title: Cell Types of Origin in the Cell Free Transcriptome in Human Health and Disease

abstract: Cell-free RNA (cfRNA) can be used to noninvasively measure dynamic and longitudinal physiological changes throughout the body. While there is considerable effort in the liquid biopsy field to determine disease tissue-of-origin, pathophysiology occurs at the cellular level. Here, we describe two approaches to identify cell type contributions to cfRNA. First we used Tabula Sapiens, a transcriptomic cell atlas of the human body to computationally deconvolve the cell-free transcriptome into a sum of cell type specific transcriptomes, thus revealing the spectrum of cell types readily detectable in the blood. Second, we used individual tissue transcriptomic cell atlases in combination with the Human Protein Atlas RNA consensus dataset to create cell type signature scores which can be used to infer the implicated cell types from cfRNA for a variety of diseases. Taken together, these results demonstrate that cfRNA reflects cellular contributions in health and disease from diverse cell types, potentially enabling determination of pathophysiological changes of many cell types from a single blood test.


title: The self-regulated response of the Wnt pathway to an oncogenic mutation in β-catenin

abstract:
Oncogenic mutations in {beta}-catenin can inhibit degradation of {beta}-catenin by preventing phosphorylation of its degron. Degron phosphorylation is mediated by the Axin scaffold, Casein Kinase 1 (CK1) and Glycogen Synthase Kinase 3 (GSK3). We studied an oncogenic form of {beta}-catenin with a deletion of serine 45 (S45), a site that is phosphorylated by the kinase CK1. When the S45 site is phosphorylated, it promotes the GSK3-mediated phosphorylations of the {beta}-catenin degron. Deletion of S45 would be expected to prevent GSK3-mediated phosphorylation of the mutant protein and thus block degradation. We found that the S45 mutant was still phosphorylatable by GSK3, and its expression increased the concentration of Axin, restoring the rate of GSK3-mediated phosphorylation to levels comparable to those observed for the wild-type {beta}-catenin. We conclude that there is one core mechanism for creating the phosphodegron for both primed and unprimed {beta}-catenin, which involves the generation of an Axin-GSK3 complex.

SIGNIFICANCEUnderstanding how the Wnt pathway responds to mutations in {beta}-catenin phosphodegron can reveal important properties of the pathway in normal and cancer cells and is valuable for the design of more effective therapeutic strategies.

category: systems biology


title: SARS CoV-2 variant B.1.617.1 is highly pathogenic in hamsters than B.1 variant

abstract:
BackgroundThe recent emergence of new SARS-CoV-2 lineage B.1.617 in India has been associated with a surge in the number of daily infections. This variant has combination of specific mutations L452R, E484Q and P681R reported to possibly enhance the transmissibility with likelihood of escaping the immunity. We investigated the viral load and pathogenic potential of B.1.617.1 in Syrian golden hamsters.

MethodsTwo groups of Syrian golden hamsters (9 each) were inoculated intranasally with SARS CoV-2 isolates, B.1 (D614G) and B.1.617.1 respectively. The animals were monitored daily for the clinical signs and body weight. The necropsy of three hamsters each was performed on 3, 5- and 7-days post-infection (DPI). Throat swab (TS), nasal wash (NW) and organ samples (lungs, nasal turbinate, trachea) were collected and screened using SARS-CoV-2 specific Real-time RT-PCR.

ResultsThe hamsters infected with B.1.617.1 demonstrated increased body weight loss compared to B.1 variant. The highest viral load was observed in nasal turbinate and lung specimens of animals infected with B.1.167.1 on 3 DPI. Neutralizing antibody (NAb) and IgG response in hamsters of both the groups were observed from 5 and 7 DPI respectively. However, higher neutralizing antibody titers were observed against B.1.167.1. Gross pathology showed pronounced lung lesions and hemorrhage with B.1.671 compared to B.1.

ConclusionsB.1617.1 and B.1 variant varied greatly in their infectiousness, pathogenesis in hamster model. This study demonstrates higher pathogenicity in hamsters evident with reduced body weight, higher viral load in lungs and pronounced lung lesions as compared to B.1 variant.

SummaryB.1.617.1 is the new SARS-CoV-2 lineage that emerged in India. Maximal body weight loss and higher viral load in hamsters infected with B.1.617.1. It caused pronounced lung lesions in hamsters compared to B.1 variant which demonstrates the pathogenic potential of B.1.617.1.

category: microbiology


title: The CTPase activity of ParB acts as a timing mechanism to control the dynamics and function of prokaryotic DNA partition complexes

abstract: DNA partitioning CTPases of the ParB family mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromeric region of target DNA molecules and then spread laterally to form large nucleoprotein complexes that serve as docking points for the DNA segregation machinery. Here, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. Moreover, we solve crystal structures of ParB in the pre- and post-hydrolysis state and provide insights into the catalytic mechanism underlying nucleotide hydrolysis. The characterization of CTPase-deficient ParB variants reveals that CTP hydrolysis serves as a timing mechanism to control the sliding time of ParB. Hyperstable clamps are trapped on the DNA, leading to excessing spreading and severe chromosome segregation defects in vivo. These findings clarify the role of the ParB CTPase cycle in partition complex dynamics and function and thus complete our understanding of this prototypic CTP-dependent molecular switch.


title: Proteomic traits vary across taxa in a coastal Antarctic phytoplankton bloom

abstract: Production and use of proteins is under strong selection in microbes, but its unclear how proteome-level traits relate to ecological strategies. We identified and quantified proteomic traits of eukaryotic and prokaryotic microbes through an Antarctic phytoplankton bloom using in situ metaproteomics. To do this, we first used simulations, cultures, and bioinformatic methods to rigorously assess our inferences about various proteomic traits and use these assessments to provide several practical recommendations for researchers using metaproteomics. Different taxa, rather than different environmental conditions, formed distinct clusters based on their ribosomal and photosynthetic proteomic proportions, and we propose that these characteristics relate to ecological differences. We defined and used a proteomic proxy for regulatory cost, which showed that SAR11 had the lowest regulatory cost of any taxa we observed at our summertime Southern Ocean study site. Haptophytes had lower regulatory cost than diatoms, which may underpin haptophyte-to-diatom bloom progression in the Ross Sea. Using metaproteomics, we have quantified several proteomic traits (ribosomal and photosynthetic proteomic proportions, regulatory cost) in eukaryotic and prokaryotic taxa, which can then be incorporated into trait-based models of microbial communities that reflect resource allocation strategies.


title: Prior aerosol infection with lineage A SARS-CoV-2 variant protects hamsters from disease, but not reinfection with B.1.351 SARS-CoV-2 variant

abstract: The circulation of SARS-CoV-2 has resulted in the emergence of variants of concern (VOCs). It is currently unclear whether previous infection with SARS-CoV-2 provides protection against reinfection with VOCs. Here, we show that low dose aerosol exposure to hCoV-19/human/USA/WA-CDC-WA1/2020 (WA1, lineage A), resulted in a productive mild infection. In contrast, low dose of SARS-CoV-2 via fomites did not result in productive infection in the majority of exposed hamsters and these animals remained non-seroconverted. After recovery, hamsters were re-exposed to hCoV-19/South African/KRISP-K005325/2020 (VOC B.1.351) via an intranasal challenge. Seroconverted rechallenged animals did not lose weight and shed virus for 3 days. They had little infectious virus and no pathology in the lungs. In contrast, shedding, weight loss and extensive pulmonary pathology caused by B.1.351 replication was observed in the non-seroconverted animals. The rechallenged seroconverted animals did not transmit virus to naive sentinels via direct contact transmission, in contrast to the non-seroconverted animals. Reinfection with B.1.351 triggered an anamnestic response that boosted not only neutralizing titers against lineage A, but also titers against B.1.351. Our results confirm that aerosol exposure is a more efficient infection route than fomite exposure. Furthermore, initial infection with SARS-CoV-2 lineage A does not prevent heterologous reinfection with B.1.351 but prevents disease and onward transmission. These data suggest that previous SARS-CoV-2 exposure induces partial protective immunity. The reinfection generated a broadly neutralizing humoral response capable of effectively neutralizing B.1.351 while maintaining its ability to neutralize the virus to which the initial response was directed against.


title: Identification of Splice Regulators of Fibronectin-EIIIA and EIIIB by Direct Measurement of Exon Usage in a Flow-Cytometry Based CRISPR Screen

abstract: The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regulators of alternative splicing in FN, we have developed an in vitro flow-cytometry based assay, using RNA-binding probes to determine alternative exon inclusion level in aortic endothelial cells. This approach allows us to detect exon inclusion in the primary transcripts themselves, rather than in surrogate splicing reporters. We validated this assay in cells with and without FN-EIIIA and -EIIIB expression. In a small-scale CRISPR KO screen of candidate regulatory splice factors, we successfully detected known regulators of EIIIA and EIIIB splicing, and detected several novel regulators. Finally, we show the potential in this approach to broadly interrogate upstream signaling pathways in aortic endothelial cells with a genome-wide CRISPR-KO screen, implicating the TNFalpha and RIG-I-like signaling pathways and genes involved in the regulation of fibrotic responses. Thus, we provide a novel means to screen the regulation of splicing of endogenous transcripts, and predict novel pathways in the regulation of FN-EIIIA inclusion.

category: cell biology


title: Inherited Human Sex Reversal due to Loss of a Water-Mediated Hydrogen Bond at a Conserved Protein-DNA Interface

abstract: Male sex determination in mammals is initiated by SRY, a Y-encoded architectural transcription factor. The protein contains a high-mobility-group (HMG) box that mediates sequence-specific DNA bending. Mutations in SRY causing XY gonadal dysgenesis (Swyer syndrome) cluster in the box. Although such mutations usually arise de novo in spermatogenesis, some are inherited: male development occurs in one genetic background (the father) but not another (the sterile XY daughter). Here, we compare de novo and inherited mutations at an invariant Tyr adjoining the motifs basic tail (consensus position 72; Y127C and Y127F in intact SRY). Crystal structures of homologous SOX-DNA complexes suggest that the wild-type side chains para-OH group anchors a water-mediated hydrogen bond to the DNA backbone. In an embryonic gonadal cell line, Y127C (de novo) led to accelerated proteasomal proteolysis and blocked transcriptional activity; activity remained low on rescue of expression by chemical proteasome inhibition. Y127F (inherited) preserved substantial transcriptional activity: 91({+/-}11)% on SRY overexpression and 65({+/-}17)% at physiological expression. Control studies indicated no change in protein lifetime or nuclear localization. Only subtle biophysical perturbations were observed in vitro. Although though inherited variants specific DNA affinity was only twofold lower than wild type, stopped-flow kinetic analysis revealed a sevenfold decrease in lifetime of the complex. Time-resolved fluorescence energy transfer (using a 15-base pair DNA site) demonstrated native mean DNA bending but with a slightly widened distribution of end-to-end DNA distances. Our findings highlight the contribution of a single water-mediated hydrogen bond to robustness of a genetic switch in human development.

category: developmental biology


title: An evolutionary perspective of suicide as a way to protect bacteria from being killed by phage

abstract: In 1984, retrons were described as DNA sequences that code for a reverse transcriptase and a unique single-stranded DNA/RNA hybrid called multicopy single-stranded DNA (msDNA). It would not be until 2020 that a function was shown, when compelling evidence was presented that retrons can protect bacteria from infections by lytic phage via an abortive infection mechanism. When infected with the virulent mutant of the phage lambda, {lambda}VIR, and to a lesser extent other phages, a retron designated Ec48 is activated, the Escherichia coli bearing this element dies and the infecting phage is lost. With the aid of a mathematical model, we explored the a priori conditions under which retrons will protect bacterial populations from invasion by phage and when retron-bearing bacteria will evolve in populations without this element. Using E. coli with and without Ec48, and {lambda}VIR we estimated the parameters of this model and tested the hypotheses generated from our analysis of its properties. Our models and experiments suggest cells expressing retron-mediated abortive infection have no advantage over cells lacking this system as a defense against phage, and we question whether the protection against phage infection is the unique selective force responsible for their evolution in bacteria.

category: evolutionary biology


title: The ecological consequences and evolution of retron-mediated suicide as a way to protect bacteria from being killed by phage


title: The ecological consequences and evolution of retron-mediated suicide as a way to protect Escherichia coli from being killed by phage


title: Amino acid sensor conserved from bacteria to humans

abstract: Amino acids are recognized as signals by various receptors in bacteria, archaea, and eukaryotes. However, no common mechanism for amino acid recognition is currently known. Here we show that a subclass of a ubiquitous extracellular domain dCache_1 contains a simple amino acid recognition motif, and it is found throughout the Tree of Life. In bacteria, this motif exclusively binds amino acids, including GABA, and it is present in all major receptor types. In humans, this motif is found in 2{delta} subunits of voltage-gated calcium channels that are implicated in neuropathic pain and neurodevelopmental disorders. Our findings suggest that GABA-derived drugs bind to the same motif in human 2{delta} subunits that binds natural GABA ligands in bacterial chemoreceptors.


title: Screening bioactive food compounds in honey bees suggests curcumin blocks alcohol-induced damage to longevity and DNA methylation

abstract: Various bioactive food compounds may confer health and longevity benefits, possibly through altering or preserving the human epigenome. While bioactive food compounds are widely being marketed as  improving health and longevity by counteracting harmful effects of poor nutrition and lifestyle, claimed effects are often not adequately documented. Using the honey bee (Apis mellifera) as a model species, we here employed a multi-step screening approach to investigate seven compounds for effects on lifespan and DNA methylation using ELISA and whole genome bisulfite sequencing (WGBS). A positive longevity effect was detected for valproic acid, isovaleric acid, and cyanocobalamin. For curcumin, we found that lifespan shortening caused by ethanol intake, was restored when curcumin and ethanol were co-administered. Furthermore, we identified region specific DNA methylation changes as a result of ethanol intake. Ethanol specific changes in DNA methylation were fully or partially blocked in honey bees receiving ethanol and curcumin together. Ethanol-affected and curcumin-blocked differentially methylated regions covered genes involved in fertility, temperature regulation and tubulin transport. Our results demonstrate fundamental negative effects of low dose ethanol consumption on lifespan and associated DNA methylation changes and present a proof-of-principle on how longevity and DNA methylation changes can be negated by the bioactive food component curcumin. Our findings provide a fundament for further studies of curcumin in mice and humans and offer an avenue to explore regarding possible prevention of health issues related to alcohol consumption.

category: genomics


title: A Kink in DWORF Helical Structure Controls the Activation of the Sarco-plasmic Reticulum Ca2+-ATPase

abstract: The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase embedded in the sarcoplasmic reticulum. For each enzymatic cycle, SERCA transports 2 Ca2+ ions per ATP hydrolyzed in exchange for 2 to 3 H+ ions. SERCA is responsible for approximately 70% of Ca2+ transport and plays a central role in muscle relaxation. SERCAs function is regulated by endogenous regulins, single-pass membrane proteins that bind the ATPase within the membrane. While most of the regulins, such as phospholamban and sarcolipin, inhibit SERCAs activity, a newly discovered protein DWarf Open Reading Frame (DWORF) has a unique activating effect. DWORF is a 3.8 kDa bitopic membrane protein expressed in cardiac muscle. In this work, we determine the structure, topology, and per-residue lipid interactions of DWORF in lipid bilayers using a combination of high-resolution oriented sample solid-state NMR (OS-ssNMR) spectroscopy and refinement by replica-averaged orientationally-restrained molecular dynamics (RAOR-MD). We found that DWORFs structural topology consists of a dynamic N-terminal domain, an amphipathic juxtamembrane helix that crosses the lipid groups at an angle of 64{degrees} and a transmembrane (TM) C-terminal helix with an angle of 32{degrees}. A kink induced by Pro15, unique to DWORF, separated the two helical domains. A single Pro15Ala mutant significantly decreases the kink and eliminates DWORFs activating effect on SERCA. Overall, our findings directly link DWORFs structural topology to its unique activating effect on SERCA.

category: biophysics


title: Transcriptional Pathology Evolves Over Time in Rat Hippocampus Following Lateral Fluid Percussion Traumatic Brain Injury

abstract: Traumatic brain injury (TBI) causes acute and lasting impacts on the brain, driving pathology along anatomical, cellular, and behavioral dimensions. Rodent models offer the opportunity to study TBI in a controlled setting, and enable analysis of the temporal progression that occurs from injury to recovery. We applied transcriptomic and epigenomic analysis, characterize gene expression and in ipsilateral hippocampus at 1 and 14 days following moderate lateral fluid percussion (LFP) injury. This approach enabled us to identify differential gene expression (DEG) modules with distinct expression trajectories across the two time points. The major DEG modules represented genes that were up- or downregulated acutely, but largely recovered by 14 days. As expected, DEG modules with acute upregulation were associated with cell death and astrocytosis. Interestingly, acutely downregulated DEGs related to neurotransmission mostly recovered by two weeks. Upregulated DEG modules related to inflammation were not necessarily elevated acutely, but were strongly upregulated after two weeks. We identified a smaller DEG module with delayed downregulation at 14 days including genes related to cholesterol metabolism and amyloid beta clearance. Finally, differential expression was paralleled by changes in H3K4me3 at the promoters of differentially expressed genes at one day following TBI. Following TBI, changes in cell viability, function and ultimately behavior are dynamic processes. Our results show how transcriptomics in the preclinical setting has the potential to identify biomarkers for injury severity and/or recovery, to identify potential therapeutic targets, and, in the future, to evaluate efficacy of an intervention beyond measures of cell death or spatial learning.

category: neuroscience


title: The tunicate metabolite 2-(3,5-diiodo-4-methoxyphenyl)ethan-1-amine targets ion channels of vertebrate sensory neurons

abstract: Marine tunicates produce defensive amino-acid derived metabolites, including 2-(3,5-diiodo-4-methoxyphenyl)ethan-1-amine (DIMTA), but their mechanisms of action are rarely known. Using an assay-guided approach, we found that out of the many different sensory cells in the mouse dorsal root ganglion (DRG), DIMTA selectively affected low-threshold cold thermosensors. Whole-cell electrophysiology experiments using DRG cells, channels expressed in Xenopus oocytes and human cell lines revealed that DIMTA blocks several potassium channels, reducing the magnitude of the afterhyperpolarization and increasing the baseline [Ca2+]i of low-threshold cold thermosensors. When injected into mice, DIMTA increased the threshold of cold sensation by >3 oC. DIMTA may thus serve as a lead in the further design of compounds that inhibit problems in the cold-sensory system, such as cold allodynia and other neuropathic pain conditions.


title: Population analysis of Vibrio cholerae in aquatic reservoirs reveals a novel sister species (Vibrio paracholerae sp. nov.) with a history of association with human infections

abstract:
Most efforts to understand the biology of Vibrio cholerae have focused on a single group, the pandemic-generating lineage harbouring the strains responsible for all known cholera pandemics. Consequently, little is known about the diversity of this species in its native aquatic environment. To understand the differences in the V. cholerae populations inhabiting in regions with varying history of cholera cases and how that might influence the abundance of pandemic strains, a comparative analysis of population composition was performed. Little overlap was found in lineage compositions between those in Dhaka (cholera endemic) located in the Ganges delta, and of Falmouth (no known history of cholera), a small coastal town on the US East Coast. The most striking difference was the presence of a group of related lineages at high abundance in Dhaka which was completely absent from Falmouth. Phylogenomic analysis revealed that these lineages form a cluster at the base of the phylogeny of V. cholerae species, sufficiently differentiated genetically and phenotypically to form a novel species. Strains from this species have been anecdotally isolated from around the world and were isolated as early as 1916 from a British soldier in Egypt suffering from choleraic diarrhoea. In 1935 Gardner and Venkatraman unofficially referred to a member of this group as Vibrio paracholerae. In recognition of this earlier designation, we propose the name Vibrio paracholerae, sp. nov. for this bacterium. Genomic analysis suggests a link with human populations for this novel species and substantial interaction with its better-known sister species.

ImportanceCholera continues to remain a major public health threat around the globe. Understanding the ecology, evolution and environmental adaptation of the causative agent Vibrio cholerae and tracking the emergence of novel lineages with pathogenic potential are essential to combat the problem. In this study, we investigated the population dynamics of Vibrio cholerae in an inland locality which is known as endemic for cholera and compared with that of a cholera free coastal location. We found the consistent presence of the pandemic generating V. cholerae in cholera-endemic Dhaka and an exclusive presence of a lineage phylogenetically distinct from other V. cholerae. Our study suggests that this lineage represents a novel species having pathogenic potential and a human link to its environmental abundance. The possible association with human population, co-existence and interaction with toxigenic V. cholerae in the natural environment make this potential human pathogen an important subject for future studies.


title: Influence of GdVO4:Eu3+ nanocrystals on growth, germination, root cell viability and oxidative stress of wheat (Triticum aestivum L.) seedlings

abstract: Increasing application of lanthanide-doped nanocrystals (LDNCs) entails a risk of a harmful impact on the natural environment. Therefore, in the presented study the influence of gadolinium orthovanadates doped with Eu3+ nanocrystals on wheat (Triticum aestivum L.), chosen as a model plant species, was investigated. The seeds were grown in Petri dishes filled with colloids of LDNCs at the concentrations of: 0, 10, 50 and 100 {micro}g/ml. The plants growth endpoints (number of roots, roots length, roots mass, hypocotyl length and hypocotyl mass) and germination rate were found to be not significantly changed after the exposure to GdVO4:Eu3+ nanocrystals at all used concentrations. The presence of LDNCs also had no effect on oxidative stress intensity determined on the basis of the amount of lipid peroxidation product (thiobarbituric acid reactive substances; TBARS) of the roots. Similarly, TTC (tetrazolium chloride) assay did not show any differences in cells viability. However, root cells of the treated seedlings contained less amount of Evans Blue (EB) when compared to the control.

category: ecology


title: Life-histories as determinants of infection prevalence for trypanosomatids: A meta-analysis

abstract: Trypanosomatids are a diverse family of protozoan parasites, some of which cause devastating human and livestock diseases. There are two distinct infection life-cycles in trypanosomatids; some species complete their entire life-cycle in a single host (monoxenous) while others infect two hosts (dixenous). Dixenous trypanosomatids are mostly vectored by insects, and the human trypanosomatid diseases are caused mainly by vectored parasites. While infection prevalence has been described for subsets of hosts and trypanosomatids, little is known about whether monoxenous and dixenous trypanosomatids differ in infection prevalence. Here, we use meta-analyses to synthesise all published evidence of trypanosomatid infection prevalence for the last two decades, encompassing 931 unique host-trypansomatid systems. In examining 581 studies that describe infection prevalence, we find, strikingly, that monoxenous species are two-fold more prevalent than dixenous species across all hosts. We also find that dixenous trypanosomatids have significantly lower infection prevalence in insects than their non-insect hosts. Within monoxenous trypanosomatids, genera infecting bees are most prevalent and infection prevalence does not vary between wild and managed bees. To our knowledge, these results reveal for the first time, a fundamental difference in infection prevalence according to host specificity where vectored species suffer from lower infection prevalence as a result of a  jack of all trades, master of none style trade-off between the vector and subsequent hosts.


title: Double trouble: having two hosts reduces infection prevalence in vectored trypanosomatids


title: Dog10K_Boxer_Tasha_1.0: A long-read assembly of the dog reference genome

abstract: The domestic dog has evolved to be an important biomedical model for studies regarding the genetic basis of disease, morphology and behavior. Genetic studies in the dog have relied on a draft reference genome of a purebred female boxer dog named "Tasha" initially published in 2005. Derived from a Sanger whole genome shotgun sequencing approach coupled with limited clone-based sequencing, the initial assembly and subsequent updates have served as the predominant resource for canine genetics for 15 years. While the initial assembly produced a good quality draft, as with all assemblies produced at the time it contained gaps, assembly errors and missing sequences, particularly in GC-rich regions, which are found at many promoters and in the first exons of protein coding genes. Here we present Dog10K_Boxer_Tasha_1.0, an improved chromosome-level highly contiguous genome assembly of Tasha created with long-read technologies, that increases sequence contiguity >100-fold, closes >23,000 gaps of the Canfam3.1 reference assembly and improves gene annotation by identifying >1200 new protein-coding transcripts. The assembly and annotation are available at NCBI under the accession GCF_000002285.5.


title: Immune priming can prevent WNV establishment in Culex quinquefasciatus mosquitoes: evidence for immune priming based reversal of WNV-mediated immune suppression.

abstract:
Mosquito-borne infectious diseases cause wide-spread loss of life and livelihood often in low-income settings. However, control of mosquito-vectored viral diseases such as West Nile virus (WNV) and Japanese encephalitis virus (JEV) remains challenging. Here we use an existing feature of the insect immune system to effectively vaccinate Culex quinquefasciatus mosquitoes against WNV infection. We find that priming mosquitoes by exposure to inactivated WNV reduces their likelihood of developing transmissible infections of WNV after live infection. We used RNA sequencing to identify gene expression in response to WNV and JEV infection, and the role of prior priming exposure on constitutive and induced expression on infection. Infection with either Flavivirus causes broad suppression of gene expression. WNV and JEV infection resulted in suppression of different suites of genes with notable immune genes, such as antimicrobial peptides, being strongly suppressed on WNV infection. We hypothesise that the increased resistance to WNV infection seen in primed mosquitoes may be the result of priming nullifying the immune suppression found in non-primed WNV-fed mosquitoes, potentially through greater expression of mRNA regulatory genes such as cap-binding proteins in primed mosquitoes.

Author summaryMosquitoes vector many devastating infectious diseases. Two such vectored viral diseases are West Nile virus (WNV) and Japanese encephalitis virus (JEV). Control of these diseases remains challenging, and no vaccine exists for WNV. Here, we tested whether we could instead vaccinate the mosquitoes against WNV. By injecting mosquitoes with dead WNV we found that we could reduce the number of infected mosquitoes by half. We then used whole-genome RNA sequencing to identify which genes are transcribed, which will help us understand genes that are important for this form of insect immune priming, and for responses to normal WNV and JEV infection. We found that WNV suppresses the expression of many immune genes but these genes are expressed normally in vaccinated mosquitoes. Our findings expand our understanding of mosquito infection with these viruses but also demonstrate how prior exposure to a disease can produce lasting protection.


title: Seasonal song variation in male Carolina Wrens (Thryothorus ludovicianus)

abstract: Birdsongs primary function is attracting and stimulating mates and repelling rivals during the breeding season. However, many species also sing during the nonbreeding season, which raises questions about the function of nonbreeding song and the proximate mechanisms underlying its production. In this study, we compared spectral and temporal measurements from a large sample of breeding (N = 267) and nonbreeding songs (N = 283) of Carolina Wrens (Thryothorus ludovicianus), a nonmigratory, temperate species that sings year-round. We found that breeding songs were longer than nonbreeding songs and had more syllables within each song. Trill rate, the number of notes per syllable, minimum and maximum frequency and frequency bandwidth did not differ detectably between the two seasons. This study is the first to examine seasonal song differences in Carolina Wrens and provides a basis for future investigations into the drivers behind this seasonal variation.

category: animal behavior and cognition


title: XENet: Using a new graph convolution to accelerate the timeline for protein design on quantum computers

abstract:
Graph representations are traditionally used to represent protein structures in sequence design protocols where the folding pattern is known. This infrequently extends to machine learning projects: existing graph convolution algorithms have shortcomings when representing protein environments. One reason for this is the lack of emphasis on edge attributes during massage-passing operations. Another reason is the traditionally shallow nature of graph neural network architectures. Here we introduce an improved message-passing operation that is better equipped to model local kinematics problems such as protein design. Our approach, XENet, pays special attention to both incoming and outgoing edge attributes.

We compare XENet against existing graph convolutions in an attempt to decrease rotamer sample counts in Rosettas rotamer substitution protocol. This use case is motivating because it allows larger protein design problems to fit onto near-term quantum computers. XENet outperformed competing models while also displaying a greater tolerance for deeper architectures. We found that XENet was able to decrease rotamer counts by 40% without loss in quality. This decreased the problem size of our use case by more than a factor of 3. Additionally, XENet displayed an ability to handle deeper architectures than competing convolutions.

Author summaryGraphs data structures are ubiquitous in the field of protein design and are at the core of the recent advances in artificial intelligence brought forth by graph neural networks (GNNs). GNNs have led to some impressive results in modeling protein interactions, but are not as common as other tensor representations.

Most GNN architectures tend to put little to no emphasis on the information stored on edges; however, protein modeling tools often use edges to represent vital geometric relationships about residue pair interactions. In this paper, we show that a more advanced processing of edge attributes can lead to considerable benefits when modeling chemical data.

We introduce XENet, a new member of the GNN family that is shown to have improved ability to model protein residue environments based on chemical and geometric data. We use XENet to intelligently simplify the optimization problem that is solved when designing proteins. This task is important to us and others because it allows larger proteins to be designed on near-term quantum computers. We show that XENet is able to train on our protein modeling data better than existing methods, successfully resulting in a dramatic decrease in protein design sample space with no loss in quality.

category: bioinformatics


title: Network Pharmacology Integrated Molecular Docking Based Prediction of Active Compounds and Potential Targets in Tinospora crispa Linn. as Insulin Sensitizer

abstract: Insulin resistance is a metabolic disorder characterized by the decreased response to insulin in muscle, liver, and adipose cells. The normal insulin levels are unable to control glucose, lipids, and energy homeostasis. This condition remains a complex phenomenon that involves several genetic defects and environmental stresses, such as obesity. A full understanding is required to understand the entire itinerary and functional consequences of the occurrence of insulin resistance to develop a potent drug in diabetes management. In the present study, we investigated the mechanism of known phytochemical constituents of Tinospora crispa and its interaction with insulin resistant target proteins by using network pharmacology and molecular docking. The insulin sensitizer activities of Tinospora crispa may be associated with the inhibition of the activation of the inflammatory pathway and the activation of insulin signaling. Tinoscorside A, Makisterone C, Borapetoside A and B, and {beta} sitosterol consider the main phytoconstituents of Tinospora crispa by its binding with active sites of main protein targets of insulin resistance potential therapy. In conclusion, Tinospora crispa was one of the promising therapeutic agent in type 2 diabetes mellitus management. Regulation in glucose homeostasis, adipolysis, cell proliferation, and antiapoptosis may be the critical mechanism of Tinospora crispa as an insulin sensitizer.


title: Designing Novel and Potent Inhibitors for Multi Drug-Resistant Tuberculosis: A Computational Approach

abstract: Tuberculosis is a major global health problem and is still among the top 10 causes of death. The increasing rate of drug resistance to infectious agents has provoked an urgent need to discover novel anti-tuberculosis agents with novel modes of action. In this study, small molecule inhibitors of the proteins encoded by the drug resistant genes, i.e., katG, gyrA, pncA and rpoB of Mycobacterium tuberculosis (M. tuberculosis), were identified using computational methods. In the ligand base pharmacophore, an already reported four ligands for the four proteins encoded by the resistant genes of M. tuberculosis were selected for the generation of pharmacophores. The validated pharmacophores model of all the four proteins, generated on the basis of ligand base, were selected for further screening of ZINC drug like database. As screening results 486 structurally diverse hits for katG, 542 for PncA, 112 for rpoB and 365 for gyrA were mapped and filtered via Lipinskis rule of five. Finally, on the basis of docking score and binding interactions, ten small molecules were selected for each protein as novel inhibitors. These selected novel inhibitors have significant interaction with the active site of the protein and a strong possibility to act as an additional starting opinion in the development of new and potential inhibitors. The result indicates that novel inhibitors could be a promising lead compound and be effective in treating sensitive as well as multi drug resistant tuberculosis.


title: A statistical test for clonal exclusivity in tumour evolution

abstract: Tumour progression is an evolutionary process in which different clones evolve over time, leading to intra-tumour heterogeneity. Interactions between clones can affect tumour evolution and hence disease progression and treatment outcome. Pairs of mutations that are overrepresented in a clonally exclusive fashion over a cohort of patient samples may be suggestive of a synergistic effect between the different clones carrying these mutations. We therefore developed a novel statistical test, called GeneAccord, to identify such gene pairs that are altered in distinct subclones of the same tumour. We analysed our test for calibration and power. By comparing its performance to baseline methods, we demonstrate that to control type I errors, it is essential to account for the evolutionary dependencies among clones. In applying GeneAccord to the single-cell sequencing of a cohort of 123 acute myeloid leukaemia patients, we find 6 clonally exclusive and 2 clonally co-occurring gene pairs. The clonally exclusive pairs mostly involve genes of the key signalling pathways.

category: cancer biology


title: The petrosal and basicranial morphology of Protoceras celer

abstract: Protoceratids are an extinct family of endemic North American artiodactyls. The phylogenetic position of protoceratids in relation to camelids and ruminants has been contentious for over a century. The petrosal morphology of basal (Leptotragulus) and derived (Syndyoceras) protoceratids has suggested that protoceratids are closely related to ruminants, whereas a prior description of a disarticulated intermediate protoceratid petrosal (Protoceras celer) indicated that protoceratids were closely related to camelids. This contradictory evidence implied that there were several character reversals within the protoceratid lineage and brought into question the utility of basicranial characters in artiodactyl phylogenetics. Here, we provide descriptions of an additional P. celer petrosal. The descriptions are based on data produced by computed tomography scans, which allowed us to image the petrosal in situ in the skull. Our results indicate that the petrosal morphology of P. celer is similar to that of other protoceratids, implying that, contrary to previous evidence, petrosal morphology is conserved within the Protoceratidae.

category: paleontology


title: CREB regulates the expression of Type 1 Inositol 1,4,5-trisphosphate receptors

abstract:
Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) play a central role in regulating intracellular calcium signals in response to a variety of internal/external cues. Dysregulation of IP3R signaling is the underlying cause for numerous pathological conditions. It is also well established that the activity of IP3Rs is governed by several post-translational modifications including phosphorylation by protein kinase A (PKA). However, the long-term effects of PKA activation on expression of IP3R sub-types, remains largely unexplored. In this report, we investigate the effect of chronic activation of PKA on expression of IP3R sub-types. We demonstrate that expression of IP3R1 is augmented upon prolonged activation of PKA or upon ectopic over-expression of CREB but does not alter IP3R2 and IP3R3 sub-type abundance. Conversely, inhibition of PKA or blocking endogenous CREB diminished IP3R1 expression. We also demonstrate that agonist-induced Ca2+-release mediated by IP3R1 is significantly attenuated upon blocking endogenous CREB. Moreover, CREB by regulating the expression of KRAS-induced actin-interacting protein (KRAP) ensures proper localization and licensing of IP3R1. Overall, we report a crucial role for CREB in governing both the expression and proper localization of IP3R1.

Summary statementWe report a critical role of CREB in regulating the expression and proper localization of IP3R1. Agonist-induced Ca2+ release and Ca2+ puffs generated by IP3R1 are diminished upon blocking the function of endogenous CREB.

category: physiology


title: No evidence that lulworthioid fungi are dark septate endophytes in the roots of the dominant Mediterranean seagrass Posidonia oceanica

abstract:
O_LIA previous study from Sicily, Italy indicated that the dominant Mediterranean seagrass Posidonia oceanica forms a dark septate endophytic (DSE) association with a lulworthioid fungus ("Lulwoana sp."), which is in conflict with several other studies from the NW Mediterranean Sea that point at the recently described pleosporalean fungus Posidoniomyces atricolor.
C_LIO_LII collected P. oceanica roots at eight sites around Sicily and checked them for fungal colonization using light microscopy. At three sites, root fungal symbionts (=mycobionts) were isolated into pure cultures and identified using sequencing of the ITS rDNA gene.
C_LIO_LIPosidoniomyces atricolor represented the most frequent mycobiont (56 isolates), closely followed by lulworthioid fungi (51). The obtained mycobiont spectrum also comprised Cladosporium (2), Alternaria (1), Corollospora (1), Fusarium (1), Penicillium (1) and Vishniacozyma (1) isolates. The characteristic DSE root colonization similar to those occurring in terrestrial plants but not known from any other seagrass was found in all investigated P. oceanica individuals. The microscopic screening suggests that P. atricolor is indeed responsible for the observed DSE colonization.
C_LIO_LIThis study extends the known range of P. atricolor and the DSE association characteristic for P. oceanica for southern Tyrrhenian Sea/Sicily. While lulworthioid fungi regularly occur in P. oceanica tissues, including terminal fine roots, their significance and functioning are unknown and beg further investigation. However, there are currently no proofs that they belong among dark septate endophytes of this seagrass.
C_LI

One-sentence summaryThis paper corrects an opinion that "Lulwoana sp." (Lulworthiales) is a dark septate endophyte of the dominant Mediterranean seagrass Posidonia oceanica, because all available evidence suggests that the dark septate endophytic association typical for this seagrass is formed by its specific root mycobiont Posidoniomyces atricolor (Pleosporales).

category: plant biology


title: Current challenges in microbiome metadata collection

abstract: While the biomedical community has embraced data sharing (e.g. results, raw data) and supported establishment of large research consortia (e.g. the Human Microbiome Project) aimed to standardize the quality of important sets of microbiome sequencing data, the reusability of most microbiome data is still limited by the quality of its associated metadata. To ensure that microbiome data is indeed FAIR (Findable, Accessible, Interoperable, and Reusable), it is necessary to consider tools and approaches that make it easier to provide high-quality metadata that is fit for purpose moving forward. Such tools and approaches could be informed by current efforts to harmonize and improve the quality of extant microbiome metadata.

category: scientific communication and education


title: Modeling the Pancreatic Cancer Microenvironment in Search of Control Targets

abstract: Pancreatic Ductal Adenocarcinoma is among the leading causes of cancer related deaths globally due to its extreme difficulty to detect and treat. Recently, research focus has shifted to analyzing the microenvironment of pancreatic cancer to better understand its key molecular mechanisms. This microenvironment can be represented with a multi-scale model consisting of pancreatic cancer cells (PCCs), pancreatic stellate cells (PSCs), as well as cytokines and growth factors which are responsible for intercellular communication between the PCCs and PSCs. We have built a stochastic Boolean network (BN) model, validated by literature and clinical data, in which we probed for intervention strategies that force this gene regulatory network (GRN) from a diseased state to a healthy state. To do so, we implemented methods from phenotype control theory to determine a procedure for regulating specific genes within the microenvironment. We identify target genes and molecules such that the application of their control drives the GRN to the desired state by suppression (or expression) and disruption of specific signaling pathways that will eventually lead to the eradication of the cancer cells. After applying well studied control methods such as stable motifs, feedback vertex set, and computational algebra, we discovered that each produces a different set of control targets that are not necessarily minimal nor unique. Yet, we were able to gain more insight about the performance of each process and the overlap of targets discovered. Nearly every control set contains cytokines, KRas, and HER2/neu which suggests they are key players in the systems dynamics. To that end, this model can be used to produce further insight into the complex biological system of pancreatic cancer with hopes of finding new potential targets.


title: An optimized protocol for neuronal assay for transposase-accessible chromatin by sequencing (ATAC-seq) library preparation using Drosophila melanogaster

abstract: Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is rapidly becoming the assay of choice to investigate chromatin-mediated gene regulation, largely because of low input requirements, a fast workflow, and the ability to interrogate the entire genome in an unbiased manner. Many studies using ATAC-seq use mammalian or human-derived tissues, and established protocols work well in these systems. However, ATAC-seq is not yet widely used in Drosophila. Vinegar flies present several advantages over mammalian systems that make them an excellent model for ATAC-seq studies, including abundant genetic tools that allow straightforward targeting, transgene expression, and genetic manipulation, and are not available in mammalian models. Because current ATAC-seq protocols are not optimized to use flies, we developed an optimized workflow that accounts for several complicating factors present in Drosophila. We examined parameters affecting nuclei isolation, including input size, freezing time, washing, and possible confounds from retinal pigments. Then, we optimized the enzymatic steps of library construction to account for the smaller Drosophila genome size. Finally, we used our optimized protocol to generate ATAC-seq libraries that meet ENCODE quality metrics. Our optimized protocol enables extensive ATAC-seq experiments in Drosophila, thereby leveraging the advantages of this powerful model system to understand chromatin-mediated gene regulation.


title: An optimized protocol for assay for transposase-accessible chromatin by sequencing (ATAC-seq) library preparation from adult Drosophila melanogaster neurons


title: A Phylogenomic Framework for Charting the Diversity and Evolution of Giant Viruses

abstract: Large DNA viruses of the phylum Nucleocytoviricota have recently emerged as important members of ecosystems around the globe that challenge traditional views of viral complexity. Numerous members of this phylum that cannot be classified within established families have recently been reported, and there is presently a strong need for a robust phylogenomic and taxonomic framework for these viruses. Here we report a comprehensive phylogenomic analysis of the Nucleocytoviricota, present a set of giant virus orthologous groups (GVOGs) together with a benchmarked reference phylogeny, and delineate a hierarchical taxonomy within this phylum. We show that the majority of Nucleocytoviricota diversity can be partitioned into 6 orders, 32 families, and 344 genera, substantially expanding the number of currently recognized taxonomic ranks for these viruses. We integrate our results within a megataxonomy that has been adopted for all viruses to establish a unifying framework for the study of Nucleocytoviricota diversity, evolution, and environmental distribution.


title: Evaluation of wound healing effects of ginsenoside Rg1 and red ginseng extract in STZ-induced diabetic wound model: an in vivo pilot study

abstract: Red ginseng is an immune-enhancing compound that exhibits anti-inflammatory action. The ginsenoside Rg1, an ingredient of red ginseng, has been shown to play an important role in tumor suppression, wound healing, and angiogenesis. This study evaluated the effects of red ginseng extract and Rg1 in a diabetic wound model. Diabetes was induced with streptozotocin (STZ) in 8-week-old male Institute of Cancer Research (ICR) mice weighing 30-35 g. A full-thickness skin defect was treated by applying a dressing every 3 days. The mice were divided into three groups. Group 1 was administered an extract of red ginseng (10 mg/kg/d, n = 27, oral) and group 2 was administered Rg1 (10 mg/kg/d, n = 27, oral). Group 3 was a control group treated with phosphate-buffered saline (0.3 mL/kg/d, n = 27, oral). Red ginseng extract and Rg1 were orally administered to mice daily for 10 days following injury in groups 1 and 2, respectively. Both increased mRNA and protein levels of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-{beta}1 compared to controls. In addition, the wounds of animals in the Rg1 group were significantly smaller between days 7 and 10 (p < 0.05). VEGF and TGF-{beta}1 were not expressed in diabetic mice in the control group. Both red ginseng extract and Rg1 promoted the production of VEGF and TGF-{beta}1, which are important in wound healing. Our results for Rg1 suggest its potential to promote diabetic wound healing by stimulating the production or activity of VEGF and TGF-{beta}1 factors involved in the wound healing process.

category: molecular biology


title: 1H, 13C and 15N resonance assignment of the SARS-CoV-2 full-length nsp1 protein and its mutants reveals its unique secondary structure features in solution.

abstract: Structural characterization of the SARS-CoV-2 full length nsp1 protein will be an essential tool for developing new target-directed antiviral drugs against SARS-CoV-2 and for further understanding of intra- and intermolecular interactions of this protein. As a first step in the NMR studies of the protein, we report the 1H, 13C and 15N resonance backbone assignment as well as the C{beta} of the apo form of the full-lengthSARS-CoV-2 nsp1 including folded domain together with the flaking N- and C-terminal intrinsically disordered fragments. The 19.8 kD protein was characterized by high-resolution NMR. Validation of assignment have been done by using two different mutants, H81P and K129E/D48E as well as by amino acid specific experiments. According to the obtained assignment, the secondary structure of the folded domain in solution was almost identical to its previously published X-ray structure, but some discrepancies have been detected. In the solution SARS-CoV-2 nsp1 exhibited disordered, flexible N- and C-termini with different dynamic characteristics. The short peptide in the beginning of the disordered C-terminal domain adopted two different conformations distinguishable on the NMR time scale. We propose that the disordered and folded nsp1 domains are not fully independent units but are rather involved in intramolecular interactions. Studies of the structure and dynamics of the SARS-CoV-2 mutant in solution are on-going and will provide important insights into the molecular mechanisms underlying these interactions.


title: Telomeric 8-oxoguanine drives rapid premature senescence in the absence of telomere shortening

abstract: Oxidative stress is a primary cause of cellular senescence and contributes to the pathogenesis of numerous human diseases. Oxidative damage to telomeric DNA is proposed to trigger premature senescence by accelerating telomere shortening. Here we tested this model directly using a precision tool to produce the common base lesion 8-oxo-guanine (8oxoG) exclusively at telomeres in human fibroblast and epithelial cells. A single induction of telomeric 8oxoG is sufficient to trigger multiple hallmarks of p53-dependent senescence. Telomeric 8oxoG activates ATM and ATR signaling, and enriches for markers of telomere dysfunction in replicating, but not quiescent cells. Acute 8oxoG production fails to shorten telomeres, but rather generates fragile sites and delayed mitotic DNA synthesis at telomeres, indicative of impaired replication. Based on our results we propose that oxidative stress promotes rapid senescence by producing oxidative base lesions which drive replication-dependent telomere fragility and dysfunction in the absence of shortening and shelterin loss.


title: YAP/TAZ Maintain the Proliferative Capacity and Structural Organization of Radial Glial Cells During Brain Development

abstract: The Hippo pathway regulates the development and homeostasis of many tissues and in many species. It controls the activity of two paralogous transcriptional coactivators, YAP and TAZ (YAP/TAZ). Although previous studies have established that aberrant YAP/TAZ activation is detrimental to mammalian brain development, whether and how endogenous levels of YAP/TAZ activity regulate brain development remain unclear. Here, we show that during mammalian cortical development, YAP/TAZ are specifically expressed in apical neural progenitor cells known as radial glial cells (RGCs). The subcellular localization of YAP/TAZ undergoes dynamic changes as corticogenesis proceeds. YAP/TAZ are required for maintaining the proliferative potential and structural organization of RGCs, and their ablation during cortical development reduces the numbers of cortical projection neurons and causes the loss of ependymal cells, resulting in hydrocephaly. Transcriptomic analysis using sorted RGCs reveals gene expression changes in YAP/TAZ-depleted cells that correlate with mutant phenotypes. Thus, our study has uncovered essential functions of YAP/TAZ during mammalian brain development and revealed the transcriptional mechanism of their action.


title: Do tigers hunt during the day? Diel Activity of the Asian tiger mosquito, Aedes albopictus (Diptera: Culicidae), in Urban and Suburban Habitats of North America

abstract:
Aedes (Stegomyia) albopictus (Skuse) impacts human outdoor activity because of its aggressive biting behavior, and as a major vector of mosquito-borne diseases, it is also of public health importance. Although most mosquito species exhibit crepuscular activity by primarily host seeking at dawn and dusk, Ae. albopictus has been traditionally characterized as a diurnal or day-biting mosquito. With the global expansion and increased involvement of Ae. albopictus in mosquito-borne diseases, it is imperative to elucidate the diel activity of this species, particularly in newly invaded areas. Human sweep netting and carbon dioxide-baited rotator traps were used to evaluate the diel activity of Ae. albopictus in two study sites. Both trapping methods were used in New Jerseys Mercer County, USA (temperate urban), while only human sweep netting was used in Floridas Volusia County, USA (subtropical suburban). Human sweep netting was performed to determine adult mosquito activity at sunrise, solar noon, sunset, and lunar midnight. Because New Jersey is in a temperate area, diel activity was investigated during the early season (3-19 July), peak season (25 July-19 September), and late season (22 September-22 October). Aedes albopictus showed the highest activity during peak and late seasons at solar noon (P < 0.05). At sunrise and sunset during the peak season, Ae. albopictus activity was similar. Lunar midnight activity was significantly lower than sunrise and solar noon (P < 0.05) but was similar to that of sunset. In the late season, the highest activity was observed during solar noon while the least activity was observed during sunrise and lunar midnight (P<0.05). Rotator traps used in conjunction with the human sweep net technique exhibited similar results. Seasonal activity was not differentiated in Florida due to the consistent subtropical weather. The highest adult activity was observed at sunrise using human sweep netting but it was not significantly different from solar noon and sunset. The lowest adult activity was observed at lunar midnight; however, it was not significantly different from solar noon and sunset. These results provide evidence that the diel activity of Ae. albopictus, contrary to the common perception of its diurnal activity, is much more varied. Because of the involvement of the species in the transmission of debilitating mosquito-borne pathogens such as chikungunya, dengue, and Zika virus, coupled with its affinity to thrive in human peridomestic environments, our findings have global implications in areas where Ae. albopictus thrives. It also highlights the importance of behavioral studies of vector species which will not only help mosquito control professionals plan the timing of their control efforts but also provide empirical evidence against conventional wisdoms that may unjustly persist within public health stewards.

Author SummaryThe Asian tiger mosquito, Aedes albopictus, is an invasive mosquito which is now established in at least 40 states in the USA. Lack of efficient surveillance and control methods against Ae. albopictus, in addition to human-aided accidental transportations, have played a great role in its rapid expansion. Although surveillance measures are becoming more systematic and effective, control of this species still poses a great challenge. Aedes albopictus is difficult to control in the larval stage because it primarily develops in artificial containers that are widespread in peridomestic habitats. These habitats are not only ubiquitous in these environments, they are also cryptic, inaccessible, and extremely difficult to control. Therefore, control of Ae. albopictus in these environments often relies on adult control measures which utilize insecticides dispersed through ultra-low volume equipment as a cold aerosol space spray. These adulticide applications are often conducted at night against endemic mosquito species which are primarily active between dawn and dusk. However, since Ae. albopictus has been traditionally classified as a day-biting mosquito, mosquito control specialists have had doubts about the efficacy of a nocturnal application against a diurnally active mosquito. These uncertainties about intervention efforts become even more important during public health outbreaks of mosquito-borne pathogens such as chikungunya, dengue, or Zika viruses when protection of public health is of paramount importance in peridomestic habitats. Our investigations provide evidence that Ae. albopictus exhibits activity throughout the day and night and that nighttime adulticide applications may indeed be effective against this species, and should not be disregarded.


title: Visualization of ecological successions in lakes subdivided by volcanic eruption at Akan Caldera, Hokkaido, Japan

abstract: It is not feasible to continuously observe ecological succession in lakes because of the long time-scales generally involved. Thus, the process has been inductively deduced by comparing many lakes with different succession states, or indirectly simulated by tracking studies of smaller water bodies, experiments using microcosms or mesocosms, and reconstruction of lake history by sediment analysis. However, the reality of succession processes in large lakes with slow succession is not well understood, and new approaches are needed. Theoretically, in a group of large and small lakes of similar ages and with similar initial and watershed environments, the rate of nutrient accumulation in each lake depends on the ratio of watershed area to lake size, and the lakes are predicted to evolve to different trophic levels over time. Here, we tested this hypothesis on the 10 lakes of varying sizes in Akan Caldera, Japan, which were formed thousands of years ago by fragmentation due to volcanic eruptions within the caldera. Topographic and water quality studies showed that the ratio of accumulated watershed area to lake size (area and volume), expressed logarithmically, had a positive linear regression with the total phosphorus concentration, an indicator of trophic level. The trophic levels of the lakes were diverse, including oligotrophic, mesotrophic, and eutrophic types in the traditional "lake type" classification based on total phosphorus and chlorophyll-a concentrations. Furthermore, 21 species of aquatic macrophytes were observed by a diving survey, and the plant species composition was classified into five groups corresponding to the trophic status of the lakes, indicating a conventional "hydrarch succession". The diversity of water quality and aquatic vegetation in a group of lakes with similar origins paves the way for new comparative studies of lakes, including large lakes.


title: Visualization of lake succession as indicated by macrophyte species composition and trophic levels in lakes subdivided by volcanic eruption at Akan Caldera, Hokkaido, Japan


title: Structural basis of RNA polymerase recycling by the Swi2/Snf2 ATPase RapA in Escherichia coli

abstract: After transcription termination, cellular RNA polymerases (RNAPs) are occasionally trapped on DNA, impounded in an undefined Post-Termination Complex (PTC), limiting free RNAP pool and making transcription inefficient. In Escherichia coli, a Swi2/Snf2 ATPase RapA is involved in countering such inefficiency through RNAP recycling. To understand its mechanism of RNAP recycling, we have determined the cryo-electron microscopy (cryo-EM) structures of two sets of E. coli RapA-RNAP complexes along with RNAP core enzyme and elongation complex (EC). The structures revealed the large conformational changes of RNAP and RapA upon their association implicated in the hindrance in PTC formation. Our study reveals that although RapA binds away from the DNA binding channel of RNAP, it can close the RNAP clamp allosterically thereby preventing its non-specific DNA binding. Together with DNA binding assays, we propose that RapA acts as a guardian of RNAP by which prevents non-specific DNA binding of RNAP without affecting the sigma factor binding to RNAP core enzyme, thereby enhancing RNAP recycling.

category: biochemistry


title: Phylogenetic profiling in eukaryotes: The effect of species, orthologous group, and interactome selection on protein interaction prediction

abstract:
Phylogenetic profiling in eukaryotes is of continued interest to study and predict the functional relationships between proteins. This interest is likely driven by the increased number of available diverse genomes and computational methods to infer orthologies. The evaluation of phylogenetic profiles has mainly focussed on reference genome selection in prokaryotes. However, it has been proven to be challenging to obtain high prediction accuracies in eukaryotes. As part of our recent comparison of orthology inference methods for eukaryotic genomes, we observed a surprisingly high performance for predicting interacting orthologous groups. This high performance, in turn, prompted the question of what factors influence the success of phylogenetic profiling when applied to eukaryotic genomes.

Here we analyse the effect of species, orthologous group and interactome selection on protein interaction prediction using phylogenetic profiles. We select species based on the diversity and quality of the genomes and compare this supervised selection with randomly generated genome subsets. We also analyse the effect on the performance of orthologous groups defined to be in the last eukaryotic common ancestor of eukaryotes to that of orthologous groups that are not. Finally, we consider the effects of reference interactome set filtering and reference interactome species.

In agreement with other studies, we find an effect of genome selection based on quality, less of an effect based on genome diversity, but a more notable effect based on the amount of information contained within the genomes. Most importantly, we find it is not merely selecting the correct genomes that is important for high prediction performance. Other choices in meta parameters such as orthologous group selection, the reference species of the interaction set, and the quality of the interaction set have a much larger impact on the performance when predicting protein interactions using phylogenetic profiles. These findings shed light on the differences in reported performance amongst phylogenetic profiles approaches, and reveal on a more fundamental level for which types of protein interactions this method has most promise when applied to eukaryotes.


title: Controlled bio-inspired self-organised criticality

abstract:
The control of extensive complex biological systems is considered to depend on feedback mechanisms. Reduced systems modelling has been effective to describe these mechanisms, but this approach does not sufficiently encompass the required complexity that is needed to understand how localised control in a biological system can provide global stable states. Self-Organised Criticality (SOC) is a characteristic property of locally interacting physical systems which readily emerges from changes to its dynamic state due to small nonlinear perturbations. Small changes in the local states, or in local interactions, can greatly affect the total system state of critical systems. It has long been conjectured that SOC is cardinal to biological systems that show similar critical dynamics and also may exhibit near power-law relations. Rate Control of Chaos (RCC) provides a suitable robust mechanism to generate SOC systems which operates at the edge of chaos. The bio-inspired RCC method requires only local instantaneous knowledge of some of the variables of the system, and is capable of adapting to local perturbations. Importantly, connected RCC controlled oscillators can maintain global multi-stable states, and domains with power-law relations may emerge. The network of oscillators deterministically stabilises into different orbits for different perturbations and the relation between the perturbation and amplitude can show exponential and power-law correlations. This is representative of a basic mechanism of protein production and control, that underlies complex processes such as homeostasis. Providing feedback from the global state, the total system dynamic behaviour can be boosted or reduced. Controlled SOC can provide much greater understanding of biological control mechanisms, that are based on distributed local producers, remote consumers of biological resources, with globally defined control.

Author summaryUsing a nonlinear control method inspired by enzymatic control, which is capable of stabilising chaotic systems into periodic orbits or steady-states, it is shown that a controlled system can be created that is scale-free and in a critical state. This means that the system can easily move from one stable orbit to another using only a small local perturbation. Such a system is known as self-organised criticality, and is shown in this system to be deterministic. Using a known perturbation, it will result in a scale-free response of the system that can be in a power law relation. It has been conjectured that biosystems are in a self-organised critical state, and these models show that this is a suitable approach to allow local systems to control a global state, such as homeostatic control. The underlying principle is based on rate control of chaos, and can be used to understand how biosystems can use localised control to ensure stability at different dynamic scales without supervising mechanisms.


title: A lipid-based parallel processor for chemical signals

abstract: A key goal of bottom-up synthetic biology is to construct cell- and tissue-like structures. Underpinning cellular life is the ability to process several external chemical signals, often in parallel. Until now, however, cell- and tissue-like structures have only been constructed with one signalling pathway. Here, we construct a dual-signal processor from the bottom up in a modular fashion. The processor comprises three aqueous compartments bounded by lipid bilayers and operates in an aqueous environment. It can receive two chemical signals from the external environment, process them orthogonally, and then produce a distinct output for each signal. It is suitable for both sensing and enzymatic processing of environmental signals with fluorescence and molecular outputs. In the future, such processors could serve as smart drug delivery vehicles or as modules within synthetic tissues to control their behaviour in response to external chemical signals.

category: synthetic biology


title: Flexible neural control of motor units

abstract: Voluntary movement requires communication from cortex to the spinal cord, where a dedicated pool of motor units (MUs) activates each muscle. The canonical description of MU function rests upon two foundational tenets. First, cortex cannot control MUs independently but supplies each pool with a common drive. Second, MUs are recruited in a rigid fashion that largely accords with Hennemans size principle. While this paradigm has considerable empirical support, a direct test requires simultaneous observations of many MUs across diverse force profiles. We developed an isometric task that allowed stable MU recordings even during rapidly changing forces. MU activity patterns were surprisingly behavior-dependent. MU activity could not be accurately described as reflecting common drive, but could be captured by assuming multiple drives. Neuropixels probe recordings revealed that, consistent with the requirements of flexible control, the motor cortex population response displays a great many degrees of freedom. Neighboring cortical sites recruited different MUs. Thus, MU activity is flexibly controlled to meet task demands, and cortex may contribute to this ability.


title: Simultaneous dimensionality reduction and integration for single-cell ATAC-seq data using deep learning

abstract: Advances in single-cell technologies enable the routine interrogation of chromatin accessibility for tens of thousands of single cells, shedding light on gene regulatory processes at an unprecedented resolution. Meanwhile, size, sparsity and high dimensionality of the resulting data continue to pose challenges for its computational analysis, and specifically the integration of data from different sources. We have developed a dedicated computational approach, a variational auto-encoder using a noise model specifically designed for single-cell ATAC-seq data, which facilitates simultaneous dimensionality reduction and batch correction via an adversarial learning strategy. We showcase both its individual advantages on carefully chosen real and simulated data sets, as well as the benefits for detailed cell type characterization via integrating multiple complex datasets.


title: ontoFAST: An R package for interactive and semi-automatic annotation of characters with biological ontologies

abstract:
O_LIThe commonly used Entity-Quality (EQ) syntax provides rich semantics and high granularity for annotating phenotypes and characters using ontologies. However, EQ syntax might be time inefficient if this granularity is unnecessary for downstream analysis.
C_LIO_LIWe present an R package ontoFAST that aid production of fast annotations of characters and character matrices with biological ontologies. Its interactive interface allows quick and convenient tagging of character statements with necessary ontology terms.
C_LIO_LIThe annotations produced in ontoFAST can be exported in csv format for downstream analysis. Additinally, OntoFAST provides: (i) functions for constructing simple queries of characters against ontologies, and (ii) helper function for exporting and visualising complex ontological hierarchies and their relationships.
C_LIO_LIOntoFAST enhances data interoperability between various applications and support further integration of ontological and phylogenetic methods. Ontology tools are underrepresented in R environment and we hope that onto-FAST will stimulate their further development.
C_LI


title: Termites have developed wider thermal limits to cope with environmental conditions in savannas

abstract: The most diverse and abundant family of termites, the Termitidae, evolved in warm, wet African tropical forests. Since then, they have colonised grassy biomes such as savannas. These environments have more extreme temperatures than tropical forests, and greater temporal fluctuations (both annually and diurnally) that are challenging for soft-bodied ectotherms. We propose that that a likely mechanism that facilitated the expansion from forest to savanna was the widening of physiological limits of savanna termite species in order to cope with more extreme environmental conditions. We sampled termites directly from mound structures across an environmental gradient in Ghana, and recorded the thermal tolerance of individual termites, both critical thermal maximum (CTmax) and critical thermal minimum (CTmin). We estimated colony thermal tolerance by taking an average of each tested individual, and modelled these data against several environmental factors (canopy cover above the mound, rainfall and temperature). We found that savanna termite species had significantly higher CTmax values, and significantly lower CTmin values, than forest species. In addition, areas with high canopy cover were significantly associated with low CTmax values, and areas with higher average daily rainfall were significantly associated with higher CTmin values. Our results suggest that the widening of thermal tolerances has occurred in savanna termite species, probably in response to the more extreme temperatures found in those environments.


title: LNetReduce: tool for reducing linear dynamicnetworks with separated time scales

abstract:
We introduce LNetReduce, a tool that simplifies linear dynamic networks. Dynamic networks are represented as digraphs labeled by integer timescale orders. Such models describe deterministic or stochastic monomolecular chemical reaction networks, but also random walks on weighted protein-protein interaction networks, spreading of infectious diseases and opinion in social networks, communication in computer networks. The reduced network is obtained by graph and label rewriting rules and reproduces the full network dynamics with good approximation at all time scales. The tool is implemented in Python with a graphical user interface. We discuss applications of LNetReduce to network design and to the study of the fundamental relation between time scales and topology in complex dynamic networks.

Availabilitythe code and application examples are available at https://github.com/oradules/LNetReduce.

category: systems biology


title: Segregation and integration of the functional connectome in neurodevelopmentally 'at risk' children

abstract:
Functional connectivity within and between Intrinsic Connectivity Networks (ICNs) transforms over development and supports high order cognitive functions. But how variable is this process, and does it diverge with altered cognitive developmental trajectories? We investigated age-related changes in integration and segregation within and between ICNs in neurodevelopmentally  at-risk children, identified by practitioners as experiencing cognitive difficulties in attention, learning, language, or memory. In our analysis we used performance on a battery of 10 cognitive tasks, alongside resting-state functional Magnetic Resonance Imaging in 175 at-risk children and 62 comparison children aged 5-16. We observed significant age-by-group interactions in functional connectivity between two network pairs. Integration between the ventral attention and visual networks and segregation of the limbic and fronto-parietal networks increased with age in our comparison sample, relative to at-risk children. Furthermore, functional connectivity between the ventral attention and visual networks in comparison children significantly mediated age-related improvements in executive function, compared to at-risk children. We conclude that integration between ICNs show divergent neurodevelopmental trends in the broad population of children experiencing cognitive difficulties, and that these differences in functional brain organisation may partly explain the pervasive cognitive difficulties within this group over childhood and adolescence.

Research HighlightsO_LIWe investigated functional brain organisation and its development in 175 children who experience neurodevelopmental difficulties in cognition and behaviour, relative to a comparison sample (n=62)
C_LIO_LIWe replicated common neurodevelopmental trends across the samples: functional connectivity increased within Intrinsic Connectivity Networks and the default-mode network increasingly segregated with age
C_LIO_LINeurodevelopmentally at-risk children also showed different age-related changes in functional connectivity between the ventral attention and visual networks and between the fronto-parietal and limbic networks
C_LIO_LIFurthermore, the integration between the ventral attention and visual networks in comparison children mediated age-related changes in cognition, relative to at-risk children
C_LI


title: Distinct interhemispheric connectivity at the level of the olfactory bulb emerges during Xenopus laevis metamorphosis

abstract:
The olfactory system of anuran tadpoles requires substantial restructuring to adapt to the lifestyle of the adult frogs. Xenopus laevis tadpoles have a single main olfactory epithelium in the principal nasal cavity associated with aquatic olfaction. After metamorphosis, this epithelial surface is transformed into the adult air-nose and a new epithelium, the adult water-nose, is present in the middle cavity. Impacts of this massive remodeling on odor processing, behavior and network structure are still unexplored.

In the present study, we used neuronal tracings, calcium imaging and a behavioral assay to examine the functional connectivity between the epithelium and the main olfactory bulb during metamorphosis. In tadpoles, olfactory receptor neurons in the principal cavity epithelium project axons to glomeruli in the ventral main olfactory bulb. During metamorphosis, these projections are gradually replaced by receptor neuron axons emerging from the newly forming middle cavity epithelium. Despite this metamorphotic reorganization in the ventral bulb, two spatially and functionally segregated odor processing streams remain undisrupted. In line with this, metamorphotic rewiring does not alter behavioral responses to waterborne odorants. Contemporaneously, newly formed receptor neurons in the remodeling principal cavity epithelium project their axons to the dorsal part of the bulb. The emerging neuronal networks of the dorsal and ventral main olfactory bulb show substantial differences. Glomeruli around the midline of the dorsal bulb are innervated from the left and right nasal epithelia. In addition, postsynaptic projection neurons in the dorsal bulb predominantly have smaller tufts and connect to multiple glomeruli, while more than half of projection neurons in the ventral bulb have a single, bigger tuft.

Our results show that during the metamorphotic reconstruction of the olfactory network the  water system remains functional. Differences of the neuronal network of the dorsal and ventral olfactory bulb imply that a higher degree of odor integration takes place in the dorsal main olfactory bulb. This is likely connected with the processing of different odorants, airborne vs. waterborne, in these two parts of the olfactory bulb.


title: No pervasive size trend in global community dynamics.

abstract: While recent meta-analyses have suggested that local taxonomic richness has on average remained invariant, potential shifts in functional traits remain underexplored at global scales. Here, by linking the largest cross-taxa community time series database to multiple trait databases, we show that within communities there is no clear trend between size traits and changes in abundance rank over time. This suggests that there is no pervasive tendency across biomes for larger species to be doing proportionally better or worse than smaller species.


title: Simultaneous Morphology, Motility and Fragmentation Analysis of Live Individual Sperm Cells for Male Fertility Evaluation

abstract: We present a new technique for sperm analysis on individual unstained live cells, measuring DNA fragmentation, morphology with virtual staining, and motility. The method relies on quantitative stain-free interferometric imaging and deep-learning frameworks, and is utilized for male fertility evaluation. In the common clinical practice, only motility evaluation is carried out on live human cells, while full morphological evaluation and DNA fragmentation assays require different staining protocols, and therefore cannot be performed on the same cell, resulting in inconsistencies in fertility evaluation. We use a clinic-ready interferometric module to acquire dynamic sperm cells without chemical staining, and deep learning to evaluate all three scores per cell. We show that the number of cells that pass each criterion separately does not accurately predict how many would pass all criteria. This stain-free evaluation method is expected to decrease uncertainty in infertility diagnosis, increasing treatment success rates.

category: bioengineering


title: Intracellular Vesicle Entrapment of Nanobubble Ultrasound Contrast Agents Targeted to PSMA Promotes Prolonged Enhancement and Stability In Vivo and In Vitro

abstract: Previous work has shown that active targeting of nanobubble (NB) ultrasound contrast agents to the prostate-specific membrane antigen (PSMA) significantly prolongs ultrasound signal enhancement in PSMA-expressing prostate cancer. However, the specific mechanism behind this effect is not well understood. Furthermore, prior studies were carried out using clinical ultrasound scanners in a single imaging plane. Because tumor heterogeneity can have a drastic effect on bubble kinetics and resulting contrast enhancement, a single region of interest in one imaging plane over time may not fully represent the contrast dynamics of the entire tumor. Accordingly, in the current work, we used high-frequency dynamic parametric contrast-enhanced ultrasound (DCE-US) imaging to gain a detailed understanding of NB kinetics in prostate tumors in mice. Specifically, we examined the differences in enhancement between the tumor periphery and tumor core in the same imaging plane. We also quantified intact nanobubble retention in the entire tumor volume. To better understand the mechanism behind prolonged tumor enhancement, intracellular retention and the acoustic activity of PSMA-NB were evaluated in cell culture. DCE-US US data suggest that both tumor wash-in and retention of PSMA-NB are delayed due to biomarker interaction and binding. The longer retention of PSMA-NB signal in tumor core supported target-driven bubble extravasation. In vitro studies demonstrated a higher level of internalization and prolonged-acoustic activity of internalized PSMA-NB. GC/MS analysis confirmed gas persistence in the cells after PSMA-NB internalization. The active-targeting of NB results in cellular internalization via receptor-mediated endocytosis, and the location with intracellular vesicles (late-stage endosomes/lysosomes) significantly prolongs gas retention within the cells. These features can enable background-free diagnostic imaging of the target cells/tissues, as well as highly focused ultrasound-modulated therapeutic interventions.


title: Vascular tropism models of blood-borne microbial dissemination

abstract: Similar to circulating tumour and immune cells, many blood-borne microbes preferentially "home" to specific vascular sites and tissues during hematogenous dissemination 1-5. For many pathogens, the "postal codes" and mechanisms responsible for tissue-specific vascular tropism are unknown and have been challenging to unravel. Members of the Lyme disease Borreliella burgdorferi species complex infect a broad range of mammalian tissues and exhibit complex strain-, species- and host-specific tissue tropism patterns. Intravenous perfusion experiments and intravital microscopy studies suggest that heterogeneous tissue tropism properties may depend on tissue-specific differences in host and microbial molecules supporting vascular interaction and extravasation. However, interpreting these studies can be complicated because of the immune-protective moonlighting (multitasking) properties of many B. burgdorferi adhesins. Here, we investigated whether B. burgdorferi vascular interaction properties measured by live cell imaging and particle tracking in aorta, bladder, brain, joint and skin microvascular flow chamber models predict strain- and tissue-specific dissemination patterns in vivo These studies identified strain- and endothelial cell type-specific interaction properties that accurately predicted in vivo dissemination of B. burgdorferi to bladder, brain, joint and skin but not aorta, and indicated that dissemination mechanisms in all of these tissues are distinct. Thus, the ability to interact with vascular surfaces under physiological shear stress is a key determinant of tissue-specific tropism for Lyme disease bacteria. The methods and model systems reported here will be invaluable for identifying and characterizing the diverse, largely undefined molecules and mechanisms supporting dissemination of Lyme disease bacteria. These methods and models may be useful for studying tissue tropism and vascular dissemination mechanisms of other blood-borne microbes.

category: microbiology


title: Mechanistic Insights of High Temperature-Interfered Meiosis in Autotetraploid Arabidopsis thaliana

abstract: Environmental temperature has a huge impact on multiple meiosis processes in flowering plants. Polyploid plants derived from whole genome duplication are believed to have an enhanced abiotic stress tolerance. In this study, the impact of high temperatures on male meiosis in autotetraploid Arabidopsis thaliana was investigated. We found that autotetraploid Columbia (Col-0) plants generate a subpopulation of aberrant meiotic products under normal temperature, which is significantly increased under heat stress. Cytological studies revealed that, as the case in diploid Arabidopsis thaliana, assembly of microtubular cytoskeleton network, pairing and segregation of homologous chromosomes, and meiotic recombination in autotetraploid Arabidopsis are compromised under the high temperatures. Immunostaining of {ramshorn}H2A.X and recombinase DMC1 suggested that heat stress inhibits formation of DNA double-strand breaks; additionally, it specifically destabilizes ASY1 and ASY4, but not SYN1 on chromosomes. The loading defects of ASY1 and ASY4 overlap in the syn1 mutant, which supports that the building of lateral element of synaptonemal complex occurs downstream of a SYN1-ASY4-ASY3 stepwise assembly of axis. Remarkably, heat-induced abnormalities of ASY1 and ASY4 co-localize on chromosomes of both diploid and autotetraploid Arabidopsis, suggesting that high temperatures interfere with ASY1-associated SC via an impacted stability of chromosome axis. Furthermore, ZYP1-dependent transverse filament of SC is disrupted by heat stress. Taken together, these findings suggest that polyploidization negatively contributes to instability of chromosome axis and meiotic recombination in Arabidopsis thaliana under heat stress.


title: Genetic Basis of Variation in Ubiquitin-Proteasome System Activity

abstract: Ubiquitin-proteasome system (UPS) protein degradation regulates protein abundance and eliminates mis-folded and damaged proteins from eukaryotic cells. Variation in UPS activity influences numerous cellular and organismal phenotypes. However, to what extent such variation results from individual genetic differences is almost entirely unknown. Here, we developed a statistically powerful mapping approach to characterize the genetic basis of variation in UPS activity. Using the yeast Saccharomyces cerevisiae, we systematically mapped genetic influences on the N-end rule, a UPS pathway that recognizes N-degrons, degradation-promoting signals in protein N-termini. We identified 149 genomic loci that influence UPS activity across the complete set of N-degrons. Resolving four loci to individual causal nucleotides identified regulatory and missense variants in ubiquitin system genes whose products process (NTA1), recognize (UBR1 and DOA10), and ubiquitinate (UBC6) cellular proteins. Each of these genes contained multiple causal variants and several individual variants had substrate-specific effects on UPS activity. A cis-acting promoter variant that modulates UPS activity by altering UBR1 expression also alters the abundance of 36 proteins without affecting levels of the corresponding mRNAs. Our results demonstrate that natural genetic variation shapes the full sequence of molecular events in protein ubiquitination and implicate genetic influences on the UPS as a prominent source of post-translational variation in gene expression.

category: genetics


title: Variation in Ubiquitin System Genes Creates Substrate-Specific Effects on Proteasomal Protein Degradation


title: Covalent Flexible Peptide Docking in Rosetta

abstract: Electrophilic peptides that form an irreversible covalent bond with their target have great potential for binding targets that have been previously considered undruggable. However, the discovery of such peptides remains a challenge. Here, we present CovPepDock, a computational pipeline for peptide docking that incorporates covalent binding between the peptide and a receptor cysteine. We applied CovPepDock retrospectively to a dataset of 115 disulfide-bound peptides and a dataset of 54 electrophilic peptides, for which it produced a top-five scoring, near-native model, in 89% and 100% of the cases, respectively. In addition, we developed a protocol for designing electrophilic peptide binders based on known non-covalent binders or protein-protein interfaces. We identified 7,154 peptide candidates in the PDB for application of this protocol. As a proof-of-concept we validated the protocol on the non-covalent complex of 14-3-3{sigma} and YAP1 phosphopeptide. The protocol identified seven highly potent and selective irreversible peptide binders. The predicted binding mode of one of the peptides was validated using X-ray crystallography. This case-study demonstrates the utility and impact of CovPepDock. It suggests that many new electrophilic peptide binders can be rapidly discovered, with significant potential as therapeutic molecules and chemical probes.


title: The dynamic interplay of PIP2 and ATP in the regulation of the KATP channel

abstract: ATP-sensitive potassium (KATP) channels couple the intracellular ATP concentration to insulin secretion. KATP channel activity is inhibited by ATP binding to the Kir6.2 tetramer and activated by phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we use molecular dynamics (MD) simulation, electrophysiology and fluorescence spectroscopy to show that ATP and PIP2 occupy different binding pockets that share a single amino acid residue, K39. When both ligands are present, K39 shows a greater preference to co-ordinate with PIP2 than ATP. A neonatal diabetes mutation at K39 (K39R) increases the number of hydrogen bonds formed between K39 and PIP2, reducing ATP inhibition. We also find direct effects on nucleotide binding from mutating E179, a residue proposed to interact with PIP2. Our work suggests PIP2 and ATP interact allosterically to regulate KATP channel activity.


title: Exploitation of sulfated glycosaminoglycan status for precision medicine of platinums in triple-negative breast cancer

abstract:
Triple negative breast cancer (TNBC) is a subtype of breast cancer lacking targetable biomarkers. TNBC is known to be most aggressive, and when metastatic is often drug resistant and uncurable. Biomarkers predicting response to therapy improve treatment decisions and allow personalized approaches for TNBC patients. This study explores sulfated glycosaminoglycan (sGAG) levels as a predictor of TNBC response to platinum therapy. sGAG levels were quantified in three distinct TNBC tumor models including cell line-derived, patient-derived xenograft (PDX) tumors, and isogenic models deficient in sGAG biosynthesis. The in vivo antitumor efficacy of Triplatin, a sGAG-directed platinum agent, was compared in these models to the clinical platinum agent, carboplatin. We determined that >40% of TNBC PDX tissue microarray samples have high levels of sGAGs. The in vivo accumulation of Triplatin in tumors as well as antitumor efficacy of Triplatin positively correlated with sGAG levels on tumor cells, whereas carboplatin followed the opposite trend. In carboplatin-resistant tumor models expressing high levels of sGAGs, Triplatin decreased primary tumor growth, reduced lung metastases and inhibited metastatic growth in lungs, liver, and ovaries. sGAG levels served as a predictor of Triplatin sensitivity in TNBC. Triplatin may be particularly beneficial in treating patients with chemotherapy-resistant tumors who have evidence of residual disease after standard neoadjuvant chemotherapy. More effective neoadjuvant and adjuvant treatment will likely improve clinical outcome of TNBC.

SignificanceTNBC is a heterogenous disease, often defined by the absence of a therapeutic target. Our recent results show sGAGs may provide a viable biomarker for Triplatin in patients with TNBC, producing a significant advantage over carboplatin in this setting. Selective precision medicine agents, such as Triplatin, that are active against chemotherapy-resistant disease and exploit molecular biomarkers like sGAGs may significantly benefit patients in this setting.

category: pharmacology and toxicology


title: Changes of the biophysical properties of voltage-gated Na+ currents during maturation of the sodium-taste cells in rat fungiform papillae

abstract:
Taste cells are a heterogeneous population of sensory receptors that undergoes a continuous turnover. Different chemo-sensitive cell lines rely on action potentials to release the neurotransmitter onto nerve endings. The electrical excitability is due to the presence of a tetrodotoxin-sensitive, voltage-gated sodium current (INa) similar to that found in neurons. Since the biophysical properties of INa change during neuronal development, we wondered whether the same also occurred in taste cells. Here, we used the patch-clamp recording technique to study INa in sodium sensing cells of rat fungiform papillae. We identified these cells by exploiting the known blocking effect of amiloride on ENaC, the sodium (salt) receptor. Then, based on the amplitude of INa and a morphological analysis, we subdivided sodium cells into two broad developmental groups, namely immature and mature cells. We found that: the voltage dependence of activation and inactivation changed in the transition from immature to mature state (depolarizing shift); the membrane capacitance significantly decreased in mature cells, enhancing the density of INa; a persistent sodium current, absent in immature cells, appeared in mature cells. mRNA expression analysis of the -subunits of voltage-gated sodium channels in fungiform taste buds supported the electrophysiological data. As a whole, our findings provide evidence for a noticeable change in membrane excitability during development, which is consistent with the key role played by electrical signaling in the release of neurotransmitter by mature sodium cells.

Key Points SummaryO_LITaste cells are sensory receptors that undergo continuous turnover while they detect food chemicals and communicate with afferent nerve fibers.
C_LIO_LIThe voltage-gated sodium current (INa) is a key ion current for generating action potentials in fully differentiated and chemo-sensitive taste cells, which use electrical signaling to release neurotransmitters.
C_LIO_LIHere we report that in rat taste cells involved in salt detection, the properties of INa, such as voltage dependence of activation and inactivation, undergo significant changes during the transition from immature to mature state.
C_LIO_LIOur results help understand how taste cells gain electrical excitability during turnover, a property critical to operate as chemical detectors that relay sensory information to nerve fibers.
C_LI

category: neuroscience