticity, pro-action, autonomy and experimentation (p > 0.05). However, the distribution of collaboration score was significantly less in DPMU of desert district in comparison to DPMU of plain and tribal district (p < 0.05). Conclusion: The lacuna in values of organisation culture should be addressed to strengthen the existing management structures at the state and district levels.

A systematic review of excess all-cause mortality estimation studies in India during COVID-19 pandemic.

Background: Mortality statistics are fundamental to understand the magnitude of the COVID-19 pandemic. Due to limitation of real-time data availability, researchers had used mathematical models to estimate excess mortality globally during COVID-19 pandemic. As they demonstrated variations in scope, assumptions, estimations, and magnitude of the pandemic, and hence raised a controversy all over the world. This paper aims to review the mathematical models and their estimates of mortality due to COVID-19 in the Indian context. Methods: The PRISMA and SWiM guidelines were followed to the best possible extent. A two-step search strategy was used to identify studies that estimated excess deaths from January 2020 to December 2021 on Medline, Google Scholar, MedRxiv and BioRxiv available until 0100 h, 16 May 2022 (IST). We selected 13 studies based on a predefined criteria and extracted data on a standardised, pre-piloted form by two investigators, independently. Any discordance was resolved through consensus with a senior investigator. Estimated excess mortality was analysed using statistical software and depicted using appropriate graphs. Results: Significant variations in scope, population, data sources, time period, and modelling strategies existed across studies along with a high risk of bias. Most of the models were based on Poisson regression. Predicted excess mortality by various models ranged from 1.1 to 9.5 million. Conclusion: The review presents a summary of all the estimates of excess deaths and is important to understand the different strategies used for estimation, and it highlights the importance of data availability, assumptions, and estimates.

The Transforming Outcomes for Patients Through Medical Home Evaluation and reDesign (TOPMED) Cluster Randomized Controlled Trial: Cost and Utilization Results.

BACKGROUND: Primary Care Medical Home (PCMH) redesign efforts are intended to enhance primary care's ability to improve population health and well-being. PCMH transformation that is focused on "high-value elements" (HVEs) for cost and utilization may improve effectiveness. OBJECTIVES: The objective of this study was to determine if a focus on achieving HVEs extracted from successful primary care transformation models would reduce cost and utilization as compared with a focus on achieving PCMH quality improvement goals. RESEARCH DESIGN: A stratified, cluster randomized controlled trial with 2 arms. All practices received equal financial incentives, health information technology support, and in-person practice facilitation. Analyses consisted of multivariable modeling, adjusting for the cluster, with difference-in-difference results. SUBJECTS: Eight primary care clinics that were engaged in PCMH reform. MEASURES: We examined: (1) total claims payments; (2) emergency department (ED) visits; and (3) hospitalizations among patients during baseline and intervention years. RESULTS: In total, 16,099 patients met the inclusion criteria. Intervention clinics had significantly lower baseline ED visits (P=0.02) and claims paid (P=0.01). Difference-in-difference showed a decrease in ED visits greater in control than intervention (ED per 1000 patients: +56; 95% confidence interval: +96, +15) with a trend towards decreased hospitalizations in intervention (-15; 95% confidence interval: -52, +21). Costs were not different. In modeling monthly outcome means, the generalized linear mixed model showed significant differences for hospitalizations during the intervention year (P=0.03). DISCUSSION: The trial had a trend of decreasing hospitalizations, increased ED visits, and no change in costs in the HVE versus quality improvement arms.

Loss of tetraspanin-7 expression reduces pancreatic ß-cell exocytosis Ca2+ sensitivity but has limited effect on systemic metabolism.

BACKGROUND: Tetraspanin-7 (Tspan7) is an islet autoantigen involved in autoimmune type 1 diabetes and known to regulate ß-cell L-type Ca2+ channel activity. However, the role of Tspan7 in pancreatic ß-cell function is not yet fully understood. METHODS: Histological analyses were conducted using immunostaining. Whole-body metabolism was tested using glucose tolerance test. Islet hormone secretion was quantified using static batch incubation or dynamic perifusion. ß-cell transmembrane currents, electrical activity and exocytosis were measured using whole-cell patch-clamping and capacitance measurements. Gene expression was studied using mRNA-sequencing and quantitative PCR. RESULTS: Tspan7 is expressed in insulin-containing granules of pancreatic ß-cells and glucagon-producing α-cells. Tspan7 knockout mice (Tspan7y/- mouse) exhibit reduced body weight and ad libitum plasma glucose but normal glucose tolerance. Tspan7y/- islets have normal insulin content and glucose- or tolbutamide-stimulated insulin secretion. Depolarisation-triggered Ca2+ current was enhanced in Tspan7y/- ß-cells, but ß-cell electrical activity and depolarisation-evoked exocytosis were unchanged suggesting that exocytosis was less sensitive to Ca2+ . TSPAN7 knockdown (KD) in human pseudo-islets led to a significant reduction in insulin secretion stimulated by 20 mM K+ . Transcriptomic analyses show that TSPAN7 KD in human pseudo-islets correlated with changes in genes involved in hormone secretion, apoptosis and ER stress. Consistent with rodent ß-cells, exocytotic Ca2+ sensitivity was reduced in a human ß-cell line (EndoC-ßH1) following Tspan7 KD. CONCLUSION: Tspan7 is involved in the regulation of Ca2+ -dependent exocytosis in ß-cells. Its function is more significant in human ß-cells than their rodent counterparts.

Asymmetry in the frequency and position of mitosis in the mouse embryo epiblast at gastrulation.

At gastrulation, a subpopulation of epiblast cells constitutes a transient posteriorly located structure called the primitive streak, where cells that undergo epithelial-mesenchymal transition make up the mesoderm and endoderm lineages. Mouse embryo epiblast cells were labelled ubiquitously or in a mosaic fashion. Cell shape, packing, organization and division were recorded through live imaging during primitive streak formation. Posterior epiblast displays a higher frequency of rosettes, some of which associate with a central cell undergoing mitosis. Cells at the primitive streak, in particular delaminating cells, undergo mitosis more frequently than other epiblast cells. In pseudostratified epithelia, mitosis takes place at the apical side of the epithelium. However, mitosis is not restricted to the apical side of the epiblast, particularly on its posterior side. Non-apical mitosis occurs specifically in the streak even when ectopically located. Posterior non-apical mitosis results in one or two daughter cells leaving the epiblast layer. Cell rearrangement associated with mitotic cell rounding in posterior epiblast, in particular when non-apical, might thus facilitate cell ingression and transition to a mesenchymal phenotype.

Peptide and protein chemistry approaches to study the tumor suppressor protein p53.

The tumor suppressor and master gene regulator protein p53 has been the subject of intense investigation for several decades due to its mutation in about half of all human cancers. However, mechanistic studies of p53 in cells are complicated by its many dynamic binding partners and heterogeneous post-translational modifications. The design of therapeutics that rescue p53 functions in cells requires a mechanistic understanding of its protein-protein interactions in specific protein complexes and identifying changes in p53 activity by diverse post-translational modifications. This review highlights the important roles that peptide and protein chemistry have played in biophysical and biochemical studies aimed at elucidating p53 regulation by several key binding partners. The design of various peptide inhibitors that rescue p53 function in cells and new opportunities in targeting p53-protein interactions are discussed. In addition, the review highlights the importance of a protein semisynthesis approach to comprehend the role of site-specific PTMs in p53 regulation.

Diverse fate of ubiquitin chain moieties: The proximal is degraded with the target, and the distal protects the proximal from removal and recycles.

One of the enigmas in the ubiquitin (Ub) field is the requirement for a poly-Ub chain as a proteasomal targeting signal. The canonical chain appears to be longer than the distance between the two Ub-binding proteasomal receptors. Furthermore, genetic manipulation has shown that one receptor subunit is sufficient, which suggests that a single Ub can serve as a degradation signal. To shed light on this mystery, we chemically synthesized tetra-Ub, di-Ub (K48-based), and mono-Ub adducts of HA-α-globin, where the distal or proximal Ub moieties were tagged differentially with either Myc or Flag. When incubated in a crude cell extract, the distal Ub moiety in the tetra-Ub adduct was mostly removed by deubiquitinating enzymes (DUBs) and reconjugated to other substrates in the extract. In contrast, the proximal moiety was most likely degraded with the substrate. The efficacy of degradation was proportionate to the chain length; while tetra-Ub globin was an efficient substrate, with mono-Ub globin, we observed rapid removal of the Ub moiety with almost no degradation of the free globin. Taken together, these findings suggest that the proximal moieties are necessary for securing the association of the substrate with the proteasome along the proteolytic process, whereas the distal moieties are important in protecting the proximal moieties from premature deubiquitination. Interestingly, when the same experiment was carried out using purified 26S proteasome, mono- and tetra-Ub globin were similarly degraded, highlighting the roles of the entire repertoire of cellular DUBs in regulating the degradation of proteasomal substrates.

CXCL11-CXCR3 Axis Mediates Tumor Lymphatic Cross Talk and Inflammation-Induced Tumor, Promoting Pathways in Head and Neck Cancers.

Tumor metastasis to the draining lymph nodes is critical in patient prognosis and is tightly regulated by molecular interactions mediated by lymphatic endothelial cells (LECs). The underlying mechanisms remain undefined in the head and neck squamous cell carcinomas (HNSCCs). Using HNSCC cells and LECs we determined the mechanisms mediating tumor-lymphatic cross talk. The effects of a pentacyclic triterpenoid, methyl 2-trifluoromethyl-3,11-dioxoolean-1,12-dien-30-oate (CF3DODA-Me), a potent anticancer agent, were studied on cancer-lymphatic interactions. In response to inflammation, LECs induced the chemokine (C-X-C motif) ligand 9/10/11 chemokines with a concomitant increase in the chemokine (C-X-C motif) receptor 3 (CXCR3) in tumor cells. CF3DODA-Me showed antiproliferative effects on tumor cells, altered cellular bioenergetics, suppressed matrix metalloproteinases and chemokine receptors, and the induction of CXCL11-CXCR3 axis and phosphatidylinositol 3-kinase/AKT pathways. Tumor cell migration to LECs was inhibited by blocking CXCL11 whereas recombinant CXCL11 significantly induced tumor migration, epithelial-to-mesenchymal transition, and matrix remodeling. Immunohistochemical analysis of HNSCC tumor arrays showed enhanced expression of CXCR3 and increased lymphatic vessel infiltration. Furthermore, The Cancer Genome Atlas RNA-sequencing data from HNSCC patients also showed a positive correlation between CXCR3 expression and lymphovascular invasion. Collectively, our data suggest a novel mechanism for cross talk between the LECs and HNSCC tumors through the CXCR3-CXCL11 axis and elucidate the role of the triterpenoid CF3DODA-Me in abrogating several of these tumor-promoting pathways.

Prevalence and burden of multiple sclerosis-related fatigue: a systematic literature review.

BACKGROUND: Multiple sclerosis (MS) is a chronic, demyelinating disease of the central nervous system that results in progressive and irreversible disability. Fatigue is one of the most common MS-related symptoms and is characterized by a persistent lack of energy that impairs daily functioning. The burden of MS-related fatigue is complex and multidimensional, and to our knowledge, no systematic literature review has been conducted on this subject. The purpose of this study was to conduct a systematic literature review on the epidemiology and burden of fatigue in people with multiple sclerosis (pwMS). METHODS: Systematic searches were conducted in MEDLINE, Embase, and Evidence-Based Medicine Reviews to identify relevant studies of fatigue in pwMS. English-language records published from 2010 to January 2020 that met predefined eligibility criteria were included. We initially selected studies that reported quality of life (QoL) and economic outcomes according to categories of fatigue (e.g., fatigued vs non-fatigued). Studies assessing associations between economic outcomes and fatigue as a continuous measure were later included to supplement the available data. RESULTS: The search identified 8147 unique records, 54 of which met the inclusion criteria. Of these, 39 reported epidemiological outcomes, 11 reported QoL, and 9 reported economic outcomes. The supplementary screen for economic studies with fatigue as a continuous measure included an additional 20 records. Fatigue prevalence in pwMS ranged from 36.5 to 78.0%. MS-related fatigue was consistently associated with significantly lower QoL. Results on the economic impact of fatigue were heterogeneous, but most studies reported a significant association between presence or severity of fatigue and employment status, capacity to work, and sick leave. There was a gap in evidence regarding the direct costs of MS-related fatigue and the burden experienced by caregivers of pwMS. CONCLUSION: Fatigue is a prevalent symptom in pwMS and is associated with considerable QoL and economic burden. There are gaps in the evidence related to the direct costs of MS-related fatigue and the burden of fatigue on caregivers. Addressing fatigue over the clinical course of the disease may improve health and economic outcomes for patients with MS.

Total Chemical Synthesis of ISGylated-Ubiquitin Hybrid Chain Assisted by Acetamidomethyl Derivatives with Dual Functions.

Interferon-stimulated gene 15 (ISG15) is a member of the ubiquitin-like modifiers (ULM) family, which adopts a ß-grasp fold domain(s) similar to ubiquitin (Ub) with only minor sequence homology. ISG15 consists of two Ub-like domains and aids the immune system in neutralizing infections by numerous pathogens and plays an important role in defending cells against many viruses including influenza A. Recently, Ub was found to be a substrate for ISG15, which can be ISGylated on Lys29 and Lys48, while the former is more dominant. The discovery of such hybrid ISG15-Ub chains brought forward various fundamental questions regarding the nature and effect of this conjugation. To further investigate the role of hybrid ISG15-Ub chains, the pure homogeneous material of these chains is needed in workable quantities. By applying advanced chemical strategies for protein synthesis, we report the total chemical synthesis of a 231-residue ISG15-Lys29-Ub hybrid chain. During the synthesis we encountered insoluble peptide fragments, and therefore we developed a new reversible Acm based solubilizing tag to efficiently tackle this hurdle. This new Acm tag was compared with the known Arg based Acm solubilizing tag and was found to be more reliable in terms of incorporation and efficiency as demonstrated in the synthesis of the native ISG15-Ub hybrid chain.

Affinity Maturation of Macrocyclic Peptide Modulators of Lys48-Linked Diubiquitin by a Twofold Strategy.

Messenger RNA display of peptides containing non-proteinogenic amino acids, referred to as RaPID system, has become one of the leading methods to express libraries consisting of more than trillion-members of macrocyclic peptides, which allows for discovering de novo bioactive ligands. Ideal macrocyclic peptides should have dissociation constants (KD ) as low as single-digit values in the nanomolar range towards a specific target of interest. Here, a twofold strategy to discover optimized macrocyclic peptides within this affinity regime is described. First, benzyl thioether cyclized peptide libraries were explored to identify tight binding hits. To obtain more insights into critical sequence information, sequence alignment was applied to guide rational mutagenesis for the improvement of their binding affinity. Using this twofold strategy, benzyl thioether macrocyclic peptide binders against Lys48-linked ubiquitin dimer (K48-Ub2) were successfully obtained that display KD values in the range 0.3-1.2 nm, which indicate binding two orders of magnitude stronger than those of macrocyclic peptides recently reported. Most importantly, this macrocyclic peptide also showed an improved cellular inhibition of the K48-Ub2 recognition by deubiquitinating enzymes and the 26S proteasome, resulting in the promotion of apoptosis in cancer cells.

Ubiquitin Signaling: Chemistry Comes to the Rescue.

Posttranslational modification of proteins by ubiquitin (Ub), i.e., ubiquitination, mediates a variety of cellular processes, including protein homeostasis, cell cycle, DNA repair, and viral infections. Understanding the molecular mechanism of ubiquitination in these events is the basis for unraveling its precise role in health and disease. However, the inherent complexity of Ub signaling due to the high atomic complexity of Ub conjugates, where Ub is attached to other Ub molecules and to protein substrates in various forms, imposes a major challenge for these studies. In this regard, the enzymatic approaches employed for the preparation of important Ub conjugates have severe limitations to deliver them in high homogeneity and in adequate amounts for the desired study. Recent developments in the area of chemical synthesis and semisynthesis of proteins offer great solutions to the enzymatic limitations and enabling the preparation of various Ub conjugates with precise control over the atomic structure. These conjugates significantly contribute to deciphering Ub signaling at the molecular level, and with the synthetic tools in hand, chemical biologists have become key players in efforts toward understanding the complexity of the Ub code. In this Perspective, we highlight the key contributions of these synthetic approaches and how the development of novel Ub-based reagents is greatly assisting in uncovering unknown aspects of Ub signaling. We also discuss future aspirations to address unresolved questions in this exciting area of research.