title: Assessment of genome packaging in AAVs using Orbitrap-based charge detection mass spectrometry

abstract: Adeno-associated viruses (AAV) represent important gene therapy vectors with several approved clinical applications and numerous more in clinical trials. Genome packaging is an essential step in the bioprocessing of AAVs and needs to be tightly monitored to ensure the proper delivery of transgenes and the production of effective drugs. Current methods to monitor genome packaging have limited sensitivity, a high demand on labour, and struggle to distinguish between packaging of the intended genome or unwanted side-products. Here we show that Orbitrap based charge detection mass spectrometry allows the ultra-sensitive quantification of all these different AAV bioprocessing products. A protocol is presented that allows the quantification of genome packed AAV preparations in under half an hour, requiring only micro-liter quantities of typical AAV preparations with ~1013 viral genome copies per millilitre. The method quickly assesses the integrity and amount of genome packed AAV particles to support AAV bioprocessing and characterization of this rapidly emerging class of advanced drug therapies.


title: Molecular Organization of the Early Stages of Nucleosome Phase Separation Visualized by Cryo-Electron Tomography

abstract: It has been proposed that the intrinsic property of nucleosome arrays to undergo liquid-liquid phase separation (LLPS) in vitro is responsible for chromatin domain organization in vivo. However, understanding nucleosomal LLPS has been hindered by the challenge to characterize the structure of resulting heterogeneous condensates. We used cryo-electron tomography and deep learning-based 3D reconstruction/segmentation to determine the molecular organization of condensates at various stages of LLPS. We show that nucleosomal LLPS involves a two-step process: a spinodal decomposition process yielding irregular condensates, followed by their unfavorable conversion into more compact, spherical nuclei that grow into larger spherical aggregates through accretion of spinodal material or by fusion with other spherical condensates. Histone H1 catalyzes more than 10-fold the spinodal-to-spherical conversion. We propose that this transition involves exposure of nucleosome hydrophobic surfaces resulting in modified inter-nucleosome interactions. These results suggest a physical mechanism by which chromatin may transition from interphase to metaphase structures.

category: biophysics


title: A technique for resolution assessment in blind-SIM experiments

abstract: Super resolution techniques are an excellent alternative to wide field microscopy, providing high resolution also in (typically fragile) biological sample. Among the various super resolution techniques, Structured Illumination Microscopy (SIM) improve resolution by employing multiple illumination patterns to be deconvolved with a dedicated software. In the case of blind SIM techniques, unknown patterns, such as speckles, are used, thus providing super resolved images, nearly unaffected by aberrations with a simplified experimental setup. Scattering Assisted Imaging, a special blind SIM technique, exploits an illumination PSF (speckle grains size), smaller than the collection PSF (defined by the collection objectives), to surpass the typical SIM resolution enhancement. However, if SAI is used, it is very difficult to extract the resolution enhancement from a priori considerations. In this paper we propose a protocol and experimental setup for the resolution measurement, demonstrating the resolution enhancement for different collection PSF values.


title: The ultrasound guide: katydid ear pinnae code for bat call detection

abstract: Early predator detection is a key component of the predator-prey arms race, and has driven the evolution of multiple animal hearing systems. Katydids (Insecta) have sophisticated ears, each consisting of paired tympana on each foreleg that receive sound directly externally, and internally via a narrowing ear canal through the acoustic spiracle. These ears are pressure-time difference receivers capable of sensitive and accurate directional hearing across a wide frequency range, despite the small size of katydids. Many katydid species have cuticular pinnae which form cavities around the outer tympanal surfaces, but their function is unknown. We investigated pinnal function in the katydid Copiphora gorgonensis by combining experimental biophysics and numerical modelling using 3D ear geometries. Results show that the pinnae in C. gorgonensis do not assist in directional hearing for specific call frequencies, but instead pinnae act as ultrasound detector devices. Pinnae induced large sound pressure gains that enhanced sound detection at high ultrasonic frequencies (> 60 kHz), matching the echolocation range of co-occurring insectivorous bats. Comparing pinnal mechanics of sympatric katydid species supports these findings, and suggests that pinnae evolved in katydids primarily for enhanced predator detection. Audiograms (both behavioural and neural) and tympanal cavity resonances obtained from living specimens corroborate our findings.


title: Ear pinnae in a neotropical katydid function as ultrasound guides for bat call detection


title: Ear pinnae in a neotropical katydid (Orthoptera: Tettigoniidae) function as ultrasound guides for bat detection


title: Horizontal mitochondrial transfer from the microenvironment increases glioblastoma tumorigenicity

abstract:
While dynamic microenvironmental interactions drive tumor growth and therapeutic resistance, the underlying direct cell-cell communication mechanisms remain poorly understood. We identified horizontal mitochondrial transfer as a mechanism that enhances tumorigenesis in glioblastoma. This transfer occurs primarily from brain-resident cells, including astrocytes, and can be appreciated in vitro and in vivo through intercellular connections between GBM cells and non-malignant host cells. The acquisition of astrocyte mitochondria drives an overall enhancement of mitochondrial membrane potential and metabolic capacity, while increasing glioblastoma cell self-renewal and tumor-initiating capacity. Collectively, our findings demonstrate that mitochondrial transfer augments the tumorigenic capacity of glioblastoma cells and reveals a previously unknown cell-cell communication mechanism that drives tumor growth.

One-Sentence SummaryMitochondrial transfer from astrocytes to glioblastoma alters metabolic profile and enhances the tumor-initiation capacity.

category: cancer biology


title: Biosynthesis of linear protein nanoarrays using the flagellar axoneme

abstract: Applications in biotechnology and synthetic biology often make use of soluble proteins, but there are many potential advantages to anchoring enzymes to a stable substrate, including stability and the possibility for substrate channeling. To avoid the necessity of protein purification and chemical immobilization, there has been growing interest in bio-assembly of protein-containing nanoparticles, exploiting the self-assembly of viral capsid proteins or other proteins that form polyhedral structures. But these nanoparticle are limited in size which constrains the packaging and the accessibility of the proteins. The axoneme, the insoluble protein core of the eukaryotic flagellum or cilium, is a highly ordered protein structure that can be several microns in length, orders of magnitude larger than other types of nanoparticles. We show that when proteins of interest are fused to specific axonemal proteins and expressed in living cells, they become incorporated into linear arrays which have the advantages of high protein loading capacity, high stability, and single-step purification with retention of biomass. The arrays can be isolated as membrane enclosed vesicle or as exposed protein arrays. The approach is demonstrated for both fluorescent proteins and enzymes, and in the latter case it is found that incorporation into axoneme arrays provides increased stability for the enzyme.

category: synthetic biology


title: Mechanism of innate immune reprogramming by a fungal meningitis pathogen

abstract:
How deadly fungal pathogens overcome mammalian innate immunity is largely unknown. Cryptococcus neoformans, the most common cause of fungal meningitis, induces a pathogenic type 2 response characterized by pulmonary eosinophilia and alternatively activated macrophages. Using forward genetics, we identified a fungal secreted protein, Cpl1, necessary and sufficient to enhance alternative activation of primary macrophages in vitro. Cpl1-enhanced polarization requires Toll-like receptor 4, a known mediator of allergen-induced type 2 responses. Cpl1 is essential for virulence, drives polarization of interstitial macrophages in vivo, and requires type 2 cytokine signaling for its impact on infectivity. C. neoformans selectively associates with polarized interstitial macrophages during infection, supporting a direct hostpathogen interaction. This work identifies a secreted effector produced by a human fungal pathogen that reprograms innate immunity to enable tissue infection.

One sentence summaryIdentification of a secreted fungal effector that promotes virulence by enhancing type 2 inflammation

category: immunology


title: Human IL-10-producing B cells have diverse states induced from multiple B cell subsets

abstract: Regulatory B cells (Bregs) can suppress immune responses through the secretion of IL-10 and other anti-inflammatory cytokines. This immunomodulatory capacity holds therapeutic potential, yet a definitional immunophenotype for enumeration and prospective isolation of B cells capable of IL-10 production remains elusive. We therefore applied mass cytometry to simultaneously quantify cytokine production and immunophenotype in human peripheral B cells across a range of stimulatory conditions and timepoints. While multiple B cell subsets produced IL-10, no phenotype uniquely identified IL-10+ B cells and a significant portion of IL-10+ B cells co-expressed the proinflammatory cytokines IL-6 and TNF. Despite this heterogeneity, we found operationally tolerant liver transplant recipients had a unique enrichment of IL-10+, but not TNF+ or IL-6+, B cells as compared to transplant recipients receiving immunosuppression. Thus, human IL-10-producing B cells constitute an induced, transient state arising from a diversity of B cell subsets that may contribute to maintenance of immune homeostasis.


title: Screening and Identification of Lujo Virus Entry Inhibitors from an FDA-Approved Drugs Library

abstract:
The Lujo virus (LUJV) belongs to the Old World (OW) genus Mammarenavirus (family Arenaviridae); it is categorized as a biosafety level (BSL) 4 agent. Currently, there are no U.S. Food and Drug Administration (FDA)-approved drugs or vaccines specifically for LUJV or other pathogenic OW mammarenaviruses. Here, a high-throughput screening of an FDA-approved drug library was conducted using pseudotype viruses bearing LUJV envelope glycoprotein (GPC) to identify inhibitors of LUJV entry. Three hit compounds, trametinib, manidipine, and lercanidipine, were identified as LUJV entry inhibitors in the micromolar range. Mechanistic studies revealed that trametinib inhibited LUJV GPC-mediated membrane fusion by targeting C410 (located in the transmembrane (TM) domain), while manidipine and lercanidipine inhibited LUJV entry by acting as calcium channel blockers. Meanwhile, all three hits extended their antiviral spectra to the entry of other pathogenic mammarenaviruses. Furthermore, all three could inhibit the authentic prototype mammarenavirus, lymphocytic choriomeningitis virus (LCMV), and could prevent infection at the micromolar level. This study shows that trametinib, manidipine, and lercanidipine are candidates for LUJV therapy, and highlights the critical role of calcium in LUJV infection. The presented findings reinforce the notion that the key residue(s) located in the TM domain of GPC provide an entry-targeted platform for designing mammarenavirus inhibitors.

IMPORTANCETo date, only one LUJV outbreak has been recorded; it occurred in 2008 and resulted in a fatality rate of 80% (4/5 cases). Pathogenesis studies and therapeutic strategies are therefore urgently needed. Repurposing approved drugs can accelerate the development of drug design and facilitate the understanding of infectious mechanisms. Here, three compounds, trametinib, manidipine, and lercanidipine, were identified as entry inhibitors against LUJV. Studying the underling mechanisms revealed that a key residue (C410) in LUJV GPC modulates its sensitivity/resistance to trametinib and demonstrated the critical role of calcium in LUJV infection.

category: microbiology


title: Wolbachia strain wAu differs in cellular perturbation and virus inhibition profiles from previously characterised Wolbachia strains

abstract:
Some strains of the inherited bacterium Wolbachia have been shown to be effective at reducing the transmission of dengue and other positive-sense RNA viruses by Aedes aegypti in both laboratory and field settings and are being deployed for dengue control. The degree of virus inhibition varies between Wolbachia strains; density and tissue tropism can contribute to these differences but there are also indications that this is not the only factor involved: for example, strains wAu and wAlbA are maintained at similar densities but only wAu produces strong dengue inhibition. We previously reported perturbations in lipid transport dynamics, including sequestration of cholesterol in lipid droplets, with strains wMel / wMelPop in Ae. aegypti. Here we show that strain wAu does not produce the same cholesterol sequestration phenotype despite displaying strong virus inhibition and moreover, in contrast to wMel, wAu antiviral activity was not rescued by cyclodextrin treatment. To further investigate the cellular basis underlying these differences, proteomic analysis of midguts was carried out on Ae. aegypti lines and revealed that wAu-carrying midguts showed a distinct proteome when compared to Wolbachia-free, wMel- or wAlbA-carrying midguts, in particular with respect to lipid transport and metabolism. The data suggest a possible role for perturbed RNA processing pathways in wAu virus inhibition. Together these results indicate that wAu shows unique features in its inhibition of arboviruses compared to previously characterized Wolbachia strains.

Author SummaryWolbachia endosymbionts can block transmission of dengue virus by Aedes aegypti mosquitoes, and Wolbachia release programs for dengue control are now being undertaken in several countries. Understanding the mechanisms of Wolbachia-mediated antiviral activity is important for maximizing the efficacy of this control approach. Using functional and proteomic analyses, this study indicates that different strains of Wolbachia perturb cellular functions in diverse ways and display different antiviral profiles. These differences raise the possibility that Wolbachia strain switching could be used to counteract viral escape mutations, should they arise and threaten the efficacy of dengue control programmes.


title: Nutrient availability shifts the biosynthetic potential of soil-derived microbial communities

abstract: Secondary metabolites produced by microorganisms are the main source of antimicrobials other pharmaceutical drugs. Soil microbes have been the primary discovery source for these secondary metabolites, often producing complex organic compounds with specific biological activities. Research suggests that secondary metabolism broadly shapes microbial ecological interactions, but little is known about the factors that shape the abundance, distribution, and diversity of biosynthetic gene clusters in the context of microbial communities. In this study, we investigate the role of nutrient availability on the abundance of biosynthetic gene clusters in soil-derived microbial consortia. We found that soil microbial consortia enriched in medium with 150 mg/L of glucose and 200 mg/L of trehalose (here defined as high sugar) had more biosynthetic gene cluster and higher inhibitory activity than soil microbial consortia enriched in medium with 15 mg/L of glucose + 20 mg/L of trehalose (here defined as low sugar). Our results demonstrate that laboratory microbial communities are a promising tool to study ecology of specialized metabolites.


title: Public human microbiome data dominated by highly developed countries

abstract: The importance of sampling from globally representative populations has been well established in human genomics. In human microbiome research, however, we lack a full understanding of the global distribution of sampling in research studies. This information is crucial to better understand global patterns of microbiome-associated diseases and to extend the health benefits of this research to all populations. Here, we analyze the country of origin of all 444,829 human microbiome samples that have been collected to date and are available from the worlds three largest genomic data repositories, including the Sequence Read Archive (SRA). We show that more than 71% of publicly available human microbiome samples with a known origin come from Europe, the United States, and Canada, including 46.8% from the United States alone, despite the country representing only 4.3% of the global population. We also find that central and southern Asia is the most underrepresented region: Countries such as India, Pakistan, and Bangladesh account for more than a quarter of the world population but make up only 1.8 percent of human microbiome samples. These results demonstrate a critical need to ensure more global representation of participants in microbiome studies.


title: Changes in antennal gene expression underlying sensory system maturation in Rhodnius prolixus

abstract:
BackgroundTriatomine bugs are the blood feeding insect vectors transmitting Chagas disease to humans, a neglected tropical disease that affects over 8 million people, mainly in Latin America. The behavioral responses to host cues and bug signals in Rhodnius prolixus are state dependent, i.e., they vary as a function of post-ecdysis age. At the molecular level, these changes in behavior are probably due to a modulation of peripheral and central processes. In the present study, we report a significant modulation of the expression of a large set of sensory-related genes. Results were generated by means of antennal transcriptomes of 5th instar larvae along the first week (days 0, 2, 4, 6 and 8) after ecdysis sequenced using the Illumina platform.

ResultsAge induced significant changes in transcript abundance were established in more than 6,120 genes (54,7 % of 11,186 genes expressed) in the R. prolixus antenna. This was especially true between the first two days after ecdysis when more than 2,500 genes had their expression significantly altered. In contrast, expression profiles were almost identical between day 6 and 8, with only a few genes showing significant modulation of their expression. A total of 86 sensory receptors, odorant carriers and odorant degrading enzymes were significantly modulated across age points and clustered into three distinct expression profiles.

ConclusionsThe set of sensory genes whose expression increased with age (profile 3) may include candidates underlying the increased responsiveness to host cues shown by R. prolixus during the first days after molting. For the first time, we describe the maturation process undergone at the molecular level by the peripheral sensory system is described in an hemimetabolous insect.

category: molecular biology


title: microRNAs as Indicators into the Causes and Consequences of Whole Genome Duplication Events

abstract: Whole genome duplications (WGDs) have long been considered the causal mechanism underlying the dramatic increase in vertebrate morphological complexity relative to invertebrates. This is due to the retention and neo-functionalization of paralogues generated during these events, evolving new regulatory circuits, and ultimately morphological novelty. Nonetheless, an alternative hypothesis suggests that behind the retention of most paralogues is not neo-functionalization, but instead the degree of the inter-connectivity of the intended gene product, as well as the mode of the WGD itself. Here, we explore both the causes and consequences of WGD by examining the distribution, expression, and molecular evolution of microRNAs (miRNAs) in both gnathostome vertebrates as well as chelicerate arthropods. We find that although the number of miRNA paralogues tracks the number of WGDs experienced within the lineage, few of these paralogues experienced changes to the seed sequence, and thus are functionally equivalent relative to their mRNA targets. Nonetheless, the paralogues generated by the gnathostome 2R allotetraploidization event are retained in higher numbers on one sub-genome relative the second, with the miRNAs found on the preferred set of paralogons showing both higher expression of mature miRNA transcripts and slower molecular evolution of the precursor miRNA sequences. Importantly, WGDs do not result in the creation of miRNA novelty, nor do WGDs correlate to increases in complexity. Instead, it is the number of miRNA seed sequences in the genome itself that not only better correlate to instances in complexification, but also mechanistically explain why complexity increases when new miRNA families are established.

category: evolutionary biology


title: Evaluation of rubber tree transcriptome and discovery of SNP and SSR from candidate genes involved in cellulose and lignin biosynthesis

abstract: Hevea brasiliensis (the rubber tree) is a well-known species with high economic value, and it is the primary source of natural rubber globally. Increasing demand for furniture and related industries has made rubberwood production as important as latex production. Molecular markers such as Single Nucleotide Polymorphisms (SNPs) and Simple Sequence Repeats (SSRs) are widely used for Marker Assisted Selection (MAS) which can be detected in large quantity by transcriptome sequencing. MAS is thought to be a useful method for the development of new rubberwood clones for its shorter breeding cycle compared to a conventional breeding procedure. In this study we performed RNA sequencing (RNA-seq) on four H. brasiliensis clones (RRIM 712, RRIM 2025, RRIM 3001 and PB 314) from three tissues including bark, latex and leaf samples to identify SSRs and SNPs associated with wood-formation related genes. The RNA sequencing using the Illumina NextSeq 500 v2 platform, generated 1,697,491,922 raw reads. A total of 101,269 transcripts over 400 bp in size were obtained and similarity search of the non-redundant (nr) protein database returned 83,748 (83%) positive BLASTx hits. The transcriptome analysis was annotated using the NCBI NR (National Center for Biotechnology Information Non-Redundant), UniProtKB/Swiss-Prot, gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Differential expression analysis between later-timber rubber clone and non-later-timber rubber clone on wood-formation related genes, showed genes encoding phenylalanine ammonia-lyase (PAL), caffeic acid O-methyltransferase (COMT) and cinnamoyl-CoA reductase (CCR) were highly up-regulated in a latex-timber rubber clone. In total, about 3,210,629 SNPs and 14,956 SSRs were detected with 1,786 SNPs and 31 SSRs were found for wood-formation biosynthesis of H. brasilensis from 11 lignin and cellulose gene toolboxes. After filtering and primer selection, 103 SNPs and 18 SSR markers were successfully amplified and could be useful as molecular tool for marker assisted breeding to produce new timber rubber clones.

category: genetics


title: Fine mapping spatiotemporal mechanisms of genetic variants underlying cardiac traits and disease

abstract: The causal variants and genes underlying thousands of cardiac GWAS signals have yet to be identified. Here, we leveraged spatiotemporal information on 966 RNA-seq cardiac samples and performed an expression quantitative trait locus (eQTL) analysis detecting eQTLs considering both eGenes and eIsoforms. We identified 2,578 eQTLs associated with a specific developmental stage-, tissue- and/or cell type. Colocalization between eQTL and GWAS signals of five cardiac traits identified variants with high posterior probabilities for being causal in 210 GWAS loci. Pulse pressure GWAS loci were enriched for colocalization with fetal- and smooth muscle- eQTLs; pulse rate with adult- and cardiac muscle- eQTLs; and atrial fibrillation with cardiac muscle- eQTLs. Fine mapping identified 79 credible sets with five or fewer SNPs, of which 15 were associated with spatiotemporal eQTLs. Our study shows that many cardiac GWAS variants impact traits and disease in a developmental stage-, tissue- and/or cell type-specific fashion.


title: Differential regulation of mouse hippocampal gene expression sex differences by chromosomal content and gonadal sex

abstract: Common neurological disorders, like Alzheimers disease (AD), multiple sclerosis (MS), and autism, display profound sex differences in prevalence and clinical presentation. However, sex differences in the brain with health and disease are often overlooked in experimental models. Sex effects originate, either directly or indirectly, from hormonal or sex chromosomal mechanisms. To delineate the contributions of genetic sex (XX v. XY) versus gonadal sex (ovaries v. testes) to the epigenomic regulation of hippocampal sex differences, we use the Four Core Genotypes (FCG) mouse model to uncouple chromosomal and gonadal sex. Transcriptomic and epigenomic analyses of [~]12-month-old FCG mice hippocampi, revealed genomic context-specific regulatory effects of genotypic and gonadal sex on X- and autosome-encoded gene expression and DNA modification patterns. X-chromosomal epigenomic patterns, classically associated with X-inactivation, were established almost entirely by genotypic sex, independent of gonadal sex. Differences in X-chromosome methylation were primarily localized to gene regulatory regions including promoters, CpG islands, CTCF binding sites, and active/poised chromatin, with an inverse relationship between methylation and gene expression. Whereas, autosomal gene expression demonstrated regulation by both genotypic and gonadal sex, particularly in immune processes. These data demonstrate an important regulatory role of sex chromosomes, independent of gonadal sex, on sex-biased hippocampal transcriptomic and epigenomic profiles. Future studies will need to further interrogate specific CNS cell types, identify the mechanisms by which sex chromosomes regulate autosomes, and differentiate organizational from activational hormonal effects.

category: genomics


title: Nanomolar, noncovalent antagonism of hedgehog cholesterolysis: exception to the irreversibility rule for protein autoprocessing inhibition.

abstract: Hedgehog (Hh) signaling ligands undergo carboxy terminal sterylation through specialized autoprocessing, called cholesterolysis. Sterylation is brought about intramolecularly in a single turn-over by an enzymatic domain, called HhC. HhC is found in precursor Hh proteins only. Through cholesterolysis, HhC is cleaved from the precursor. Attempts to identify molecules that inhibit intramolecular cleavage/sterylation activity of HhC have resulted in antagonists that bind HhC irreversibly through covalent mechanisms, as is commonplace for protein autoprocessing inhibitors. Here we report an exception to the "irreversibility rule" for protein autoprocessing inhibition. Using a FRET-based activity assay for HhC, we screened a focused library of sterol-like analogs for HhC cholesterolysis inhibitors. We identified and validated four structurally related noncovalent inhibitors, which were then used for SAR studies. The most effective derivative, tBT-HBT, binds HhC reversibly with an IC50 of 300 nM. An allosteric binding site for tBT-HBT, encompassing interactions from the two subdomains of HhC, is suggested by kinetic analysis, mutagenesis studies, and photoaffinity labeling. A striking resemblance is found between the inhibitors described here and a family of noncovalent, allosteric activators of HhC, which we described previously. The inhibitor/activator duality appears to be mediated by the same allosteric site, which displays sensitivity to subtle differences in the structure of a heterocycle substituent on the effector molecule.

category: biochemistry


title: Redox-dependent Structure and Dynamics of Macrophage Migration Inhibitory Factor Reveal Sites of Latent Allostery

abstract: Macrophage migration inhibitory factor (MIF) is a multifunctional immunoregulatory protein that is a key player in the innate immune response. Given its overexpression at sites of inflammation in a wide range of diseases marked by increasingly oxidative cellular environment, a comprehensive structural understanding of how cellular redox conditions may impact the structure and function of MIF is necessary. We used solution NMR spectroscopy and mass spectrometry to investigate structural and dynamic signatures of MIF under varied solution redox conditions. Our results indicate that the MIF structure is modified and becomes increasingly dynamic in an oxidative environment, which may be a means to alter the MIF functional response in a redox-dependent manner. We identified latent allosteric sites within MIF that are redox-sensitive and mutational analysis reveals that loss of redox-responsive residues attenuates activation of the coreceptor CD74. Leveraging sites of redox-sensitivity therefore reveals an avenue to modulate MIF function in its "disease state" via structure-based drug design.


title: Multiplex spatial systems analysis of local nanodose drug responses predicts effective treatment combinations of immunotherapies and targeted agents in mammary carcinoma

abstract: Better methods are needed to identify effective combinations of immunotherapies with chemotherapies and targeted anti-cancer agents. Here we present a Multiplex Implantable Microdevice Assay (MIMA) system for rapid in vivo assessment of the effects of multiple, spatially separate anticancer drugs directly in the complex tumor microenvironment. In prototypic experiments, olaparib, lenvatinib, palbociclib, venetoclax, panobinostat, doxorubicin, and paclitaxel and combinations thereof were administered simultaneously to murine mammary tumor models. Quantitative multiplex immunohistochemistry and spatial systems analyses of each local drug response defined cellular relations of fibroblasts, endothelial cells, immune lineages, immunogenic cell death, tumor proliferation and/or cancer stem cells that were used to predict effective drug combinations. A predicted combination of panobinostat, venetoclax and anti-CD40 showed long-term anti-tumor efficacy in multiple mouse models with no observable toxicity when administered systemically. Future MIMA use promises to design effective drug combinations for tumor cell control and immune activation on a personalized basis.


title: Offspring of first-generation hatchery steelhead trout (Oncorhynchus mykiss) grow faster in the hatchery than offspring of wild fish, but survive worse in the wild: possible mechanisms for inadvertent domestication and fitness loss in hatchery salmon

abstract: Salmonid fish raised in hatcheries often have lower fitness (number of returning adult offspring) than wild fish when both spawn in the wild. Body size at release from hatcheries is positively correlated with survival at sea. So one explanation for reduced fitness is that hatcheries inadvertently select for trait values that enhance growth rate under the unnatural environment of a hatchery, but that are maladaptive in the wild environment. A simple prediction of this hypothesis is that juveniles of hatchery origin should grow more quickly than fish of wild origin under hatchery conditions, but should have lower survival under wild conditions. We tested that hypothesis using multiple full sibling families of steelhead (Oncorhynchus mykiss) that were spawned using either two wild parents (WxW) or two first-generation hatchery (HxH) parents. Offspring from all the families were grown together under hatchery conditions and under semi-natural conditions in artificial streams. HxH families grew significantly faster in the hatchery, but had significantly lower survival in the streams. That we see this tradeoff after only a single generation of selection suggests that the traits involved are under very strong selection. We also considered one possible alteration to the hatchery environment that might reduce the intensity of selection among families in size at release. Here we tested whether reducing the fat content of hatchery feed would reduce the variance among families in body size. Although fish raised under a low-fat diet were slightly smaller, the variation among families in final size was unchanged. Thus, there is no evidence that reducing the fat content of hatchery feed would reduce the opportunity for selection among families on size at release.

category: zoology


title: Concomitant Pyroptotic and Apoptotic Cell Death Triggered in Monocytes Infected by Zika Virus

abstract: Zika virus (ZIKV) is a positive-sense RNA flavivirus and can cause serious neurological disorders including microcephaly in infected fetus. As a mosquito-borne arbovirus, ZIKV enters bloodstream and is transmitted into the fetus through the placenta in pregnant women. Monocytes are considered one of the earliest blood cell types to be infected by ZIKV. As a first line defence, monocytes are crucial components in innate immunity and host responses and may impact viral pathogenesis in humans. Previous studies have shown that ZIKV infection can activate inflammasomes and induce proinflammatory cytokines in monocytes. In this report, we showed that ZIKV carried out a productive infection, which lead to cell death in human and murine monocytic cells. In addition to the presence of cleaved caspase-3, indicating that apoptosis was involved, we identified the cleaved caspase-1 and gasdemin D (GSDMD) as well as increased secretion of IL-1{beta} and IL-18, suggesting that the inflammasome was activated that may lead to pyroptosis in infected monocytes. The pyroptosis was NLRP3-dependent and could be suppressed in the monocytes treated with shRNA to target and knockdown caspase-1, or an inhibited for caspase-1, indicating that the pyroptosis was triggered via a canonical approach. Our findings in this study demonstrate a concomitant occurrence of apoptosis and pyroptosis in ZIKV-infected monocytes, with multiple mechanisms involved in the cell death, which may have potentially significant impacts on viral pathogenesis in humans.


title: Tracking transitional probabilities and segmenting auditory sequences are dissociable processes in adults and neonates

abstract: Since speech is a continuous stream with no systematic boundaries between words, how do pre-verbal infants manage to discover words? A proposed solution is that they might use the transitional probability between adjacent syllables, which drops at word boundaries. Here, we tested the limits of this mechanism by increasing the size of the word-unit to 4 syllables, and its automaticity by testing asleep neonates. Using markers of statistical learning in neonates EEG, compared to adult behavioral performances in the same task, we confirmed that statistical learning is automatic enough to be efficient even in sleeping neonates. But we also revealed that: 1) Successfully tracking transition probabilities in a sequence is not sufficient to segment it 2) Prosodic cues, as subtle as subliminal pauses, enable to recover segmenting capacities 3) Adults and neonates capacities are remarkably similar despite the difference of maturation and expertise. Finally, we observed that learning increased the similarity of neural responses across infants, providing a new neural marker to monitor learning. Thus, from birth, infants are equipped with adult-like tools, allowing to extract small coherent word-like units within auditory streams, based on the combination of statistical analyses and prosodic cues.

category: neuroscience


title: Neurodevelopmental clustering of gene expression identifies lipid metabolism genes associated with neuroprotection and neurodegeneration.

abstract: APOE variants present the strongest association with sporadic Alzheimers Disease. APOE is also highly expressed during neurodevelopment in the Central Nervous System (CNS) and has been shown to be neuroprotective in infancy and gestation. We explored other lipid metabolism genes to determine whether they show a similar neurodevelopmental expression trajectory and are associated with neurodegeneration. APOE was by far the most highly expressed of the lipid metabolism genes in the CNS. Two other genes, Apolipoprotein C1 (APOC1) Glutamate-Ammonia Ligase (GLUL--also known as glutamine synthetase), co-clustered with Apolipoprotein E (APOE) in its developmental trajectory in late gestation through early childhood. These three genes highlight brain structures and developmental time-windows distinct from other lipid metabolism genes. In the CNS they are primarily expressed in astrocytes and are implicated in neuroprotection and neurodegeneration.


title: Mate choice enhances post-zygotic barriers to gene flow via ancestry bundling

abstract: Hybridization and subsequent genetic introgression are now known to be common features of the histories of many species, including our own. Following hybridization, selection often purges introgressed DNA genome-wide. While mate choice can prevent hybridization in the first place, it is also known to play an important role in post-zygotic selection against hybrids, and thus the purging of introgressed DNA. However, this role is usually thought of as a direct one: a mating preference for conspecifics reduces the sexual fitness of hybrids, reducing the transmission of introgressed ancestry. Here, we explore a second, indirect role of mate choice as a barrier to gene flow. Under assortative mating, parents covary in their ancestry, causing ancestry to be "bundled" in their offspring and later generations. This bundling effect increases ancestry variance in the population, enhancing the efficiency with which post-zygotic selection purges introgressed DNA. Using whole-genome simulations, we show that the bundling effect can comprise a substantial portion of mate choices overall effect as a post-zygotic barrier to gene flow, and that it is driven by ancestry covariances between and within maternally and paternally inherited genomes. We derive a simple method for estimating the impact of the bundling effect from standard measures of assortative mating. Applying this method to data from a diverse set of hybrid zones, we find that the bundling effect increases the purging of introgressed DNA by between 1.2-fold (in a baboon system with weak assortative mating) and 14-fold (in a swordtail system with strong assortative mating). Thus, the bundling effect of mate choice contributes substantially to the genetic isolation of species.


title: Microtubule retrograde flow retains neuronal polarization in a fluctuating state

abstract: In developing vertebrate neurons, a neurite is formed by more than a hundred microtubules. While individual microtubules are dynamic, the microtubule array itself has been regarded as stationary. Using live cell imaging in combination with photoconversion techniques and pharmacological manipulations, we uncovered that the microtubule array flows retrogradely within neurites to the soma. This microtubule retrograde flow drives cycles of microtubule density, a hallmark of the fluctuating state before axon formation. Shortly after axon formation, microtubule retrograde flow slows down in the axon, which stabilizes microtubule density cycles and thereby functions as a molecular wedge to enable axon extension. We propose microtubule retrograde flow and its specific slowdown in the axon to be the long-sought mechanism to single one neurite out to drive neuronal polarization.


title: Elucidating the neural mechanisms of Learning-to-Learn

abstract: Learning-to-learn, a progressive acceleration of learning while solving a series of similar problems, represents a core process of knowledge acquisition that draws attention in both neuroscience and artificial intelligence. To investigate its underlying brain mechanism, we trained a recurrent neural network model on arbitrary sensorimotor mappings. The network displayed an exponential speedup in learning. The neural substrate of a schema emerges within a low-dimensional subspace of population activity. Its reuse in new problems facilitates learning by limiting connection weight changes. Since the population trajectory of a recurrent network produces behavior, learning is determined by the vector field changes. We propose a novel analysis of weight-driven vector field changes, which showed that novel stimuli in new problems can distort the schema representation. Weight changes eliminate such distortions and improve the invariance of the reused representations in future learning. The accumulation of such weight changes across problems underlies the learning-to-learn dynamics.


title: RORγt-Expressing Pathogenic CD4+T Cells Cause Brain Inflammation During Chronic Colitis

abstract: Neurobehavioral disorders and brain abnormalities have been extensively reported in both Crohns Disease (CD) and Ulcerative Colitis (UC) patients. However, the mechanism causing neuropathological disorders in inflammatory bowel disease (IBD) patients remains unknown. Studies have linked the Th17 subset of CD4+T cells to brain diseases associated with neuroinflammation and cognitive impairment, including multiple sclerosis (MS), ischemic brain injury and Alzheimers disease. To better understand how CD4+T lymphocytes, contribute to brain pathology in chronic intestinal inflammation, we investigated the development of brain inflammation in the T cell transfer model of chronic colitis. Our findings demonstrate that CD4+T cells infiltrate the brain of colitic Rag1-/- mice in proportional levels to colitis severity. Colitic mice developed hypothalamic astrogliosis that correlated with neurobehavioral disorders. Moreover, the brain-infiltrating CD4+T cells expressed Th17 cell transcription factor ROR{gamma}t and displayed a pathogenic Th17 cellular phenotype similar to colonic Th17 cells. Adoptive transfer of ROR{gamma}t-deficient naive CD4+T cells failed to cause brain inflammation and neurobehavioral disorders in Rag1-/- recipients, with significantly less brain infiltration of CD4+T cells. These findings suggest that pathogenic ROR{gamma}t+CD4+T cells that aggravate colitis migrate preferentially into the brain, contributing to brain inflammation and neurobehavioral disorders, thereby linking colitis severity to neuroinflammation.


title: Mpe1 senses the polyadenylation signal in pre-mRNA to control cleavage and polyadenylation

abstract: Most eukaryotic messenger RNAs (mRNAs) are processed at their 3-end by the cleavage and polyadenylation factor (CPF/CPSF). CPF mediates endonucleolytic cleavage of the pre-mRNA and addition of a polyadenosine (poly(A)) tail, which together define the 3-end of the mature transcript. Activation of CPF is highly regulated to maintain fidelity of RNA processing. Here, using cryoEM of yeast CPF, we show that the Mpe1 subunit directly contacts the polyadenylation signal sequence in nascent pre- mRNA. This RNA-mediated link between the nuclease and polymerase modules promotes activation of the CPF endonuclease and controls polyadenylation. Mpe1 rearrangement is antagonized by another subunit, Cft2. In vivo, depletion of Mpe1 leads to widespread defects in transcription termination by RNA Polymerase II, resulting in transcription interference on neighboring genes. Together, our data suggest that Mpe1 plays a major role in selecting the cleavage site, activating CPF and ensuring timely transcription termination.


title: Glial TGFβ activity promotes axon survival in peripheral nerves

abstract:
Axons can represent the majority of the volume of a neuron and are energetically very demanding. Specialized glia ensheathe axons and are believed to support axon function and maintenance throughout life, but molecular details of glia-neuron support mechanisms remain poorly defined. Here we identify a collection of secreted and transmembrane genes that are required in glia for long-term axon survival in vivo. We show that key components of the TGF{beta} superfamily are required cell-autonomously in glia for peripheral nerve maintenance, although their loss does not disrupt glial morphology. We observe age-dependent neurodegeneration in the absence of glial TGF{beta} signaling that can be rescued by genetic blockade of Wallerian degeneration. Our data argue that glial TGF{beta} signaling normally acts to promote axon survival and suppress neurodegeneration.

Significance StatementAxon maintenance is critical to preserving the functional integrity of the nervous system across animal lifespan. Glia contribute to axon maintenance, but their precise roles remain to be fully characterized. We identify glial genes that regulate axon support and provide new molecular insight into the means by which glia promote axon survival, which may help explain why neurodegeneration occurs when glia are lost in disease. We show that TGF{beta} signaling in mature glia is essential for long-term maintenance of axons, and that loss of TGF{beta} signaling activates an axon death signaling pathway.


title: Unmasking the cryptic immunopeptidome of EZH2 mutated diffuse large B-cell lymphomas through combination drug treatment

abstract:
Exploring the repertoire of peptides presented on major histocompatibility complexes (MHC) has been utilized to identify targets for immunotherapy in many hematological malignancies. However, such data have not been described systematically for diffuse large B-cell lymphomas (DLBCL), which might be explained by the profound downregulation of MHC expression in many DLBCLs, and in particular in the EZH2-mutated subgroup. Epigenetic drug treatment, especially in the context of interferon gamma (IFNg), restored MHC expression in DLBCL. DLBCL MHC-presented peptides were identified via mass spectrometry following tazemetostat or decitabine treatments alone, or in combination with IFNg. Such treatment synergistically increased MHC class I surface protein expression up to 50-fold and class II expression up to 3-fold. Peptides presented on MHC complexes increased to a similar extent for MHC class I and remained constant for class II. Overall, these treatments restored the diversity of the immunopeptidome to levels described in healthy B cells and allowed the systematic search for new targets for immunotherapy. Consequently, we identified multiple MHC ligands from regulator of G protein signaling 13 (RGS13) and E2F transcription factor 8 (E2F8) on different MHC alleles, none of which have been described in healthy tissues and therefore represent tumor-specific MHC ligands, which are unmasked only after drug treatment. Overall, our results show that EZH2 inhibition in combination with decitabine and IFNg can expand the repertoire of MHC ligands presented on DLBCLs by revealing cryptic epitopes, thus allowing the systematic analysis and identification of new potential immunotherapy targets.

Key pointsO_LICombination therapy of interferon gamma with epigenetic regulators leads to large increases in the immunopeptidome of DLBCL.
C_LIO_LIHLA ligands from proteins RGS13 and E2F8 may provide DLBCL-specific targets for immunotherapy.
C_LI


title: SHOOT: phylogenetic gene search and ortholog inference

abstract: Determining the evolutionary relationships between genes is fundamental to comparative biological research. Here we present the phylogenetic search, SHOOT. SHOOT searches a user query sequence against a database of phylogenetic trees and returns a tree with the query sequence correctly placed within it. We show that SHOOT performs this analysis with comparable speed to a BLAST search. We demonstrate that SHOOT phylogenetic placements are as accurate as conventional tree inference and it can identify orthologs with high accuracy. In summary, SHOOT is a fast and accurate tool for phylogenetic analyses of novel query sequences. It is available online at www.shoot.bio.

category: bioinformatics


title: Increased pathogenicity of the nematophagous fungus Drechmeria coniospora following long-term laboratory culture.

abstract: Domestication provides a window into adaptive change. Over the course of 2 decades of laboratory culture, a strain of the nematode-specific fungus Drechmeria coniospora became more virulent during its infection of Caenorhabditis elegans. Through a close comparative examination of the genome sequences of the original strain and its more pathogenic derivative, we identified a small number of non-synonymous mutations in protein-coding genes. In one case, the mutation was predicted to affect a gene involved in hypoxia resistance and we provide direct corroborative evidence for such an effect. The mutated genes with functional annotation were all predicted to impact the general physiology of the fungus and this was reflected in an increased in vitro growth, even in the absence of C. elegans. While most cases involved single nucleotide substitutions predicted to lead to a loss of function, we also observed a predicted restoration of gene function through deletion of an extraneous tandem repeat. This latter change affected the regulatory subunit of a cAMP-dependent protein kinase. Remarkably, we also found a mutation in a gene for a second protein of the same, protein kinase A, pathway. Together, we predict that they result in a stronger repression of the pathway for given levels of ATP and adenylate cyclase activity. Finally, we also identified mutations in a few lineage-specific genes of unknown function that are candidates for factors that influence virulence in a more direct manner.


title: A network-based approach to integrate nutrient environment in the prediction of synthetic lethality in cancer metabolism

abstract: Synthetic Lethality (SL) is a promising concept in cancer research. A number of computational methods have been developed to predict SL in cancer metabolism, among which our network-based computational approach, based on genetic Minimal Cut Sets (gMCSs), can be found. A major challenge of these approaches to SL is to systematically consider tumor environment, which is particularly relevant in cancer metabolism. Here, we propose a novel definition of SL for cancer metabolism that integrates genetic interactions and nutrient availability in the environment. We extend our gMCSs approach to determine this new family of metabolic synthetic lethal interactions. A computational and experimental proof-of-concept is presented for predicting the lethality of dihydrofolate reductase inhibition in different environments. Finally, our novel approach is applied to identify extracellular nutrient dependences of tumor cells, elucidating cholesterol and myo-inositol depletion as potential vulnerabilities in different malignancies.

category: systems biology


title: Defective Desmosomal Adhesion Causes Arrhythmogenic Cardiomyopathy by involving an Integrin-αVβ6/TGF-β Signaling Cascade

abstract:
BackgroundArrhythmogenic Cardiomyopathy (ACM) is characterized by progressive loss of cardiomyocytes with fibrofatty replacement, systolic dysfunction and life-threatening arrhythmias. A substantial proportion of ACM is caused by mutations in genes of the desmosomal cell-cell adhesion complex, but the underlying mechanisms are not well understood. So far, treatment options are only symptomatic. Here, we investigate the relevance of defective desmosomal adhesion for ACM development and progression.

MethodsWe mutated the binding site of desmoglein-2 (DSG2), a crucial desmosomal adhesion molecule in cardiomyocytes. This DSG2-W2A mutation abrogates the tryptophan swap, a central interaction mechanism of DSG2 based on structural data. Impaired adhesive function of DSG2-W2A was confirmed by cell-cell dissociation assays and force spectroscopy measurements by atomic force microscopy. We next generated a DSG2-W2A knock-in mouse model, which was analyzed by echocardiography and histological and bio-molecular techniques including RNA sequencing, transmission electron and super-resolution microscopy. The results were compared to ACM patient samples and their relevance was confirmed in cardiac slice cultures.

ResultsThe DSG2-W2A mutation induced impaired binding and desmosomal adhesion dysfunction on cellular and molecular level. Mice bearing this mutation develop a severe cardiac phenotype recalling the characteristics of ACM, including cardiac fibrosis, impaired systolic function and arrhythmia. A comparison of the transcriptome of mutant mice with ACM patient data suggested deregulated integrin-V{beta}6 and subsequent TGF-{beta} signaling as driver of cardiac fibrosis. Accordingly, blocking antibodies targeting integrin-V{beta}6 or inhibition of TGF-{beta} receptor signaling both led to reduced expression of pro-fibrotic markers in cardiac slice cultures.

ConclusionsHere, we show that disruption of desmosomal adhesion is sufficient to induce ACM, which confirms the dysfunctional adhesion hypothesis. Mechanistically, deregulation of integrin-V{beta}6 signaling was identified as a central step towards fibrosis. This highlights the value of this model to discern mechanisms of cardiac fibrosis and to identify and test novel treatment options for ACM.

category: cell biology


title: ERM-1 phosphorylation and NRFL-1 redundantly control lumen formation in the C. elegans intestine

abstract: Reorganization of the plasma membrane and underlying actin cytoskeleton into specialized domains is essential for the functioning of most polarized cells in animals. Proteins of the ezrin-radixin-moesin (ERM) and Na+/H+ exchanger 3 regulating factor (NHERF) family are conserved regulators of cortical specialization. ERM proteins function as membrane-actin linkers and as molecular scaffolds that organize the distribution of proteins at the membrane. NHERF proteins are PDZ-domain containing adapters that can bind to ERM proteins and extend their scaffolding capability. Here, we investigate how ERM and NHERF proteins function in regulating intestinal lumen formation in the nematode Caenorhabditis elegans. C. elegans has single ERM and NHERF family proteins, termed ERM-1 and NRFL-1, and ERM-1 was previously shown to be critical for intestinal lumen formation. Using CRISPR/Cas9-generated nrfl-1 alleles we demonstrate that NRFL-1 localizes at the intestinal microvilli, and that this localization is depended on an interaction with ERM-1. However, nrfl-1 loss of function mutants are viable and do not show defect in intestinal development. Interestingly, combining nrfl-1 loss with erm-1 mutants that either block or mimic phosphorylation of a regulatory C-terminal threonine causes severe defects in intestinal lumen formation. These defects are not observed in the phosphorylation mutants alone, and resemble the effects of strong erm-1 loss of function. The loss of NRFL-1 did not affect the localization or activity of ERM-1. Together, these data indicate that ERM-1 and NRFL-1 function together in intestinal lumen formation in C. elegans. We postulate that the functioning of ERM-1 in this tissue involves actin-binding activities that are regulated by the C-terminal threonine residue and the organization of apical domain composition through NRFL-1.


title: Changes to the mtDNA copy number during the course of yeast culture growth

abstract: We show that the mtDNA copy number in growing cultures of the yeast Saccharomyces cerevisiae increases by a factor of up to 4, being lowest ([~]10 per haploid genome) and stable during rapid fermentative growth, and highest at the end of the respiratory phase. When yeast are grown on glucose, the onset of the mtDNA copy number increase coincides with the early stages of the diauxic shift, and the increase continues through respiration. A lesser yet still substantial copy number increase occurs when yeast are grown on a nonfermentable carbon source, i.e. when there is no diauxic shift. The mtDNA copy number increase during and for some time after the diauxic shift is not driven by an increase in cell size. The copy number increase occurs in both haploid and diploid strains, but is markedly attenuated in a diploid wild isolate that is a ready sporulator. Strain-to-strain differences in mtDNA copy number are least apparent in fermentation and most apparent in late respiration or stationary phase. While changes in mitochondrial morphology and function were previously known to accompany changes in physiological state, it had not been previously shown that the mtDNA copy number changes substantially over time in a clonal growing culture. The mtDNA copy number in yeast is therefore a highly dynamic phenotype.


title: Changes to the mtDNA copy number during yeast culture growth


title: Learning to embed lifetime social behavior from interaction dynamics

abstract:
Interactions of individuals in complex social systems give rise to emergent behaviors at the group level. Identifying the functional role that individuals take in the group at a specific time facilitates understanding the dynamics of these emergent processes. An individuals behavior at a given time can be partially inferred by common factors, such as age, but internal and external factors also substantially influence behavior, making it difficult to disentangle common development from individuality. Here we show that such dependencies on common factors can be used as an implicit bias to learn a temporally consistent representation of a functional role from social interaction networks. Using a unique dataset containing lifetime trajectories of multiple generations of individually-marked honey bees in two colonies, we propose a new temporal matrix factorization model that jointly learns the average developmental path and structured variations of individuals in the social network over their entire lives. Our method yields inherently interpretable embeddings that are biologically relevant and consistent over time, allowing one to compare individuals functional roles regardless of when or in which colony they lived. Our method provides a quantitative framework for understanding behavioral heterogeneity in complex social systems, and is applicable to fields such as behavioral biology, social sciences, neuroscience, and information science.

Author summaryGroup-level emergent behaviors are the result of interactions between individual group members. To understand these social dynamics, one must objectively measure the function of an individual in their group at any given time. Ideally, one would also like to compare individuals from different groups, for example, to measure how specific environmental conditions or other external factors influence group behavior. Unfortunately, such an objective measure is hard to obtain because the group and its dynamics constantly change, making it challenging to define an individuals role in the group as a function of its actions and interactions. We propose a principled approach to model individuals in complex social systems by considering that function often depends, at least partially, on common factors such as age. The model learns a meaningful and interpretable descriptor for all individuals, and can be used to understand how complex social systems function and the emergence of group behavior.

category: animal behavior and cognition


title: A Generalizable Speech Emotion Recognition Model Reveals Depression and Remission

abstract:
ObjectiveAffective disorders are associated with atypical voice patterns; however, automated voice analyses suffer from small sample sizes and untested generalizability on external data. We investigated a generalizable approach to aid clinical evaluation of depression and remission from voice using transfer learning: we train machine learning models on easily accessible non-clinical datasets and test them on novel clinical data in a different language.

MethodsA Mixture-of-Experts machine learning model was trained to infer happy/sad emotional state using three publicly available emotional speech corpora in German and US English. We examined the models predictive ability to classify the presence of depression on Danish speaking healthy controls (N = 42), patients with first-episode major depressive disorder (MDD) (N = 40), and the subset of the same patients who entered remission (N = 25) based on recorded clinical interviews. The model was evaluated on raw, de-noised, and speaker-diarized data.

ResultsThe model showed separation between healthy controls and depressed patients at the first visit, obtaining an AUC of 0.71. Further, speech from patients in remission was indistinguishable from that of the control group. Model predictions were stable throughout the interview, suggesting that 20-30 seconds of speech might be enough to accurately screen a patient. Background noise (but not speaker diarization) heavily impacted predictions.

ConclusionA generalizable speech emotion recognition model can effectively reveal changes in speaker depressive states before and after remission in patients with MDD. Data collection settings and data cleaning are crucial when considering automated voice analysis for clinical purposes.

Significant outcomes- Using a speech emotion recognition model trained on other languages, we predicted the presence of MDD with an AUC of 0.71.
- The speech emotion recognition model could accurately detect changes in voice after patients achieved remission from MDD.
- Preprocessing steps, particularly background noise removal, greatly influenced classification performance.

Limitations- No data from non-remitters, meaning that changes to voice for that group could not be assessed.
- It is unclear how well the model would generalize beyond Germanic languages.

Data availability statementDue to the nature of the data (autobiographical interviews in a clinical population), the recordings of the participants cannot be shared publicly. The aggregated model predictions and code used to run the analyses is available at https://github.com/HLasse/SERDepressionDetection.


title: Targeting the translational machinery in gastrointestinal stromal tumors (GIST) - a new therapeutic vulnerability

abstract: Although KIT-mutant GISTs can be effectively treated with tyrosine kinase inhibitors (TKIs), many patients develop resistance to imatinib mesylate (IM) as well as the FDA-approved later-line agents sunitinib, regorafenib and ripretinib. Resistance mechanisms mainly involve secondary mutations in the KIT receptor tyrosine kinase gene indicating continued dependency on the KIT signaling pathway. The fact that the type of secondary mutation confers either sensitivity or resistance towards TKIs and the notion that secondary mutations exhibit intra- and intertumoral heterogeneity complicates the optimal choice of treatment in the imatinib-resistant setting. Therefore, new strategies that target KIT independently of its underlying mutations are urgently needed. Homoharringtonine (HHT) is a first-in-class inhibitor of protein biosynthesis and is FDA-approved for chronic myeloid leukemia (CML) that is resistant to at least two TKIs. HHT has also shown activity in KIT-mutant mastocytosis models, which are intrinsically resistant to imatinib and most other TKIs. We hypothesized that HHT could be effective in GIST through downregulation of KIT expression and subsequent decrease of KIT activation and downstream signaling. Testing several GIST cell line models, HHT led to a significant reduction in nascent protein synthesis and was highly effective in the nanomolar range in IM-sensitive and IM-resistant GIST cell lines. HHT treatment resulted in a rapid and complete abolishment of KIT expression and activation, while KIT mRNA levels were minimally affected. The response to HHT involved induction of apoptosis as well as cell cycle arrest. The antitumor activity of HHT was confirmed in a GIST xenograft model. Taken together, inhibition of protein biosynthesis is a promising strategy to overcome TKI resistance in GIST.


title: A Molecular Docking Study Of Human STEAP2 For The Discovery Of New Potential Anti-Prostate Cancer Chemotherapeutic Candidates

abstract:
Prostate cancer refers to uncontrolled abnormal cell growth (Cancer) within the prostate gland. The disease is a rising health concern and accounts for 3.8% of all cancer deaths globally. Uganda has one of the highest incidence rates of the disease in Africa being 5.2% and many of the diagnosed patients are found to have advanced stage prostate cancer. This study aimed to use STEAP2 protein (prostate cancer specific biomarker) for the discovery of new lead compounds against prostate cancer.

To determine the most likely compound that can bind to STEAP2 protein, we docked the modelled STEAP2 3 Dimension structure against 2466 FDA (Food and Drug Administration) approved drug candidates using Autodock vina. Protein Basic Local Alignment Search Tool (BLASTp) search, Multiple Sequence Alignment (MSA), and phylogenetics were further carried out to analyse the diversity of this marker and determine its conserved domains as suitable target regions.

Six promising drug candidates (ligands) were identified of which Triptorelin had the highest binding energy (-12.1 kcal/mol). Leuprolide was the second most promising candidate with a docking energy of -11.2 kcal/mol. All the top 3 of 6 drugs interacted with highly conserved residues Ser-372 and Gly-369 in close proximity with the iron binding domain that was shown to be important for catalysis of metal reduction.

The two drugs had earlier been approved for treatment of advanced prostate cancer but with an elusive mode of action. Thanks to this study we now have an insight on how their interaction with STEAP2 might be important during treatment.

Author summaryPrevious studies on prostate cancer prevalence have shown that Uganda among other low to middle income countries has a high prostate cancer incidence rate. Majority of the patients at the time of diagnosis have advanced stage disease. While there has been great improvement in therapeutic options for prostate cancer over the last decade, the drugs currently used are still limited and not 100% effective making cure elusive. This is further compounded by undesirable side-effects from some of the treatments. Altogether this creates a need for the discovery of novel, safe, and efficacious chemotherapeutic agents with minimal or no side effects. In this study, we used a prostate cancer specific protein biomarker; STEAP2 that is highly expressed at all disease stages and is androgen independent and FDA approved drugs from the drugbank to help improve drug specificity, efficacy, and reduce undesirable side effects through in-silico methods. The results from the study give an insight on mechanisms of action of current therapy for advanced disease and suggest these very drugs be used even at early stage disease.


title: Functional connectivity of fMRI using differential covariance predicts structural connectivity and behavioral reaction times

abstract: Recordings from resting state functional magnetic resonance imaging (rs-fMRI) reflect the influence of pathways between brain areas. A wide range of methods have been proposed to measure this functional connectivity (FC), but the lack of "ground truth" has made it difficult to systematically validate them. Most measures of FC produce connectivity estimates that are symmetrical between brain areas. Differential covariance (dCov) is an algorithm for analyzing FC with directed graph edges. Applied to synthetic datasets, dCov-FC was more effective than covariance and partial correlation in reducing false positive connections and more accurately matching the underlying structural connectivity. When we applied dCov-FC to resting state fMRI recordings from the human connectome project (HCP) and anesthetized mice, dCov-FC accurately identified strong cortical connections from diffusion Magnetic Resonance Imaging (dMRI) in individual humans and viral tract tracing in mice. In addition, those HCP subjects whose rs-fMRI were more integrated, as assessed by a graph-theoretic measure, tended to have shorter reaction times in several behavioral tests. Thus, dCov-FC was able to identify anatomically verified connectivity that yielded measures of brain integration causally related to behavior.


title: Sleep spindles track cortical learning patterns for memory consolidation

abstract: Memory consolidation, the transformation of labile memory traces into stable long-term representations, is facilitated by post-learning sleep. Computational and biophysical models suggest that sleep spindles may play a key mechanistic role for consolidation, igniting structural changes at cortical sites involved in prior learning. Here we tested the resulting prediction that spindles are most pronounced over learning-related cortical areas and that the extent of this learning-spindle overlap predicts behavioural measures of memory consolidation. Using high-density scalp Electroencephalography (EEG) and Polysomnography (PSG) in healthy volunteers, we first identified cortical areas engaged during a temporospatial associative memory task (power decreases in the alpha/beta frequency range, 6-20 Hz). Critically, we found that participant-specific topographies (i.e., spatial distributions) of post-learning sleep spindle amplitude correlated with participant-specific learning topographies. Importantly, the extent to which spindles tracked learning patterns further predicted memory consolidation across participants. Our results provide empirical evidence for a role of post-learning sleep spindles in tracking learning networks, thereby facilitating memory consolidation.


title: Motor learning in reaching tasks leads to homogenization of task space error distribution

abstract:
A human arm, up to the wrist, is often modelled as a redundant 7 degree-of-freedom serial robot. Despite its inherent nonlinearity, we can perform point-to-point reaching tasks reasonably fast and with reasonable accuracy in the presence of external disturbances and noise. In this work, we take a closer look at the task space error during point-to-point reaching tasks and learning during an external force-field perturbation. From experiments and quantitative data, we confirm a directional dependence of the peak task space error with certain directions showing larger errors than others at the start of a force-field perturbation, and the larger errors are reduced with repeated trials implying learning. The analysis of the experimental data further shows that a) the distribution of the peak error is made more uniform across directions with trials and the error magnitude and distribution approaches the value when no perturbation is applied, b) the redundancy present in the human arm is used more in the direction of the larger error, and c) homogenization of the error distribution is not seen when the reaching task is performed with the non-dominant hand. The results support the hypothesis that not only magnitude of task space error, but the directional dependence is reduced during motor learning and the workspace is homogenized possibly to increase the control efficiency and accuracy in point-to-point reaching tasks. The results also imply that redundancy in the arm is used to homogenize the workspace, and additionally since the bio-mechanically similar dominant and non-dominant arms show different behaviours, the homogenizing is actively done in the central nervous system.

SignificanceThe human arm is capable of executing point-to-point reaching tasks reasonably accurately and quickly everywhere in its workspace. This is despite the inherent nonlinearities in the mechanics and the sensorimotor system. In this work, we show that motor learning enables homogenization of the task space error thus overcoming the nonlinearities and leading to simpler internal models and control of the arm movement. It is shown, across subjects, that the redundancy present in the arm is used to homogenize the task space. It is further shown, across subjects, that the homogenization is not an artifact of the biomechanics of the arm and is actively performed in the central nervous system since homogenization is not seen in the non-dominant hand.


title: Response outcome gates the effect of spontaneous cortical state fluctuations on perceptual decisions

abstract: Sensory responses of cortical neurons are more discriminable when evoked on a base-line of desynchronized spontaneous activity, but cortical desynchronization has not generally been associated with more accurate perceptual decisions. Here we show that mice perform more accurate auditory judgements when activity in the auditory cortex is elevated and desynchronized before stimulus onset, but only if the previous trial was an error, and that this relationship is occluded if previous outcome is ignored. We confirmed that the outcome-dependent effect of brain state on performance is neither due to idiosyncratic associations between the slow components of either signal, nor to the existence of specific cortical states evident only after errors. Instead, errors appear to gate the effect of cortical state fluctuations on discrimination accuracy. Neither facial movements nor pupil size during the baseline were associated with accuracy, but they were predictive of measures of responsivity, such as the probability of not responding to the stimulus or of responding prematurely. These results suggest that the functional role of cortical state on behavior is dynamic and constantly regulated by performance monitoring systems.


title: Analysis and extension of exact mean-field theory with dynamic synaptic currents

abstract:
Neural mass models such as the Jansen-Rit or Wendling systems provide a practical framework for representing and interpreting electro-physiological activity (1-6) in both local and global brain models (7, 8). However, they are only partly derived from first principles. While the post-synaptic potential dynamics are inferred from data and can be grounded on diffusion physics (9-11), Freemans "wave to pulse" (W2P) sigmoid function (12-14), used to transduce mean population membrane potential into firing rate, rests on a weaker theoretical standing. On the other hand, Montbrio et al (15, 16) derive an exact mean-field theory (MPR) from a quadratic integrate and fire neuron model under some simplifying assumptions, thereby connecting microscale neural mechanisms and meso/macroscopic phenomena. The MPR model can be seen to replace Freemans W2P sigmoid function with a pair of differential equations for the mean membrane potential and firing rate variables--a dynamical relation between firing rate and membrane potential--, providing a more fundamental interpretation of the semi-empirical NMM sigmoid parameters. In doing so, we show it sheds light on the mechanisms behind enhanced network response to weak but uniform perturbations. In the exact mean-field theory, intrinsic population connectivity modulates the steady-state firing rate W2P relation in a monotonic manner, with increasing self-connectivity leading to higher firing rates. This provides a plausible mechanism for the enhanced response of densely connected networks to weak, uniform inputs such as the electric fields produced by non-invasive brain stimulation. This new, dynamic W2P relation also endows the neural mass model with a form of "inertia", an intrinsic delay to external inputs that depends on, e.g., self-coupling strength and state of the system. Next, we complete the MPR model by adding the second-order equations for delayed post-synaptic currents and the coupling term with an external electric field, bringing together the MPR and the usual NMM formalisms into a unified exact mean-field theory (NMM2) displaying rich dynamical features. In the single population model, we show that the resonant sensitivity to weak alternating electric field is enhanced by increased self-connectivity and slow synapses.

O_TEXTBOXSignificance

Several decades of research suggest that weak electric fields influence neural processing. A long-standing question in the field is how networks of neurons process spatially uniform weak inputs that barely affect a single neuron but that produce measurable effects in networks. Answering this can help implement electric field coupling mechanisms in neural mass models of the whole brain, and better represent the impact of electrical stimulation or ephaptic communication. This issue can be studied using local detailed computational models, but the use of statistical mechanics methods can deliver "mean-field models" to simplify the analysis. Following the steps of Montbrio et al (15, 16), we show that the sensitivity to inputs such a weak alternating electric field can be modulated by the intrinsic self-connectivity of a neural population, and produce a more grounded set of equations for neural mass modeling to guide further work.

C_TEXTBOX


title: The serine/threonine kinase MINK1 directly regulates the function of promigratory proteins

abstract:
Upregulation of the developmental Wnt/planar cell polarity pathway is observed in many cancers and is associated with cancer development at early and late stages. We recently showed that PRICKLE1 and VANGL2, two core Wnt/PCP components, are overexpressed in triple negative breast cancer and associated with poor prognosis. PRICKLE1 is a cytoplasmic protein phosphorylated by the poorly described serine/threonine kinase MINK1 which triggers its localization at the plasma membrane, a key step for its function. Knockdown experiments have demonstrated that MINK1 and PRICKLE1 contribute to TNBC cell motility and spreading in vitro and in vivo. However, the identity of MINK1 substrates and the role of MINK1 enzymatic activity in this process have not yet been addressed issues.

We carried out a phosphoproteomic strategy and identified novel MINK1 substrates including LL5{beta}. LL5{beta} is a membrane scaffold molecule that anchors microtubules at the cell cortex through its association with the plus-end MT proteins CLASPs to trigger focal adhesion disassembly. LL5{beta} is a prominent member of the MINK1-PRICKLE1 protein complex and is directly phosphorylated by MINK1 that promotes its interaction with CLASP. Using a kinase inhibitor, we demonstrate that the enzymatic activity of MINK1 is involved in the protein complex assembly and localization, and cell migration. Analysis of gene expression data show that the concomitant up-regulation of PRICKLE1 and LL5{beta} mRNA levels encoding MINK1 substrates is associated with a poor metastasis-free survival for TNBC patients. Altogether, our results suggest that MINK1 may represent a potential target in TNBC.


title: Single cell RNA Sequencing Identifies G-protein Coupled Receptor 87 as a Novel Basal Cell Marker of Distal Honeycomb Cysts in Idiopathic Pulmonary Fibrosis

abstract: Idiopathic Pulmonary Fibrosis (IPF) is a progressive and fatal lung disease with limited therapeutic options. Epithelial reprogramming and honeycomb cysts are key pathological features of IPF, however, the IPF distal bronchiole cell subtypes and their potential contribution to IPF development and progression still remain poorly characterized. Here, we utilized single-cell RNA sequencing on enriched EpCAM+ cells of the distal IPF and Donor lung. Using the 10x Genomics platform, we generated a dataset of 47,881 cells and found distinct cell clusters, including rare cell types, such as suprabasal cells recently reported in the healthy lung. We identified G-protein coupled receptor (GPR) 87 as a novel surface marker of distal Keratin (KRT)5+ basal cells. GPR87 expression was localized to distal bronchioles and honeycomb cysts in IPF in situ by RNA Scope and immunolabeling. Modulation of GPR87 in primary human bronchial epithelial cells cultures resulted in impaired airway differentiation and ciliogenesis. Thus, GPR87 is a novel marker and potentially druggable target of KRT5+ basal progenitor cells likely contributing to bronchiole remodeling and honeycomb cyst development in IPF.


title: The Chromatin Remodeling Protein CHD-1 and the EFL-1/DPL-1 Transcription Factor Cooperatively Down Regulate CDK-2 to Control SAS-6 Levels and Centriole Number

abstract:
Centrioles are submicron-scale, barrel-shaped organelles typically found in pairs, and play important roles in ciliogenesis and bipolar spindle assembly. In general, successful execution of centriole-dependent processes is highly reliant on the ability of the cell to stringently control centriole number. This in turn is mainly achieved through the precise duplication of centrioles during each S phase. Aberrations in centriole duplication disrupt spindle assembly and cilia based signaling and have been linked to cancer, primary microcephaly and a variety of growth disorders. Studies aimed at understanding how centriole duplication is controlled have mainly focused on the post-translational regulation of two key components of this pathway: the master regulatory kinase ZYG-1/Plk4 and the scaffold component SAS-6. In contrast, how transcriptional control mechanisms might contribute to this process have not been well explored. Here we show that the chromatin remodeling protein CHD-1 contributes to the regulation of centriole duplication in the C. elegans embryo. Specifically, we find that loss of CHD-1 or inactivation of its ATPase activity can restore embryonic viability and centriole duplication to a strain expressing insufficient ZYG-1 activity. Interestingly, loss of CHD-1 is associated with increases in the levels of two ZYG-1-binding partners: SPD-2, the centriole receptor for ZYG-1 and SAS-6. Finally, we explore transcriptional regulatory networks governing centriole duplication and find that CHD-1 and a second transcription factor, EFL-1/DPL-1 cooperate to down regulate expression of CDK-2, which in turn promotes SAS-6 protein levels. Disruption of this regulatory network results in the overexpression of SAS-6 and the production of extra centrioles.

Author SummaryCentrioles are cellular constituents that play an important role in cell reproduction, signaling and movement. To properly function, centrioles must be present in the cell at precise numbers. Errors in maintaining centriole number result in cell division defects and diseases such as cancer and microcephaly. How the cell maintains proper centriole copy number is not entirely understood. Here we show that two transcription factors, EFL-1/DPL-1 and CHD-1 cooperate to reduce expression of CDK-2, a master regulator of the cell cycle. We find that CDK-2 in turn promotes expression of SAS-6, a major building block of centrioles. When EFL-1/DPL-1 and CHD-1 are inhibited, CDK-2 is overexpressed. This leads to increased levels of SAS-6 and excess centrioles. Our work thus demonstrates a novel mechanism for controlling centriole number and is thus relevant to those human diseases caused by defects in centriole copy number control.


title: Uip4p modulates nuclear pore complex function in Saccharomyces cerevisiae

abstract: A double membrane bilayer perforated by nuclear pore complexes (NPCs) governs the shape of the nucleus, the prominent distinguishing organelle of a eukaryotic cell. Despite the absence of lamins in yeasts, the nuclear morphology is stably maintained and shape changes occur in a regulated fashion. In a quest to identify factors that contribute to regulation of nuclear shape and function in Saccharomyces cerevisiae, we used a fluorescence imaging based approach. Here we report the identification of a novel protein, Uip4p, that is required for regulation of nuclear morphology. Loss of Uip4 compromises NPC function and loss of nuclear envelope (NE) integrity. Our localisation studies show that Uip4 localizes to the NE and endoplasmic reticulum (ER) network. Furthermore, we demonstrate that the localization and expression of Uip4 is regulated during growth, which is crucial for NPC distribution.


title: Seasonal migration as a life history trait facilitating adaptation to climate change

abstract: Predicting the geographic range of species and their response to variation in climate are entwined goals in conservation and evolutionary ecology. Species distribution models (SDMs) are foundational in this effort and used to visualize the geographic range of species as the spatial representation of its realized niche, or when based only on climate, its climate niche. SDMs can also forecast shifts in species range given climate change, but often lack of empirical support for causal links between climate and demography, yielding uncertain predictions. We addressed such uncertainties whilst also exploring the role of migration and resident life-histories in climate adaptation in mobile animals using 48 years of detailed demographic and climate data for song sparrows (Melospiza melodia), a polytypic species that varies in migratory life history. We developed SDMs representing demographic and climate niches of migratory and resident populations in western North America from California (CA) to Alaska (AK) using data from a focal population in British Columbia (BC) and 1.2 million citizen science observations. Distributions of resident and migrant populations predicted by each model agreed strongly (72.8%) in the region of our focal population, but less well in regions with dissimilar climates. Mismatches were largest in CA, smaller in AK, but in all cases supported the hypothesis that climate influences the evolution of migration and limits year-round residency. Our results imply that migrants predominated in our focal population a century ago, but that climate change has favored range expansions by non-migratory phenotypes and facilitated an upward shift in the elevational range of residents. We suggest long-term studies are crucial to evaluating the predictions of SDMs positing causal links between climatic conditions and species demography. We found such links to be robust regionally and particularly useful to elucidating the potential for migration or residence to facilitate adaptation to climate change.

category: ecology


title: HiCImpute: A Bayesian Hierarchical Model for Identifying Structural Zeros and Enhancing Single Cell Hi-C Data.

abstract: Single cell Hi-C techniques enable one to study cell to cell variability in chromatin interactions. However, single cell Hi-C (scHi-C) data suffer severely from sparsity, that is, the existence of excess zeros due to insufficient sequencing depth. Complicate things further is the fact that not all zeros are created equal, as some are due to loci truly not interacting because of the underlying biological mechanism (structural zeros), whereas others are indeed due to insufficient sequencing depth (sampling zeros), especially for loci that interact infrequently. Differentiating between structural zeros and sampling zeros is important since correct inference would improve downstream analyses such as clustering and discovery of subtypes. Nevertheless, distinguishing between these two types of zeros has received little attention in the single cell Hi-C literature, where the issue of sparsity has been addressed mainly as a data quality improvement problem. To fill this gap, in this paper, we propose HiCImpute, a Bayesian hierarchy model that goes beyond data quality improvement by also identifying observed zeros that are in fact structural zeros. HiCImpute takes spatial dependencies of scHi-C 2D data structure into account while also borrowing information from similar single cells and bulk data, when such are available. Through an extensive set of analyses of synthetic and real data, we demonstrate the ability of HiCImpute for identifying structural zeros with high sensitivity, and for accurate imputation of dropout values in sampling zeros. Downstream analyses using data improved from HiCImpute yielded much more accurate clustering of cell types compared to using observed data or data improved by several comparison methods. Most significantly, HiCImpute-improved data has led to the identification of subtypes within each of the excitatory neuronal cells of L4 and L5 in the prefrontal cortex.


title: Sex differences in the reward value of familiar mates in prairie voles

abstract: The rewarding properties of social interactions facilitate relationship formation and maintenance. Prairie voles are one of the few laboratory species that form selective relationships, manifested as "partner preferences" for familiar partners versus strangers. While both sexes exhibit strong partner preferences, this similarity in outward behavior likely results from sex-specific neurobiological mechanisms. We recently used operant conditioning to demonstrate that females work harder for access to a familiar versus unfamiliar conspecific of either sex, while males worked equally hard for access to any female, indicating a key sex difference in social motivation. As tests were performed with one social target at a time, males might have experienced a ceiling effect, and familiar females might be more relatively rewarding in a choice scenario. Here we performed a social choice operant task in which voles could repeatedly lever-press to gain temporary access to either the chamber containing their mate or one containing a novel opposite-sex vole. Females worked hardest to access their mate, while males pressed at similar rates for either female. Individual male behavior was heterogeneous, congruent with multiple mating strategies in the wild. Voles exhibited preferences for favorable over unfavorable environments in a non-social operant task, indicating that lack of social preference does not reflect lack of discrimination between chambers. Oxytocin receptor genotype at the intronic SNP NT213739 replicated a prior association with stranger-directed aggression within the test. These findings suggest that convergent preference behavior in male and female voles results from sex-divergent pathways, particularly in the realm of social motivation.


title: Engineering a thixotropic and biochemically tunable hyaluronan and collagen bioink for biofabrication of multiple tissue construct types

abstract: The field of three-dimensional (3D) bioprinting has advanced rapidly in recent years. Significant reduction in the costs associated with obtaining functional 3D bioprinting hardware platforms is both a cause and a result of these advances. As such, there are more laboratories than ever integrating bioprinting methodologies into their research. However, there is a lack of standards in the field of biofabrication governing any requirements or characteristics to support cross-compatibility with biomaterial bioinks, hardware, and different tissue types. Here we describe a modular extracellular matrix (ECM) inspired bioink comprised of collagen and hyaluronic acid base components that: 1) employ reversible internal hydrogen bonding forces to generate thixotropic materials that dynamically reduce their elastic moduli in response to increased shear stress, thus enabling increased compatibility with printing hardware; and 2) modular addons in the form of chemically-modified fibronectin and laminin that when covalently bound within the bioink support a variety of tissue types, including liver, neural, muscle, pancreatic islet, and adipose tissue. These features aim to accelerate the deployment of such bioinks for tissue engineering of functional constructs in the hands of various end users.

category: bioengineering


title: Single-cell transcriptomics elucidates in vitro reprogramming of human intestinal epithelium cultured in a physiodynamic gut-on-a-chip

abstract: The microphysiological human gut-on-a-chip has demonstrated in vivo-relevant cellular fidelity of intestinal epithelium compared to its cultures in a static condition1, 2. Microfluidic control of morphogen gradients and mechanical cues robustly induced morphological histogenesis with villi-like three-dimensional (3D) microarchitecture, lineage-associated cytodifferentiation, and physiological functions of a human intestinal Caco-2 epithelium3, 4. However, transcriptomic dynamics that orchestrates morphological and functional reprogramming of the epithelium in a microphysiological culture remains elusive. Single-cell transcriptomic analysis revealed that a gut-on-a-chip culture that offers physiological motions and flow drives three distinctive subclusters that offer distinct gene expression and unique spatial representation in 3D epithelial layers. The pseudotemporal trajectory of individual cells visualized the evolutionary transition from ancestral genotypes in static cultures into more heterogeneous phenotypes in physiodynamic cultures on cell cycles, differentiation, and intestinal functions including digestion, absorption, drug transport, and metabolism of xenobiotics. Furthermore, the inversed transcriptomic signature of oncogenes and tumor-suppressor genes of Caco-2 cells verified that a gut-on-a-chip culture drives a postmitotic reprogramming of cancer-associated phenotypes. Thus, we discovered that a physiodynamic on-chip culture is necessary and sufficient for a cancer cell line to be reprogrammed to elicit in vivo-relevant heterogeneous cell populations with restored normal physiological signatures.


title: pSiteExplorer: Visualization of the mass spectrometric data underlying phospho-site quantification

abstract: Mass spectrometry based phospho-proteomics is a widely used approach to assess protein phosphorylation. Intensities of phospho-peptide ions are obtained by integrating the MS signal over their chromatographic peaks. How individual peptide measurements mapping to the same phospho-site are combined for the quantification of the given site is, however, in most cases hidden from researchers conducting, reviewing, and reading these studies. I here describe pSiteExplorer, an R script that visualizes the peak intensities associated with phospho-sites in MaxQuant output tables. Barplots of MS intensities originating from phospho-peptides with distinct amino acid sequences due to missed cleavages, different numbers of phosphates and from all off-line chromatographic fractions and charge states are displayed. This tool will help gaining a deeper insight into phospho-site quantifications by contrasting individual and summed phospho-peptide intensities with the site-level values derived by MaxQuant. This will support the validation of quantification results, for example, for the selection of candidates for follow-up studies.


title: Multiplexed triage of candidate biomarkers in plasma using internal standard triggered-parallel reaction monitoring mass spectrometry

abstract: Despite advances in proteomic technologies, clinical translation of plasma biomarkers remains low, partly due to a major bottleneck between the discovery of candidate biomarkers and downstream costly clinical validation studies. Due to a dearth of multiplexable assays, generally only a few candidate biomarkers are tested, and the validation success rate is accordingly low. Here, we demonstrate the capability of internal standard triggered-parallel reaction monitoring (IS-PRM) to prioritize candidate biomarkers for validation studies. A 5,176-plex assay coupling immunodepletion and fractionation with IS-PRM was developed and implemented in human plasma to quantify peptides representing 1,314 breast cancer biomarker candidates. Compared to prior approaches using data-dependent analysis, IS-PRM showed improved sensitivity (912 vs 295 proteins quantified) and precision (CV 0.1 vs 0.27) enabling rank-ordering of candidate biomarkers for validation studies. The assay greatly expands capabilities for quantification of large numbers of proteins and is well suited for prioritization of viable candidate biomarkers.

category: cancer biology


title: Identification and validation of putative target genes regulated by miR-34 in cervical cancer

abstract:
The emergence of large-scale transcriptomic data provides the opportunity for identifying novel putative targets of microRNAs (miRNAs). In this study, we followed a computational pipeline to predict the candidate gene targets of the miR-34 family. This approach integrates the expressions of miR-34 with genes of heterogeneous primary cervical epithelial squamous cell carcinomas (CESC). Integration of miR-34b and epithelial-mesenchymal transition (EMT) regulated genes has also been focussed, EMT being a reversible process that fuels cancer metastasis. An in-silico approach involving three processes was carried out with CESC datasets of the cancer atlas genome (TCGA), which includes correlation analysis, target prediction database lookup, functional enrichment, network analysis, survival analysis, and EMT score derivation. The results indicate that the miR-34 family may regulate the candidate genes of the mTOR pathway, cell cycle (CCND2) and cell adhesion functions (FZD4). Further, the study reveals the possible regulation of EMT signature genes, namely BMP7, CAV1 and ID2by miR-34b. Further, these transcriptomic signatures were validated in a subset of CESC from the South Asian Indian population (n = 10) and in non-cancerous cervical tissues (n = 5). Upon stably expressing miR-34b in cervical cancer cells (C33A and HeLa), we found repression of these candidate genes and a low negative correlation (r2 = -0.07) between miR-34b and EMT score indicating FN1 as its putative target. Together, these studies revealed the potential targets of the miR-34 family, especially miR-34b, with the hope that they would emerge as potential biomarkers and/or promising therapeutic targets in CESC.

Brief DescriptionA combined analysis of miR-34 and gene expression in heterogeneous primary CESC, along with the integration of miR-34b and EMT regulated genes, was used to predict the candidate gene targets of the miR-34 family. The results show that the miR-34 family may regulate the mTOR pathway, cell cycle, and cell adhesion functions. Further, we showed that EMT signature genes (BMP7, CAV1, ID2, FN1) were regulated by miR-34b in CESC and cervical cancer cells.