Identification of the three zinc-binding sites on Tau protein Tau protein has been extensively studied due to its key roles in microtubular cytoskeleton regulation and in the formation of aggregates found in some neurodegenerative diseases. Recently it has been shown that zinc is able to induce tau aggregation by interacting with several binding sites. However, the precise location of these sites and the molecular mechanism of zinc-induced aggregation remain unknown. Here we used Nuclear Magnetic Resonance (NMR) to identify zinc binding sites on hTau40 isoform. These experiments revealed three distinct zinc binding sites on tau, located in the N-terminal part (H14, H32, H94, and H121), the repeat region (H299, C322, H329 and H330) and the C-terminal part (H362, H374, H388 and H407). Further analysis enabled us to show that the C-terminal and the N-terminal sites are independent of each other. Using molecular simulations, we modeled the structure of each site in a complex with zinc. Given the clinical importance of zinc in tau aggregation, our findings pave the way for designing potential therapies for tauopathies.

HighlightsO_LIZinc is known to induce tau aggregation in neurodegenerative diseases
C_LIO_LIZinc binding locations and mechanism are not yet clear
C_LIO_LIUsing NMR we localized 3 zinc binding site on tau
C_LIO_LIBy molecular simulations, we proposed a modeled structure of each site
C_LIO_LIOur findings pave the way for designing potential therapies for tauopathies
C_LI
Maf/ham1-like pyrophosphatases of non-canonical nucleotides are host-specific partners of viral RNA-dependent RNA polymerases Cassava brown streak disease (CBSD), dubbed the "Ebola of plants", is a serious threat to food security in Africa caused by two viruses of the family Potyviridae: cassava brown streak virus (CBSV) and Ugandan (U)CBSV. Intriguingly, U/CBSV, along with another member of this family and one secoviridae, are the only known RNA viruses encoding a protein of the Maf/ham1-like family, a group of widespread pyrophosphatase of non-canonical nucleotides (ITPase) expressed by all living organisms. Despite the socio-economic impact of CDSD, the relevance and role of this atypical viral factor has not been yet established. Here, using an infectious cDNA clone and reverse genetics, we demonstrate that UCBSV requires the ITPase activity for infectivity in cassava, but not in the model plant Nicotiana benthamiana. HPLC MS/MS experiments showed that, quite likely, this host-specific constraint is due to an unexpected high concentration of non-canonical nucleotides in cassava. Finally, protein analyses and experimental evolution of mutant viruses indicated that keeping a fraction of the yielded UCBSV ITPase covalently bound to the viral RNA-dependent RNA polymerase (RdRP) optimizes viral fitness, and this seems to be a feature shared by the other members of the Potyviridae family expressing Maf/ham1-like proteins. All in all, our work (i) reveals that the over-accumulation of non-canonical nucleotides in the host might have a key role in antiviral defense, and (ii) provides the first example of an RdRP-ITPase partnership, reinforcing the idea that RNA viruses are incredibly versatile at adaptation to different host setups.
A noble TGF beta biogenesis inhibitor exhibits both potent anti-fibrotic and anti-inflammatory capabilities AbstractsIdiopathy pulmonary fibrosis (IPF) is an intractable and fatal human disorder. Our previous study showed that eupatilin exerted a potent anti-fibrotic effect on both in vitro fibrogenesis and bleomycin-induced lung fibrosis model (BLM). Subsequently, an analog called ONG41008 had been identified as a more potent anti-fibrotic than eupatilin and also showed a potent anti-inflammatory capability. Orally administered ONG41008 significantly improved onset of BLM in both prophylactic and therapeutic model and its therapeutic efficacy was similarly compared to or better than pirfenidone by measuring production of collagen and hydroxyproline. Staining collagen or SMA corroborated these results.

As in vitro fibrogenesis models, DHLF (Diseased Human Lung Fibroblasts from IPF patients) and HSC (hepatic stellate cells) were used for direct effects of ONG41008 on pivotal cellular and molecular functions associated with pathogenic myofibroblasts; ONG41008 dismantled latent TGFb complex (LTC), generating inactive forms of TGF{beta}, likely limiting TGF{beta} to TGF{beta} receptor via depolymerization of F-actin and this blunted SMAD2/SMAD3 phosphorylation, thereby reprogramming EMT. A set of cell imaging studies and transcriptomic analysis were conducted to explore how ONG41008 elicited both anti-fibrotic and anti-inflammatory capabilities. Elastin (ELN) seemed to be a pioneering pharmacodynamic marker. It was also found that NOX4 played an important role in anti- fibrosis because it was functionally connected to major central nod proteins such as lysyl- oxidase (LOX) and numerous collagen family members in an ONG41008-specific fibrogenic interactome. Human NOX4 was significantly induced by TGF{beta} and completely knocked down by ONG41008. It has been shown that production of reactive oxygen species (ROS) led to activation of inflammasome. ONG41008 may be likely related to anti-inflammation, leading to a key protective effect on fibrogenesis. Concomitant with downregulation of NOX4, expression of macrophages homing chemokines, CCL2 and CCL7 were significantly attenuated by ONG41008. In vitro anti-inflammatory activities of ONG41008 were investigated in RAW264.7 cells, a mouse monocytic cell line stimulated with LPS. ONG41008 substantially attenuated TNF, CXCL10, CCL2 and CCL7, which are proinflammatory cytokine and important chemokines influencing T cells or macrophages. TNF was situated at the central nod in LPS-treated macrophages via an ONG41008-specific interactome analysis.

Taken together, ONG41008 is a TGF{beta} biogenesis inhibitor, being a potent drug for a broad range of fibrotic diseases and could antagonize inflammatory diseases as well. Evolution of thermal physiology alters the projected range of threespine stickleback under climate change Species distribution models (SDMs) are widely used to predict range shifts but could be unreliable under climate change scenarios because they do not account for evolution. The thermal physiology of a species is a key determinant of range and thus incorporating thermal trait evolution into SDMs might be expected to alter projected ranges. We identified a genetic basis for physiological and behavioural traits that evolve in response to temperature change in natural populations of threespine stickleback (Gasterosteus aculeatus). Using these data, we created geographic range projections using a mechanistic niche area approach under two climate change scenarios. Under both scenarios, trait data was either static ( no evolution models), allowed to evolve at observed evolutionary rates ( evolution models), or allowed to evolve at a rate of evolution scaled by the trait variance that is explained by quantitative trait loci (QTL;  scaled evolution models). We show that incorporating these traits and their evolution substantially altered the projected ranges for a widespread panmictic marine population, with over 7-fold increases in area under climate change projections when traits are allowed to evolve. Evolution-informed SDMs should improve the precision of forecasting range dynamics under climate change, and aid in their application to management and the protection of biodiversity. Balancing sensitivity and specificity in preclinical research The success of scientific discovery in preclinical drug development is based on the different roles of exploration and confirmation. Exploration requires sensitivity while confirmation needs to be specific. Based on published empirical data, we simulated two preclinical research trajectories to examine their effectiveness to detect true effects while keeping animal numbers low. We compare a conventional p-value based approach with a method based on an a priori determined smallest effect size of interest (SESOI). Using a SESOI increases transition rates from exploration to confirmation and results in higher detection rates across the trajectory. Specificity in the SESOI trajectory increases if the prior probability of true effects is low. We conclude that employing a SESOI is superior to a p-value based approach. Based on our findings, we propose a reconsideration of planning and conducting preclinical experiments, especially when the prior probability of true hypotheses is low. RNAi screen of RING/U-box domain ubiquitin ligases identifies critical regulators of tissue regeneration in planarians Regenerative processes depend on the interpretation of signals to coordinate cell behaviors. The role of ubiquitin-mediated signaling is known to be important in many cellular and biological contexts, but its role in regeneration is not well understood. To investigate how ubiquitylation impacts tissue regeneration in vivo, we are studying planarians that are capable of regenerating after nearly any injury using a population of stem cells. Here we used RNAi to screen RING/U-box E3 ubiquitin ligases that are highly expressed in planarian stem cells and stem cell progeny. RNAi screening identified nine genes with functions in regeneration, including the spliceosomal factor prpf19 and histone modifier rnf2; based on their known roles in developmental processes, we further investigated these two genes. We found that prpf19 was required for animal survival but not for stem cell maintenance, suggesting a role in promoting cell differentiation. Because RNF2 is the catalytic subunit of the Polycomb Repressive Complex 1 (PRC1), we also examined other putative members of this complex (CBX and PHC). We observed a striking phenotype of regional tissue misspecification in cbx and phc RNAi planarians. To identify genes regulated by PRC1, we performed RNA-seq after knocking down rnf2 or phc. Although these proteins are predicted to function in the same complex, we found that the set of genes differentially expressed in rnf2 versus phc RNAi were largely non-overlapping. Using in situ hybridization, we showed that rnf2 regulates gene expression levels within a tissue type, whereas phc is necessary for the spatial restriction of gene expression, findings consistent with their respective in vivo phenotypes. This work not only uncovered roles for RING/U-box E3 ligases in stem cell regulation and regeneration, but also identified differential gene targets for two putative PRC1 factors required for maintaining cell-type-specific gene expression in planarians. An NLR Integrated Domain toolkit to identify plant pathogen effector targets Plant resistance genes (or NLR "Nod-like Receptors") are known to contain atypical domains procuring them with a decoy capacity. Some of these integrated domains (or ID) allow the plant to lure the virulence determinants ("effectors") of pathogens and triggering a specific NLR immune reaction.

In this work, our goal was to generate a library of known IDs that could be screened with plant pathogen effectors in order to identify putative new effector virulence targets and NLR-effector pairs.

We curated the IDs contained in NLRs from seven model and crop plant species. We cloned 52 IDs representing 31 distinct Pfam domains. This library was screened for interaction by yeast-two-hybrid with a set of 31 conserved Ralstonia solanacearum type III effectors. This screening and the further in planta interaction assay allowed us to identify three interactions, involving different IDs (kinase, DUF3542, WRKY) and two type III effectors (RipAE and PopP2).

PopP2 was found to physically interact with ID#85, an atypical WRKY domain integrated in the GmNLR-ID85 NLR protein from Soybean. Using a imaging method in living plant cells, we showed that PopP2 associates with ID#85 in the nucleus. But unlike the known WRKY-containing Arabidopsis RRS1-R NLR receptor, this newly identified soybean WRKY domain could not be acetylated by PopP2 and its atypical sequence (WRKYGKR) also probably renders it inefficient in plant immunity triggering.

This ID toolkit is available for screening with other plant pathogen effectors and should prove useful to discover new effectors targets and potentially engineer new plant resistance genes. Warmer temperatures result in maladaptive learning of sexual preferences O_LIEmpirical studies predicting the effects of the climate crisis on biodiversity loss that take into account learning ability and sexual selection are currently lacking.
C_LIO_LIUsing the African butterfly Bicyclus anynana, which shows strong phenotypic plasticity (i.e., polyphenism) in response to temperature, we find that learning produces a maladaptive mate preference under climate warming.
C_LIO_LIWe first modelled climate warming scenarios and then showed experimentally that as temperature becomes an unreliable cue to the onset of the dry season, adult butterflies displayed the wet season rather than the dry season form.
C_LIO_LIFemale learning further suppressed their innate, adaptive sexual preference for dry season males. Instead, females learned to prefer a phenotype transiently present during the seasonal transition.
C_LIO_LIFemale fertility and longevity were also indirectly affected by learning, likely reducing female fitness following climate warming.
C_LIO_LIOur results emphasize the importance of sexual selection, learning, and their fitness consequences for understanding (mal)adaptation of natural populations to climate warming.
C_LI Plasma proteome analyses in individuals of European and African ancestry identify cis-pQTLs and models for proteome-wide association studies Improved understanding of genetic regulation of proteome can facilitate the identification of causal mechanisms for complex traits. We analyzed data on 4,657 plasma proteins from 7,213 European American (EA) and 1,871 African American (AA) individuals from the ARIC study, and further replicated findings on 467 AA individuals from the AASK study. Here we identified 2,004 proteins in EA and 1,618 in AA, with majority overlapping, which showed associations with common variants in cis-regions. Availability of AA samples led to smaller credible sets and significant number of population-specific cis-pQTLs. Elastic-net produced powerful models for protein prediction in both populations. An application of proteome-wide association studies (PWAS) to serum urate and gout, implicated several proteins, including IL1RN, revealing the promise of the drug anakinra to treat acute gout flares. Our study demonstrates the value of large and diverse ancestry study for genetic mechanisms of molecular phenotypes and their relationship with complex traits. Hydrophobic mismatch is a key factor in protein transport on the Tat pathway The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoid, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits, TatA, TatB and TatC. TatA and TatB from all bacterial species possess short transmembrane alpha-helices (TMHs), both of which are only fifteen residues long in E. coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer. Here, by modifying the length of the TMHs of E. coli TatA and TatB, we assess the functional importance of the hydrophobic mismatch in the Tat transport mechanism. Surprisingly, both TatA and TatB with as few as 11 residues in their respective TMHs are still able to insert into the membrane bilayer, albeit with a decline in membrane integrity. Three different assays, both qualitative and quantitative, were conducted to evaluate the Tat activity of the TMH length mutants. Our experiments indicate that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off with any modification of this length. We believe our study supports a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation, and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes. A prediction: granule cells can perform linearly non-separable computations Multiple studies show how dendrites might extend some neurons computational capacity. Past works most often focus on pyramidal cells which have an extended dendritic tree where voltage can vary independently; pyramidal neurons dendrites emit local spikes turning dendritic branches into local non-linear subunits. However, these studies leave a large fraction of the nervous system unexplored. Here, we study a neuron with a modest dendritic and non-spiking dendrites. Granule cells dendrites do not spike and these cells membrane voltage remain constant over the neuron. This study uses a conjunction of Boolean algebra and biophysical modelling to predict that Granule cells can perform linearly non-separable computations. In a previous study, we found a linearly non-separable Boolean function possible to implement without dendritic spikes, we coined this computation the feature binding problem. In the present work, we use the intrinsic saturation of synaptic conductance to implement this computation in a biophysical model. An integrate and fire, cannot perform such type of computations. Confirming our prediction would change how we understand the nervous system. Barcode aware adaptive sampling for GridION and PromethION Oxford Nanopore sequencers. Adaptive sampling enables selection of individual DNA molecules from sequencing libraries, a unique property of nanopore sequencing. Here we develop our adaptive sampling tool readfish to become "barcode-aware" enabling selection of different targets within barcoded samples or filtering out individual barcodes. We show that multiple human genomes can be assessed for copy number and structural variation on a single sequencing flow cell using sample specific customised target panels on both GridION and PromethION devices. Molecular Dynamics Simulations of Phytopthora sojae Avirulence Factor 5, PsAvh5, Define Membrane Binding, Inositolphosphate-3'-phosphate Interactions and Protein Mechanics Phytopthora Avirulence proteins are a primary target for development of rational chemical and biological control of some of the most devastating plant pathogens. Despite the sequencing of entire genomes, and characterization of many of these proteins at the chemical level, many questions remain regarding actual chemical and biological interactions involved. In addition, disputed roles of ligands, such as Inositolphosphate-3-phosphate and amino acids of important function remain unclear. To address some of these issues, we developed molecular models from structural elements and published data for Phytopthora sojae avirulence protein 5. Molecular dynamics simulations are used to study protein function, interactions involved primarily with lipids and membranes, and inositol derivatives. Our findings indicate that the protein is stable as a monomer, and in a dimeric form. Also, that these proteins interact with Inositolphosphate-3phosphate as a necessary membrane element, in binding. We identified several amino acids of importance, additional to defining the mechanical features of the protein within the binding process to different membranes. A high affinity, comparable to other membrane surface binding molecules of -219.54 Kcal for the dimer, and -176.61 for the monomer were determined. With either form, we found the inositolphosphate-3-phosphate to be essential in the membrane binding process. Our findings answer some of the debated questions while creating a point to further test avirulence proteins in general for functional aspects. Additionally, the structures and data can be utilized to provide a better starting point for rational design approaches to control this pathogen. Chromosome breaks in breast cancers occur near herpes tumor virus sequences and explain why the cancer comes back One explanation for why breast cancer has a relentless tendency to come back after treatment is that breast cancer phenomes are fluid, so it is difficult for therapy to target new phenotypes that arise. In breast cancer, viral interactions cause chromosome breakage and fusion, producing anaphase bridges containing two centromeres. Cell division then leads to more chromosome breaks, more mutations, and new cancer phenotypes. Analyses of public data from about 2100 breast cancers implicate Epstein-Barr virus (EBV or Human Herpes Virus 4) in the cycles that break chromosomes and produce anaphase bridges. EBV generates breakage cycles with highly fragmented chromosomes (chromothripsis) as an inherent feature. These episodes continually produce new cancer driver mutations. DNA at chromothripsis boundaries in breast cancers consistently matches EBV tumor variants. Breakpoints are often near known EBV binding sites, and breast cancer shares an EBV methylation signature with other known EBV cancers. Breast cancer breakpoints on all chromosomes cluster around the same positions as in cancers definitively associated with EBV infection (nasopharyngeal cancer and Burkitts lymphoma). The status of EBV in the microbiome and the presence of anaphase bridges are phenotypic markers of breast cancer that are not explicitly targeted by chemotherapy. The breast cancer phenome continually supplies new cancer driver genes that foster breast cancer recurrence.

SummaryBreaks in breast cancer chromosomes and in fragments of anaphase bridges occur mainly near regions that resemble Epstein-Barr virus (EBV) tumor variants

Extensive breaks are an inherent outcome of anaphase bridges and the reassembly of highly fragmented chromosomes continually generates new cancer driver mutations and new cancer phenotypes.

Treatment does not explicitly target the underlying breakage and reassembly cycles so breast cancer often comes back.

The breast cancer phenome is not fixed and can continuously generate new phenotypes

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=154 HEIGHT=200 SRC="FIGDIR/small/467751v4_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@6537cforg.highwire.dtl.DTLVardef@132fa50org.highwire.dtl.DTLVardef@1cfcb04org.highwire.dtl.DTLVardef@11979aa_HPS_FORMAT_FIGEXP  M_FIG C_FIG On-pathway multivalency of a broadly neutralizing evolution drug candidate A promising approach to prevent antibiotic resistance is design of multivalent antibiotics that constrain bacterial evolvability by blocking multiple escape routes. Previously we discovered that an "evolution drug" lead designed to broadly neutralize bacterial dihydrofolate reductase (DHFR) and its trimethoprim escape variants is multivalent in E. coli cells. In this work we identified the second target of this new lead. We analysed antibiotic induced global metabolic perturbations and deployed metabolic-network analysis and structural similarity search of the putative targets and narrowed down the search to a few enzymes. Further, combining in vivo and in vitro analyses, we identified HPPK (folK), an essential protein in bacterial folate-metabolism, as the non-DHFR target of the lead. We observed that compounds with interaction specificity against WT do not develop multivalency against related proteins. This multivalent-design strategy is effective in blocking the emergence of drug-resistant phenotypes.

HighlightsO_LIAn evolution drug lead designed to block bacterial evolvability manifested behaviours suggesting an intra-cellular multivalent activity.
C_LIO_LIA multi-layered metabolomic and structure guided approach unravelled the second intra-cellular target of the evolution-drug lead, showing folate-pathway inhibitions at two metabolic points.
C_LIO_LIThe on-pathway anti-folate activity is exhibited by inhibiting two essential proteins viz. DHFR and its upstream protein HPPK.
C_LIO_LIMultivalent drug development schemes present a promising anti-evolution strategy to address the problem of "drug-resistance" by inhibiting more than one metabolic protein.
C_LI Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation ObjectiveUltraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKC-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKC nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation.

MethodsWe studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKC nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation.

ResultsOur time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKC in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKC nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNF, IFN{gamma}, and IFN in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency.

ConclusionsROCK regulated NETosis through modulation of PKC nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB- induced skin inflammation by attenuation of NETosis and NET-associated cytokines. Ecology of active viruses and their bacterial hosts in frozen Arctic peat soil revealed with H218O stable isotope probing metagenomics Winter carbon loss in northern ecosystems is estimated to be greater than the average growing season carbon uptake. However, most ecosystem carbon measurements neglect winter months since carbon losses (primarily driven by microbial decomposers) are assumed to be negligible at low temperatures. We used stable isotope probing (SIP) targeted metagenomics to reveal the genomic potential of active soil microbial populations under winter conditions, with an emphasis on viruses and virus-host dynamics. Peat soils from the Bonanza Creek LTER site in Alaska were incubated under subzero anoxic conditions with H218O for 184 and 370 days. We identified 46 bacterial populations (MAGs; spanning 9 bacterial phyla) and 243 viral populations (vOTUs) that actively took up 18O and produced significant CO2 throughout the incubation. Active hosts, predicted for 33% of the active vOTUs, were some of the most abundant MAGs and capable of fermentation and organic matter degradation. Approximately three-quarters of the active vOTUs carried auxiliary metabolic genes that spanned five functional categories, including carbon utilization, highlighting the potential impact of viruses in this peat soils microbial biogeochemistry. These results illustrate significant bacterial and viral activity and interactions occur in frozen soils, revealing viruses are a major community-structuring agent throughout winter months. Multi-omics investigation of tacrolimus nephrotoxicity BackgroundTacrolimus, an immunosuppressive drug prescribed to a majority of transplanted patients is nephrotoxic, through still unclear mechanisms. This study aims to evaluate the impact of tacrolimus on a lineage of proximal tubular cells using a multi-omics approach.

MethodsLLC-PK1 cells were exposed to 5 M of tacrolimus for 24h. Intracellular proteins and metabolites, and extracellular metabolites were extracted and analysed by LC-MS/MS. The transcriptional expression of the dysregulated proteins PCK-1, FBP1 and FBP2 was measured using RT-qPCR.

ResultsIn our cell model, tacrolimus impacted different metabolic pathways including those of arginine (e.g., citrulline, ornithine) (p < 0.0001), amino acids (e.g., valine, isoleucine, aspartic acid) (p < 0.0001) and pyrimidine (p<0.01). In addition, it induced oxidative stress (p < 0.01) as shown by a decrease in total cell glutathione quantity. It impacted cell energy through an increase in Krebs cycle intermediates (e.g., citrate, aconitate, fumarate) (p < 0.01) and down-regulation of PCK-1 (p < 0.05) and FPB1 (p < 0.01), which are key enzymes in gluconeogenesis. Apart from glucose synthesis, gluconeogenesis is an important process in kidney mediated acid-base balance control.

ConclusionThe variations found using this multi-omics approach clearly point towards a dysregulation of energy production in epithelial cells of the renal tubule, and potentially of their functions, that may be implicated in tacrolimus nephrotoxicity in the clinics. GEM-DeCan: Improved tumor immune microenvironment profiling through novel gene expression and DNA methylation signatures predicts immunotherapy response Quantifying the proportion of the different cell types present in tumor biopsies remains a priority in cancer research. So far, a number of deconvolution methods have emerged for estimating cell composition using reference signatures, either based on gene expression or on DNA methylation from purified cells. These two deconvolution approaches could be complementary to each other, leading to even more performant signatures, in cases where both data types are available. However, the potential relationship between signatures based on gene expression and those based on DNA methylation remains underexplored.

Here we present five new deconvolution signature matrices, based on DNA methylation or RNAseq data, which can estimate the proportion of immune cells and cancer cells in a tumour sample. We test these signature matrices on available datasets for in-silico and in-vitro mixtures, peripheral blood, cancer samples from TCGA, bone marrow from multiple myeloma patients and a single-cell melanoma dataset. Cell proportions estimates based on deconvolution performed using our signature matrices, implemented within the EpiDISH framework, show comparable or better correlation with FACS measurements of immune cell-type abundance and with various estimates of cancer sample purity and composition than existing methods.

Using publicly available data of 3D chromatin structure in haematopoietic cells, we expanded the list of genes to be included in the RNAseq signature matrices by considering the presence of methylated CpGs in gene promoters or in genomic regions which are in 3D contact with these promoters. Our expanded signature matrices have improved performance compared to our initial RNAseq signature matrix. Finally, we show the value of our signatures in predicting patient response to immune checkpoint inhibitors in three melanoma and one bladder cancer cohorts, based on bulk tumour sample gene expression.

We also provide GEM-DeCan: a snakemake pipeline, able to run an analysis from raw sequencing data to deconvolution based on various gene expression signature matrices, both for bulk RNASeq and DNA methylation data. The code for producing the signature matrices and reproducing all the figures of this paper is available on the GEM-DeCan repository. Data-Driven Network Models for Genetic Circuits From Time-Series Data with Incomplete Measurements Synthetic gene networks are frequently conceptualized and visualized as static graphs. This view of biological programming stands in stark contrast to the transient nature of biomolecular interaction, which is frequently enacted by labile molecules that are often unmeasured. Thus, the network topology and dynamics of synthetic gene networks can be difficult to verify in vivo or in vitro, due to the presence of unmeasured biological states. Here we introduce the dynamical structure function as a new mesoscopic, data-driven class of models to describe gene networks with incomplete measurements. We introduce a network reconstruction algorithm and a code base for reconstructing the dynamical structure function from data, to enable discovery and visualization of graphical relationships in a genetic circuit diagram as time-dependent functions rather than static, unknown weights. We prove a theorem, showing that dynamical structure functions can provide a data-driven estimate of the size of crosstalk fluctuations from an idealized model. We illustrate this idea with numerical examples. Finally, we show how data-driven estimation of dynamical structure functions can explain failure modes in two experimentally implemented genetic circuits, a historical genetic circuit and a new E. coli based transcriptional event detector. 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. Darwin's small and medium ground finches might have taste preferences, but not for human foods Urbanization is rapidly changing ecological niches. On the inhabited Galapagos Islands, Darwins finches consume human-introduced foods preferentially; however, it remains unclear why. Here we presented pastry with flavour profiles typical of human foods (oily, salty, sweet) to small ground finches (Geospiza fuliginosa) and medium ground finches (Geospiza fortis) to test if latent taste preferences might drive selection of human foods. If human-food flavours were consumed more than a neutral or bitter control only at sites with human foods, then we predicted tastes were acquired after urbanization; however, if no site-differences were found then this would indicate latent taste preferences. Contrary to both predictions, we found little evidence that human-food flavours were preferred compared to control flavours at any site. Instead, finches showed a weak aversion to oily foods, but only at remote (no human foods present) sites. This was further supported by behavioural responses, with beak wiping occurring more often at remote sites after finches tasted flavours associated with human foods. Our results suggest, therefore, that while Darwins finches may have acquired a tolerance to the flavours of human food, latent taste preferences are unlikely to have played a major role in their dietary response to increased urbanization. Modular biosynthesis of plant hemicellulose and its impact on yeast cells BackgroundThe carbohydrate polymers that encapsulate plants cells have benefited humans for centuries and have valuable biotechnological uses. In the past five years, exciting possibilities have emerged in the engineering of polysaccharide-based biomaterials. Despite impressive advances on bacterial cellulose-based hydrogels, comparatively little is known about how plant hemicelluloses can be reconstituted and modulated in cells suitable for biotechnological purposes.

ResultsHere, we assembled cellulose synthase-like A (CSLA) enzymes using an optimized Pichia pastoris platform to produce tunable heteromannan (HM) polysaccharides in yeast. By swapping the domains of plant mannan and glucomannan synthases, we engineered chimeric CSLA proteins that made {beta}-1,4-linked mannan in quantities surpassing those of the native enzymes while minimizing the burden on yeast growth. Prolonged expression of a glucomannan synthase from Amorphophallus konjac was toxic to yeast cells: reducing biomass accumulation and ultimately leading to compromised cell viability. However, an engineered glucomannan synthase as well as CSLA pure mannan synthases and a CSLC glucan synthase did not inhibit growth. Interestingly, Pichia cell size could be increased or decreased depending on the composition of the CSLA protein sequence. HM yield and glucose incorporation could be further increased by co-expressing chimeric CSLA proteins with a MANNAN-SYNTHESIS-RELATED (MSR) co-factor from Arabidopsis thaliana.

ConclusionThe results provide novel routes for the engineering of polysaccharide-based biomaterials that are needed for a sustainable bioeconomy. The characterization of chimeric cellulose synthase-like enzymes in yeast offers an exciting avenue to produce plant polysaccharides in a tunable manner. Furthermore, cells modified with non-toxic plant polysaccharides such as {beta}-mannan offer a modular chassis to produce and encapsulate sensitive cargo such as therapeutic proteins. Ensovibep, a novel trispecific DARPin candidate that protects against SARS-CoV-2 variants SARS-CoV-2 has infected millions of people globally and continues to undergo evolution. Emerging variants can be partially resistant to vaccine induced immunity and therapeutic antibodies, emphasizing the urgent need for accessible, broad-spectrum therapeutics. Here, we report a comprehensive study of ensovibep, the first trispecific clinical DARPin candidate, that can simultaneously engage all three units of the spike protein trimer to potently inhibit ACE2 interaction, as revealed by structural analyses. The cooperative binding of the individual modules enables ensovibep to retain inhibitory potency against all frequent SARS-CoV-2 variants, including Omicron BA.1 and BA.2, as of February 2022. Moreover, viral passaging experiments show that ensovibep, when used as a single agent, can prevent development of escape mutations comparably to a cocktail of monoclonal antibodies (mAb). Finally, we demonstrate that the very high in vitro antiviral potency also translates into significant therapeutic protection and reduction of pathogenesis in Roborovski dwarf hamsters infected with either the SARS-CoV-2 wild-type or the Alpha variant. In this model, ensovibep prevents fatality and provides substantial protection equivalent to the standard of care mAb cocktail. These results support further clinical evaluation and indicate that ensovibep could be a valuable alternative to mAb cocktails and other treatments for COVID-19. Discovery and functional assessment of a novel adipocyte population driven by intracellular Wnt/β-catenin signaling in mammals Wnt/{beta}-catenin signaling has been well established as a potent inhibitor of adipogenesis. Here, we identified a population of adipocytes that exhibit persistent activity of Wnt/{beta}-catenin signaling, as revealed by the Tcf/Lef-Gfp reporter allele, in embryonic and adult mouse fat depots, named as Wnt+ adipocytes. We showed that the {beta}-catenin-mediated signaling activation in these cells is Wnt ligand- and receptor-independent but relies on AKT/mTOR pathway and is essential for cell survival. Such adipocytes are distinct from classical ones in transcriptomic and genomic signatures and can be induced from various sources of mesenchymal stromal cells including human cells. Genetic lineage-tracing and targeted cell ablation studies revealed that these adipocytes convert into beige adipocytes directly and are also required for beige fat recruitment under thermal challenge, demonstrating both cell autonomous and non-cell autonomous roles in adaptive thermogenesis. Furthermore, mice bearing targeted ablation of these adipocytes exhibited glucose intolerance, while mice receiving exogenously supplied such cells manifested enhanced glucose utilization. Our studies uncover a unique adipocyte population in regulating beiging in adipose tissues and systemic glucose homeostasis. Morphology of immatures of the thelytokous ant, Monomorium triviale Wheeler (Formicidae: Myrmicinae: Solenopsidini) with descriptions of the extraordinary last-instar queen larvae The ant genus Monomorium is one of the most species-rich but taxonomically problematic groups in the hyperdiverse subfamily Myrmicinae. An East Asian species, M. triviale Wheeler, produces both reproductive queens and sterile workers via obligate thelytokous parthenogenesis. Here, we describe the immature forms of M. triviale based on light and scanning electron microscopy observations, with a note on the striking caste dimorphism in the last larval instar. The last-instar queen larvae were easily recognized by their large size, "aphaenogastroid" body shape, and rows of doorknob-like tubercles on the lateral and dorsal body surface. This type of queen-specific structure has not been found in ants in general, let alone congeneric species found in Japan. In stark contrast to the queen larvae, worker larvae showed a "pheidoloid" body shape and a body surface similar to other ants. The worker larvae were estimated to have three instars, consistent with previously described congeners. The pupae of both castes had no cocoon, a characteristic commonly described in other Myrmicinae species. In total, the developmental period from egg to adult worker averaged 59 days under 25{degrees}C. We discuss possible functions of the tubercles of queen larvae based on previous studies.