High glucose enhances antigen-independent CTL killing via TRAIL Cytotoxic T lymphocytes (CTLs) are involved in development of diabetes. However, the impact of excessive glucose on CTL-mediated antigen-independent killing remains elusive. Here, we report that TNF-related apoptosis inducing ligand (TRAIL) is substantially up- regulated in CTLs in environments with high glucose (HG) both in vitro and in vivo. The PI3K- Akt-NF{kappa}B axis and non-mitochondrial reactive oxygen species are essential in HG-induced TRAIL upregulation in CTLs. TRAILhigh CTLs induce apoptosis of pancreatic beta cell line 1.4E7. Metformin and Vitamin D synergistically reduce HG-enhanced expression of TRAIL in CTLs and coherently protect 1.4E7 cells from TRAIL-mediated apoptosis. Notably, in patients with diabetes, correlation between Vitamin D concentrations in plasma and glucose levels is linked to HG-enhanced TRAIL expression on CTLs. Microarray data reveal that OXCT2, an important enzyme in ketone body catabolism, is a promising target in response to vitamin D. Our work not only reveals a novel mechanism of CTL involvement in progression of diabetes, but also establishes CTLs as a target for combined metformin and vitamin D therapy to protect pancreatic beta cells of diabetic patients.
Coronavirus-associated molecular mimicry through homology to a SARS-CoV-2 peptide could be leading to susceptibility in patients with HLA-A*02:01 and HLA-A*24:02 serotypes This study aims to predict autoimmunity-related pathological mechanisms that possess risk for individuals with specific human leukocyte antigen (HLA) serotypes and shared by certain coronaviruses including SARS-CoV-2, based on homology to a SARS-CoV-2 peptide. With the given aim, 1-) coronavirus-associated sequences, which are homologous to the 15mer SARS-CoV-2 peptide CFLGYFCTCYFGLFC, are obtained. 2-) Human peptides that have at least 7 residue matches with those coronavirus sequences, and the SARS-CoV-2 15mer, are found. 3-) Epitope pairs, which are sourced by those aligned coronavirus and human sequences are identified. 4-) Epitope pairs that are predicted to bind strongly not only to the same HLA allele with each other but also to the same HLA allele as those of the respective alignment of the SARS-CoV-2 peptide are selected. Following are the identified proteins or peptides (with HLA-A*02:01 or HLA-A*24:02 epitopes), as described in 1-to-4: Immunoglobulin heavy chain junction regions, CRB1 isoform I precursor, slit homolog 2 protein, hCG1995581, hCG2028737, phospholipid phosphatase-related protein type 2. Among those, CRB1 isoform I precursor sequence with the predicted HLA-A*24:02 epitope aligns with the highest number of different sequences. Results imply autoimmunity risk in COVID-19 patients with HLA-A*02:01 and HLA-A*24:02 serotypes, through molecular mimicry, as a shared pathogenicity risk that can be prevalent upon getting infected with certain coronaviruses. These can pave way to improved risk groups assessment and autoimmunity treatment options, for COVID-19 and its associated diseases. Also, the approach in this study can be used to predict prospective pathologies of the transmissible variants in susceptible humans.
The Skeletal Muscle Circadian Clock Regulates Titin Splicing Through RBM20 Circadian rhythms are maintained by a cell autonomous, transcriptional-translational feedback loop known as the molecular clock. While previous research suggests a role of the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomere filaments. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMSBmal1-/-) mice, we showed that knocking out skeletal muscle clock function alters titin isoform expression through both RNAseq and LC-MS. This alteration in titins spring length resulted in sarcomere length heterogeneity. We demonstrate the direct link between altered titin splicing and sarcomere length in vitro using U7 snRNPs that truncate the region of titin altered in iMSBmal1-/- muscle. Finally, we identified a mechanism whereby the skeletal muscle clock regulates titin isoform expression through transcriptional regulation of Rbm20, a potent splicing regulator of titin. Our findings demonstrate the importance of the skeletal muscle circadian clock in maintaining titin isoform through regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many chronic diseases, our results highlight a novel pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies. Hematopoietic differentiation is characterized by a transient peak of entropy at a single cell level. Hematopoietic differentiation has been metaphorically represented as linear trajectories with discrete steps from hematopoietic stem cells to mature cells. While the transcriptional state of cells at the beginning or at the end of these trajectories are well described from bulk analysis, what happens in the intermediate states has remained elusive until the use of single cell approaches. Applying Shannon entropy to measure cell-to-cell variability among cells at the same stage of differentiation, we observed a transient peak of gene expression variability in all the hematopoietic differentiation pathways. Strikingly, genes with the highest entropy variation in a given differentiation pathway matched genes known as pathway-specific, whereas genes with the highest expression variation were common to all pathways. Finally, we showed that the level of cell-to-cell variation is increased in the most immature compartment of hematopoiesis in myelodysplastic syndromes. These data suggest that differentiation could be better conceptualized as a dynamical stochastic process with a transient stage of cellular indetermination. The Functional Nanopore Screen: A Versatile High-throughput Assay to Study and Engineer Protein Nanopores in Escherichia coli Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly developed for different biotechnological applications. Yet, a capacity to study and engineer protein nanopores by combinatorial means has so far been hampered by a lack of suitable assays that combine sufficient experimental resolution with throughput. Addressing this technological gap, the Functional Nanopore (FuN) screen now provides a quantitative and dynamic read-out of nanopore assembly and function in E. coli. The assay is based on genetically-encoded fluorescent protein sensors that resolve the nanopore-dependent influx of Ca2+ across the inner membrane of E. coli. Illustrating its versatile capacity, the FuN screen is first applied to dissect the molecular features that underlie the assembly and stability of nanopores formed by the S2168 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S2168 with different ring-shaped oligomeric protein structures that feature defined hexa-, hepta- and octameric geometries. Library screening highlights substantial plasticity in the ability of the S2168 transmembrane module to oligomerize in alternative geometries while the functional properties of the resultant nanopores can be fine-tuned through the identity of the connecting linkers. Overall, the FuN screen is anticipated to facilitate both fundamental studies and complex nanopore engineering endeavors with many potential applications in biomedicine, biotechnology and synthetic biology. Venomics and antivenomics of Indian spectacled cobra (Naja naja) from the Western Ghats Venom proteome profiling of Naja naja from the Western Ghats region in Kerala was achieved through SDS-PAGE and RP-HPLC followed by Q-TOF LC-MS/MS analysis, incorporating PEAKS and Novor assisted de novo sequencing methodologies. A total of 115 proteins distributed across 17 different enzymatic and non-enzymatic venom protein families were identified through conventional and 39 peptides through homology-driven proteomics approaches. Fourteen peptides derived through de novo complements the Mascot data indicating the importance of homology-driven approaches in improving protein sequence information. Among the protein families identified, glutathione peroxidase and endonuclease were reported for the first time in the Indian cobra venom. Immunological cross-reactivity assessed using Indian polyvalent antivenoms suggested that VINS showed better EC50 (2.48 g/mL) value than that of PSAV (6.04 g/mL) and Virchow (6.03 g/mL) antivenoms. Western blotting experiments indicated that all the antivenoms elicited poor binding specificities, especially towards low molecular mass proteins. Second-generation antivenomics studies revealed that VINS antivenom was less efficient to detect many low molecular mass proteins such as three-finger toxins and Kunitz-type serine protease Inhibitors. Taken together, the present study enabled a large-scale characterization of the venom proteome of Naja naja from the Western Ghats and emphasized the need for developing more efficient antivenoms.

HighlightsO_LIProteomics of cobra venom resulted in the identification of 115 proteins representing 17 snake venom protein families.
C_LIO_LIDe novo approaches exclusively yielded 39 peptides harbouring multiple amino acid mutations.
C_LIO_LIGlutathione peroxidase and endonuclease were identified for the first time in Indian cobra venom.
C_LIO_LIIndian polyvalent antivenoms showed varying cross-reactivity towards cobra venom.
C_LIO_LIVINS antivenom was less efficient to detect many low molecular mass proteins (< 20 kDa).
C_LI SMC protein RecN drives RecA filament translocation and remodelling for in vivo homology search While the molecular repertoire of the homologous recombination pathway is well-studied, the search mechanism that enables recombination between distant homologous regions is poorly understood. Earlier work suggests that the recombinase RecA, an essential component for homology search, forms an elongated filament, nucleating at the break site. How this RecA structure carries out long distance search remains unclear. Here, we follow the dynamics of RecA after induction of a single double-strand break on the Caulobacter chromosome. We find that the RecA-nucleoprotein filament, once formed, rapidly translocates in a directional manner in the cell, undergoing several pole-to-pole traversals, until homology search is complete. Concomitant with translocation, we observe dynamic remodelling of the filament. Importantly in vivo, the RecA filament alone is incapable of such long distance movement; both translocation and dynamic remodelling are contingent on action of SMC-like protein RecN, via its ATPase cycle. We provide a stochastic description of RecN-regulated changes in filament length during translocation via modulation of RecA assembly-disassembly. In summary, we have uncovered the three key elements of homology search driven by RecN: mobility of a finite segment of RecA, filament remodelling and ability to conduct multiple pole-to-pole traversals, which together point to a novel optimal search strategy. Systematic temporal analysis of transcriptomes using TrendCatcher identifies early and persistent neutrophil activation as an early hallmark of severe COVID-19 Studying temporal gene expression shifts during disease progression provides important insights into the biological mechanisms that distinguish adaptive and maladaptive responses. Existing tools for the analysis of time course transcriptomic data are not designed to optimally identify distinct temporal patterns when analyzing dynamic differentially expressed genes (DDEGs). Moreover, there is a lack of methods to assess and visualize the temporal progression of biological pathways mapped from time course transcriptomic datasets. In this study, we developed an open-source R package TrendCatcher (https://github.com/jaleesr/TrendCatcher), which applies the smoothing spline ANOVA model and break point searching strategy to identify and visualize distinct dynamic transcriptional gene signatures and biological processes from longitudinal datasets. We used TrendCatcher to perform a systematic temporal analysis of COVID-19 peripheral blood transcriptomes, including bulk RNA-seq and scRNA-seq time course data. TrendCatcher uncovered the early and persistent activation of neutrophils and coagulation pathways as well as impaired type I interferon (IFN-I) signaling in circulating cells as a hallmark of patients who progressed to severe COVID-19, whereas no such patterns were identified in individuals receiving SARS- CoV-2 vaccinations or patients with mild COVID-19. These results underscore the importance of systematic temporal analysis to identify early biomarkers and possible pathogenic therapeutic targets. Frameshift and wild-type proteins are highly similar because the genetic code and genomes were optimized for frameshift tolerance Frameshift protein sequences encoded by alternative reading frames of coding genes have been considered meaningless, and frameshift mutations have been considered of little importance for the molecular evolution of coding genes and proteins. However, functional frameshifts have been found widely existing. It was puzzling how a frameshift protein kept its structure and functionality while its amino-acid sequence was changed substantially. Here we show that frame similarities between frameshifts and wild types are higher than random similarities and are defined at the genetic code, gene, and genome levels. In the standard genetic code, frameshift codon substitutions are more conservative than random substitutions. The frameshift tolerability of the standard genetic code ranks in the top 2.0-3.5% of alternative genetic codes, showing that the genetic code is nearly optimal for frameshift tolerance. Furthermore, frameshift-resistant codons (codon pairs) appear more frequently than expected in many genes and certain genomes, showing that the frameshift optimality is reflected not only in the genetic code but more importantly, in its allowance of further optimizing the frameshift tolerance of a particular gene or genome, which shed light on the role of frameshift mutations in molecular and genomic evolution. Selection towards lighter seeds in Danish grasslands over an eight-year period Time-series analysis of cover data from 236 Danish grassland sites demonstrated community selection on seed mass. Across four grassland habitat types and during an eight-year period there was significant community selection towards lighter seeds. In the analysis, the continuous seed size variable was used to group plant species into functional types. This method was chosen in order to account for the sampling process of the abundance data, and is in contrast to most other analyses of trait selection, where the community weighted mean of the traits is used as the dependent variable. Generally, little is known on the underlying causes of possible plant trait community selection in semi-natural grasslands that are not subjected to successional processes. Sablefish (Anoplopoma fimbria Pallas, 1814) plasma biochemistry and hematology reference intervals including blood cell morphology Plasma biochemistry and hematology reference intervals are integral health assessment tools in all medical fields, including aquatic animal health. As sablefish (Anoplopoma fimbria) are becoming aquaculturally and economically more important, this manuscript provides essential reference intervals (RI) for their plasma biochemistry and hematology along with reference photomicrographs of blood cells in healthy, fasted sablefish. Blood cell morphology can differ between fish species. In addition, blood cell counts and blood chemistry can vary between fish species, demographics, water conditions, seasons, diets, and culture systems, which precludes the use of RIs from other fish species. For this study, blood was collected for plasma biochemistry and hematology analysis between June 20 and July 18, 2019, from healthy, yearling sablefish, hatched and reared in captivity on a commercial diet. Overnight fast of 16-18 hours did not sufficiently reduce lipids in the blood, which led to visible lipemia and frequent rupture of blood cells during analysis. Therefore, sablefish should be fasted for 24 to 36 hours before blood is collected to reduce hematology artifacts or possible reagent interference in plasma biochemistry analysis. Lymphocytes were the most dominant leukocytes (98%), while eosinophils were rare, and basophils were not detected in sablefish. Neutrophils were very large cells with Dohle bodies. In mammals and avian species, Dohle bodies are usually signs of toxic change from inflammation, but no such association was found in these fish. In conclusion, lipemia can interfere with sablefish blood analysis, and available removal methods should be evaluated as fasting for up to 36 h might not always be feasible. Also, more studies are required to establish RI for different developmental stages and rearing conditions. Glucose inhibits haemostasis and accelerates diet-induced hyperlipidaemia in zebrafish larvae Hyperglycaemia damages the microvasculature in part through the reduced recruitment of immune cells and interference with platelet signalling, leading to poor wound healing and accelerated lipid deposition in mammals. We investigated the utility of zebrafish larvae to model the effect of glucose on neutrophil and macrophage recruitment to a tail wound, wound-induced haemostasis, and chicken egg yolk feed challenge-induced hyperlipidaemia by supplementing larvae with exogenous glucose by immersion or injection. Neither method of glucose supplementation affected the recruitment of neutrophils and macrophages following tail transection. Glucose injection reduced thrombocyte retention and fibrin plug formation while only thrombocyte retention was reduced by glucose immersion following tail transection. We observed accelerated lipid accumulation in glucose-injected larvae challenged with high fat chicken egg yolk feeding. Our study identifies conserved and divergent effects of high glucose on inflammation, haemostasis, and hyperlipidaemia in zebrafish larvae compared to mammals. Rational Exploration of Fold Atlas for Human Solute Carrier Proteins Solute carrier (SLC) superfamily is the largest group responsible for transmembrane transport of substances in human cells. It includes more than 400 members which are organized into 65 families according to their physiological function and sequence similarity. Different families of solute transporters can adopt the same or different folds that determine the working mechanism and reflect the evolutionary relationship between SLC members. Analysis of structural data in literatures shows that there are 13 different folds in the solute carrier superfamily covering 40 families and 343 members. To further study their working mechanism, we systematically explored the SLC superfamily to look for more folds. Based on our results, at least three new folds were found for SLC superfamily, and one was experimentally verified in the SLC44 family. Our work has laid a foundation and provided important insights for the systematic and comprehensive study on the structure and function of solute carriers. Affordable Caenorhabditis elegans tracking system for classroom use For decades the nematode C. elegans has served as an outstanding research organism owing to its unsurpassed experimental amenability. This advantage has also made this tiny worm an attractive vehicle for science instruction across higher learning institutions. However, the prohibitive cost associated with the automated behavioral assessment of these animals remains an obstacle preventing their full adoption in undergraduate and high school settings. To improve this situation, we developed an inexpensive worm tracking system for use by high school interns and undergraduate students. Over the past two years this tracker has been successfully used by undergraduate students in our introductory Cell and Molecular lab (BSC220) at Illinois State University. Here we describe and demonstrate the use of our inexpensive worm tracking system. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Host phenology can drive the evolution of intermediate virulence strategies in some parasites AO_SCPLOWBSTRACTC_SCPLOWThe traditional mechanistic trade-offs resulting in a negative correlation between transmission and virulence are the foundation of nearly all current theory on the evolution of parasite virulence. Several ecological factors have been shown to modulate the optimal virulence strategies predicted from mechanistic trade-off models, but these ecological factors have not yet been shown to be sufficient to explain the intermediate virulence strategies observed in any natural system. The timing of seasonal activity, or phenology, is a common factor that influences the types and impact of many ecological interactions but is difficult to incorporate into virulence evolution studies. We develop a mathematical model of a disease system with seasonal host activity to study the evolutionary consequences of host phenology on the virulence of obligate-killer parasite. Results from this model demonstrated that seasonal host activity is sufficient to drive the evolution of intermediate parasite virulence in some types of natural disease systems, even when a traditional mechanistic trade-off between transmission and virulence is not assumed in the modeling framework. The optimal virulence strategy in these systems can be determined by both the duration of the host activity period as well as the variation in the host emergence timing. Parasites with low virulence strategies are favored in environments with long host activity periods and in environments in which hosts emerge synchronously. The results demonstrate that host phenology can be sufficient to select for intermediate optimal virulence strategies, providing an alternative mechanism to account for virulence evolution in some natural systems. Neprosin belongs to a new family of glutamic peptidase family based on in silico evidence Neprosin was first discovered in the insectivorous tropical pitcher plants of Nepenthes species as a novel protease with prolyl endopeptidase (PEP) activity. Neprosin has two uncharacterized domains of neprosin activation peptide and neprosin. A previous study has shown neprosin activity in hydrolyzing proline-rich gliadin, a gluten component that triggers celiac disease. In this study, we performed in silico structure-function analysis to investigate the catalytic mechanism of neprosin. Neprosin sequences lack the catalytic triad and motifs of PEP family S9. Protein structures of neprosins from Nepenthes x ventrata (NvNpr) and N. rafflesiana (NrNpr1) were generated by ab initio methods and comparatively assessed to obtain high-quality models. Structural alignment of models to experimental structures in the Protein Data Bank (PDB) found a high structural similarity to glutamic peptidases. Further investigations reveal other resemblances to the glutamic peptidases with low optimum pH that activates the enzyme via autoproteolysis for maturation. Two highly conserved glutamic acid residues, which are stable according to the molecular dynamics simulation, can be found at the active site of the substrate cleft. Protein docking demonstrated that mature neprosins bind well with potent antigen I-gliadin at the putative active site. Taken together, neprosins represent a new glutamic peptidase family, with a putative catalytic dyad of two glutamic acids. This study illustrates a hypothetical enzymatic mechanism of the neprosin family and demonstrates the useful application of an accurate ab initio protein structure prediction in the structure-function study of a novel protein family. Environment dependent benefits of inter-individual variation in honey bee recruitment Inter-individual differences in behaviour within the members of a social group can affect the groups productivity. In eusocial insects, individual differences amongst workers in a colony play a central role division of labour and task allocation. Extensive empirical and theoretical work has highlighted variation in response thresholds as a proximate mechanism underlying individual behavioural differences and hence division of labour. However, other response parameters, like response probability and intensity, can affect these differences. In this study, we first extended a previously published agent-based model on honey bee foraging to understand the relative importance of response (dance) probability and response (dance) intensity in the task of recruitment. Comparing variation obtained from the simulations with previously published empirical data, we found that response intensity plays a more important role than probability in producing consistent inter-individual differences in recruitment behaviour. We then explored the benefits provided by this individual variation in recruitment behaviour to the colonys collective foraging effort under different environmental conditions. Our results revealed that individual variation leads to a greater energetic yield per forager, but only when food is abundant. Our study highlights the need to consider all response parameters while studying division of labour and adds to the growing body of evidence linking individual variation in behavioural responses to the success of social groups. Cell trajectory modeling identifies a primitive trophoblast state defined by BCAM enrichment In early placental development, progenitor cytotrophoblasts (CTBs) differentiate along one of two cellular trajectories: the villous or extravillous pathways. CTBs committed to the villous pathway fuse with neighboring CTBs to form the outer multinucleated syncytiotrophoblast (SCT), while CTBs committed to the extravillous pathway differentiate into invasive extravillous trophoblasts (EVT). Unfortunately, little is known about the processes controlling human CTB progenitor maintenance and differentiation. To address this, we established a single cell RNA sequencing (scRNA-seq) dataset from first trimester placentas to identify cell states important in trophoblast progenitor establishment, renewal, and differentiation. Multiple distinct trophoblast states were identified, representing progenitor CTBs, column CTBs, SCT precursors, and EVT. Lineage trajectory analysis identified a progenitor origin that was reproduced in human trophoblast stem cell organoids. Heightened expression of basal cell adhesion molecule (BCAM) defined this primitive state, where BCAM enrichment or gene silencing resulted in enhanced or diminished organoid growth. Together, this work describes at high-resolution trophoblast heterogeneity within the first trimester, resolves gene networks within human CTB progenitors, and identifies BCAM as a primitive progenitor marker and possible regulator.

Summary StatementLineage trajectory modeling identifies multiple human progenitor trophoblast states and defines trophoblast differentiation kinetics, where BCAM-expressing progenitors demonstrate enhanced regenerative ability. Spontaneous Eye Blink Rate during the Working Memory Delay Period Predicts Task Accuracy Spontaneous eye blink rate (sEBR) has been linked to attention and memory, specifically working memory (WM). sEBR is also related to striatal dopamine (DA) activity with schizophrenia and Parkinsons disease showing increases and decreases respectively in sEBR. A weakness of past studies of sEBR and WM is that correlations have been reported using blink rates taken at baseline either before or after performance of the tasks used to assess WM. The goal of the present study was to understand how fluctuations in sEBR during different phases of a visual WM task predict task accuracy. In two experiments, with recordings of sEBR collected inside and outside of a magnetic resonance imaging bore, we observed sEBR to be positively correlated with WM task accuracy during the WM delay period. We also found task-related modulation of sEBR, including higher sEBR during the delay period compared to rest, and lower sEBR during task phases (e.g., stimulus encoding) that place demands on visual attention. These results provide further evidence that sEBR could be an important predictor of WM task performance with the changes during the delay period suggesting a role in WM maintenance. The relationship of sEBR to DA activity and WM maintenance is discussed. Mayaro Virus Infection Elicits an Innate Immune Response in Anopheles stephensi Mayaro virus (MAYV) is an arboviral pathogen in the genus Alphavirus that is circulating in South America with potential to spread to naive regions. MAYV is also one of the few viruses with the ability to be transmitted by mosquitoes in the genus Anopheles, as well as the typical arboviral transmitting mosquitoes in the genus Aedes. Few studies have investigated the infection response of Anopheles mosquitoes. In this study we detail the transcriptomic and small RNA responses of An. stephensi to infection with MAYV via infectious bloodmeal at 2, 7, and 14 days post infection (dpi). 487 unique transcripts were significantly regulated, 78 putative novel miRNAs were identified, and an siRNA response is observed targeting the MAYV genome. Gene ontology analysis of transcripts regulated at each timepoint suggested activation of the Toll pathway at 7 dpi and repression of pathways related to autophagy and apoptosis at 14 dpi. These findings provide a basic understanding of the infection response of An. stephensi to MAYV and help to identify host factors which might be useful to target to inhibit viral replication in Anopheles mosquitoes.

AUTHOR SUMMARYMayaro virus (MAYV) is a mosquito-borne Alphavirus responsible for outbreaks in South America and the Caribbean. In this study we infected Anopheles stephensi with MAYV and sequenced mRNA and small RNA to understand how MAYV infection impacts gene transcription and the expression of small RNAs in the mosquito vector. Genes involved with innate immunity and signaling pathways related to cell death are regulated in response to MAYV infection of An. stephensi, we also discover novel miRNAs and describe the expression patterns of miRNAs, siRNAs, and piRNAs following bloodmeal ingestion. These results suggest that MAYV does induce a molecular response to infection in its mosquito vector species. Suppression of sost/sclerostin and dickkopf-1 promote intervertebral disc structure in mice Intervertebral disc (IVD) degeneration is a leading cause of low back pain and characterized by accelerated extracellular matrix breakdown and IVD height loss but there is no approved pharmacological therapeutic. Deletion of Wnt signaling receptor Lrp5 induces IVD degeneration and suggests that Wnt signaling in the IVD may be responsive to inhibition of Wnt signaling inhibitors sost(gene)/sclerostin(protein) or dickkopf-1 (dkk1). Anti-sclerostin antibody (Scl-Ab) is an FDA-approved bone therapeutic that activates Wnt signaling. We (1) determined if pharmacological neutralization of sclerostin, dkk1 or their combination stimulate Wnt signaling and promote IVD structure and (2) determined the extent of the response of the IVD to global, persistent deletion of sost. Nine-week-old C57Bl/6J female mice (n=6-7/grp) were subcutaneously injected 2x/wk for 5.5 wk with scl-Ab (25 mg/kg), dkk1-Ab (25 mg/kg), 3:1 scl-Ab/dkk1-Ab (18.75:6.25 mg/kg) or vehicle (Veh). Separately, IVD of sost KO and WT (wildtype) mice (n=8, grp) were harvested at 16 weeks of age. First, compared to vehicle, scl-Ab, dkk1-Ab and 3:1 scl-Ab/dkk1-Ab similarly increased lumbar IVD height and {beta}-catenin gene expression. Despite these similarities, scl-Ab decreased cellular stress-related heat shock protein gene expressions while neither dkk1-Ab nor scl-Ab/dkk1-Ab altered the same. Genetically and compared to WT, sost KO increased MRI-determined hydration and proteoglycan staining in the IVD. Notably, persistent deletion of sost was compensated by upregulation of dkk1, which consequently reduced the cell nuclear fraction for Wnt signaling transcription factor {beta}-catenin in whole IVD. Lastly, RNA-sequencing pathway analysis confirmed the compensatory suppression of Wnt signaling and determined a reduction of cellular stress pathways. Together, suppression of sost/sclerostin or dkk1 each promote IVD structure by stimulating Wnt signaling, but sclerostin and dkk1 may differentially regulate cellular stress pathways. Ultimately, postmenopausal women prescribed scl-Ab injections to prevent vertebral fracture may also benefit from a restoration of IVD height and health.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/449486v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@db2e17org.highwire.dtl.DTLVardef@1af1bbborg.highwire.dtl.DTLVardef@145cc4corg.highwire.dtl.DTLVardef@167a19e_HPS_FORMAT_FIGEXP  M_FIG C_FIG Suppression of Wnt signaling inhibitors by genetic or pharmacological approaches promotes intervertebral disc structure and hydration by Wnt signaling. However, persistent activation of Wnt signaling induces a compensatory reduction of Wnt signaling that shifts IVD cells toward a chondrocyte-like (CLC) phenotype. AF: annulus fibrosus, NC: notochordal cell, NP: nucleus pulposus, PG: proteoglycan TNFa-induced LDL cholesterol accumulation involve elevated LDLR cell surface levels and SR-B1 downregulation in human arterial endothelial cellsan arterial endothelial cells Excess lipid droplets are frequently observed in arterial endothelial cells at sites of advanced atherosclerotic plaques. Here, the role of tumor necrosis factor alpha (TNF) in modulating low density lipoprotein (LDL) content in confluent primary human aortic endothelial cells (pHAECs) was investigated. TNF promoted up to 2 folds increase in cellular cholesterol, which was resistant to ACAT inhibition. The cholesterol increase was associated with increased 125I-LDL surface binding. Using the non-hydrolysable label, Dil, TNF could induce a massive increase in Dil-LDL by over 200 folds. The elevated intracellular Dil-LDL was blocked with excess unlabeled LDL and PCSK9, but not oxidized LDL (oxLDL), receptor associated protein (RAP), or apolipoprotein (apoE) depletion. Moreover, TNF-induced increase of LDL-derived lipids was elevated through lysosome inhibition. Using specific LDLR antibody, the Dil-LDL accumulation was reduced by over 99%. Effects of TNF included LDLR cell surface increase by 138%, and very large increases in ICAM-1 total and surface proteins, respectively. In contrast, that of scavenger receptor B1 (SR-B1) was reduced. Additionally, LDLR antibody bound rapidly in TNF-treated cells by about 30 folds, inducing a migrating shift in the LDLR protein. The effect of TNF on Dil-LDL accumulation was inhibited by the antioxidant tetramethythiourea (TMTU) dose-dependently, but not by inhibitors against NF-{kappa}B, stress kinases, ASK1, JNK, p38, or apoptosis caspases. Grown on transwell inserts, TNF did not enhance apical to basolateral LDL cholesterol or Dil release. It is concluded that TNF promotes LDLR functions through combined increase at the cell surface and SR-B1 downregulation. Designing viral diagnostics with model-based optimization Diagnostics, particularly for rapidly evolving viruses, stand to benefit from a principled, measurement-driven design that harnesses machine learning and vast genomic data--yet the capability for such design has not been previously built. Here, we develop and extensively validate an approach to designing viral diagnostics that applies a learned model within a combinatorial optimization framework. Concentrating on CRISPR-based diagnostics, we screen a library of 19,209 diagnostic-target pairs and train a deep neural network that predicts, from RNA sequence alone, diagnostic signal better than contemporary techniques. Our model then makes it possible to design assays that are maximally sensitive over the spectrum of a viruss genomic variation. We introduce ADAPT (https://adapt.guide), a system for fully-automated design, and use ADAPT to design optimal diagnostics for the 1,933 vertebrate-infecting viral species within 2 hours for most species and 24 hours for all but 3. We experimentally show ADAPTs designs are sensitive and specific down to the lineage level, including against viruses that pose challenges involving genomic variation and specificity. ADAPTs designs exhibit significantly higher fluorescence and permit lower limits of detection, across a viruss entire variation, than the outputs of standard design techniques. Our model-based optimization strategy has applications broadly to viral nucleic acid diagnostics and other sequence-based technologies, and, paired with clinical validation, could enable a critically-needed, proactive resource of assays for surveilling and responding to pathogens. Targeted biallelic integration of an inducible Caspase 9 suicide gene for safer cellular therapies prevents development of drug-resistant escapees in human iPSCs Drug-inducible suicide systems may help to minimize risks of cellular therapies due to the tumor forming potential of human induced pluripotent stem cells (hiPSCs). Recent research challenged the usefulness of such systems since rare drug-resistant subclones were observed that showed elimination or silencing of the transgene.

We have introduced a drug-inducible Caspase9 suicide system (iCASP9) into the AAVS1 safe harbor locus of hiPSCs. In these cells, apoptosis could be efficiently induced in vitro. In mice, drug treatment generally led to rapid elimination of teratomas, but individual animals subsequently formed tumor tissue from monoallelic iCASP9 hiPSCs. Very rare drug-resistant subclones of monoallelic iCASP9 hiPSCs appeared in vitro with frequencies of ~ 3x10-8. Transgene elimination, presumably via Loss of Heterozygosity (LoH), or methylation of the CAG promoter but not methylation of the O_SCPLOWPPPC_SCPLOWO_SCPCAP1C_SCPCAPO_SCPLOWRC_SCPLOWO_SCPCAP12C_SCPCAPO_SCPLOWCC_SCPLOW locus were identified as underlying mechanisms. In contrast, we never observed any escapees from biallelic iCASP9 hiPSCs, even after treatment of up to 0.8 billion hiPSCs.

In conclusion, biallelic integration of an iCASP9 system in the AAVS1 locus may substantially contribute to the safety level of iPSC-based therapies, which should be calculated by relating clonal escapee frequencies to the cell number in tumors of a size that is readily detectable during routine screening procedures. Developing High-Affinity Decoy Receptor Optimized for Multiple Myeloma and Diffuse Large B Cell Lymphoma Treatment Recent T Cell therapies have been effective in the treatment of hematological cancers. However, immunotoxicity and treatment relapse pose significant clinical challenges. Here, we revealed distinctive requirement for neutralizing TNF receptor ligands APRIL and BAFF in MM and DLBCL. Furthermore, we investigated the use of BCMA decoy receptor (sBCMA-Fc) as a therapeutic inhibitor of ARPIL and BAFF. While wild-type sBCMA-Fc successfully blocked APRIL signaling with picomolar binding affinity, inhibiting tumor growth in MM models, it lacked efficacy in inhibiting DLBCL progression due to its weak binding for BAFF. To expand the therapeutic utility of sBCMA-Fc, using a protein engineering approach, we generated an affinity enhanced mutant sBCMA-Fc fusion molecule (sBCMA-Fc V3) with 4-folds and 500-folds enhancement in binding to APRIL and BAFF respectively. The sBCMA-Fc V3 clone significantly enhanced antitumor activity against both MM and DLBCL. Importantly, sBCMA-Fc V3 was proven to be a viable clinical candidate by showing adequate toxicity profile and on-target mechanism of action in nonhuman primates.

SUMMARYThis study demonstrates the dichotomous function of APRIL and BAFF in MM and DLBCL, that can be safely targeted by an engineered fusion protein designed to trap APRIL and BAFF with ultra-high binding affinity.