title: Combination treatment can hinder the evolution of resistance to antimicrobial peptides

abstract: Antibiotic resistant microbial pathogens are becoming a major threat to human health. Therefore, there is an urgent need to develop new alternatives to conventional antibiotics. One such promising alternative is antimicrobial peptides (AMPs), which are produced by virtually all organisms and typically inhibit bacteria via membrane disruption. However, previous studies demonstrated that bacteria can rapidly develop AMP resistance. Here, we study whether combination therapy, known to be able to inhibit the evolution of resistance to conventional antibiotics, can also hinder the evolution of AMP resistance. To do so, we evolved the opportunistic pathogen S. aureus in the presence of individual AMP, AMP pairs, and a combinatorial antimicrobial peptide library. Treatment with some AMP pair indeed hindered the evolution of resistance compared with individual AMPs. In particular, resistance to pairs was delayed when resistance to the individual AMPs came at a cost of impaired bacterial growth, and did not confer cross-resistance to other tested AMPs. The lowest level of resistance evolved during treatment with the combinatorial antimicrobial peptide library termed random antimicrobial peptide mixture, which contains more than a million different peptides. A better understanding of how AMP combinations affect the evolution of resistance is a crucial step in order to design  resistant proof AMPs cocktails that will offer a sustainable treatment option for antibiotic resistant pathogens.


title: STOmicsDB: a database of Spatial Transcriptomic data

abstract: Recent technological development in spatial transcriptomics allows researchers to measure gene expression of cells and their spatial locations at the almost single-cell level, which generates detailed biological insight into biological processes. However, specialized spatial transcriptomics databases are rare. Here, we present the Spatial TranscriptOmics DataBase (STOmicsDB), a user-friendly database with multifunctions including search of relevant publications and tools, public dataset visualization, customized specialized databases, new data archive, and online analysis. The current version of STOmicsDB consists of 141 curated spatial transcript datasets covering 12 species, and includes 5,618 spatial multi-omics publications and 674 tools. STOmicsDB is freely accessible at https://db.cngb.org/stomics/.

category: genomics


title: Two types of hand withdraw movement to place food in the mouth mediated by somatosensation in 22-species of strepsirrhines

abstract: The evolution of visual control of the hand to assist feeding by primates is uncertain but in anthropoid primates vision contributes not only to reaching for food and grasping it but also to the withdraw movement that brings food to the mouth. The strepsirrhines are a relatively large monophyletic group of Euarchontoglires near the base of the primate cladogram that are described as using vision to reach for food, but it is not known whether they use vision to assist the withdraw movement. The present study answere this question in 22 species of captive strepsirrhines from 6 of the seven strepsirrhine families, Daubentoniidae, Cheirogaleidae, Indriidae, Lemuridae, Lorisidae and Galagidae. Animals were videorecorded as they ate their normal food provisions. Dependent measures for analyses were ground withdraw movements, bringing grasped food to the mouth, and inhand withdraw movements, brining food held in the hand to the mouth, as well as the posture and head movements associated with each type of withdraw. Frame-by-frame scores from the video record showed that there were large differences between and within strepsirrhine families in these movements. Nevertheless, for all species, the withdraw movement was mediated by somatosensation, with mouth reaching and perioral contact with food determining how food was eventually eaten. Nonvisual behavior also contributed to food grasping as many species sniffed food before or during grasping. Even amongst species that made most use of the hand for their withdraws, the insectivores Loris lydekkerianus and Galago senegalensis, and herbivores, Hapalemur simus and Eulemur flavifrons, perioral contact was used to orient food for biting. The use of somatosensation and the absence of vision in mediating getting food in strepsirrhines suggests that visual mediation of the withdraw is an anthropoid innovation.

category: animal behavior and cognition


title: Do males know? Evidence-driven rainmaking of male bonobos to meet fertile phase of females

abstract:
Female bonobos exhibit prolonged receptivity, one suggested function of which is to lower male mating competition. However, it is questionable whether easier access to receptive females can reduce male-male competition, given the exclusive nature of male reproductive success. We tested whether males could determine the fertile phase of females. We found that ovulation probability predicted male mating effort. High-ranking males copulated with females with higher fertility, and male-male agonistic interactions increased when there were fertile females in the party. When there were multiple females with maximal swelling, males concentrated their mating effort on females with an older infant whose maximal swelling started earlier, and they continued mating efforts until detumescence (rainmaking). These findings suggest that male bonobos distinguish between fertile and non-fertile phases of females and that having more receptive females in the party does not reduce male-male competition for fertile females.

TeaserMales use the rainmakers rule to meet the periovulatory phase of the female bonobo for better reproductive success.


title: Do males know? Evidence-driven rainmaking mating strategy by male bonobos to meet the fertile window of females


title: In vitro engineering of the lung alveolus

abstract: Therapeutic lung regeneration is predicated upon successful reconstitution of lung alveoli, the functional units of gas exchange. Here, we identify requisite multimodal cues that are critical to reconstructing the alveolar epithelium and alveoli in lung scaffolds. Alveolar reconstruction in vitro is divided into several distinct phases. In the first phase, endothelial cells coordinate with fibroblasts and select exogenous factors to promote alveolar scaffold population with surfactant-secreting alveolar epithelial type 2 cells (AEC2s). After formation of organized epithelial alveolar-like structures, subsequent withdrawal of Wnt and FGF agonism synergizes with tidal-level mechanical strain to induce differentiation of AEC2s to squamous type 1 AECs (AEC1s) in cultured alveoli, in situ. These results outline a rational strategy to engineer an alveolus of AEC2s and AEC1s contained within epithelial-mesenchymal-endothelial units, and reveal the critical interplay amongst biochemical, cellular, and mechanical niche cues within the reconstituting alveolus.

category: bioengineering


title: Step length and the extrapolated center of mass co-vary to stabilize the margin of stability in the anterior-posterior direction during unobstructed and obstructed walking

abstract: During steady-state gait, the instantaneous passive dynamics, quantified using the margin of stability in the anterior-posterior (AP) direction (MOSAP), are unstable. Although the instability indicates that substantial control will be required to reject a perturbation, the instability also allows for forward progression with low energy expenditure when the gait is unperturbed. Here, we examined whether humans control MOSAP to exploit the passive dynamics, and whether they adjust MOSAP when the threat to AP balance is increased. We computed MOSAP for six consecutive steps when healthy young adults performed unobstructed gait trials, and when they approached and crossed an obstacle. Uncontrolled manifold analysis indicated that the extrapolated center of mass and step length co-varied to maintain MOSAP for all steps during unobstructed and obstructed gait. Furthermore, the MOSAP values indicated unstable passive dynamics for all steps except while crossing the obstacle with the leading limb. Based on these results, we concluded that the MOSAP is controlled to exploit the passive dynamics and achieve forward progression at low energetic cost. Finally, MOSAP increased (indicating more stable dynamics) while approaching and crossing the obstacle with the leading limb, and then declined for later steps, indicating a fluid tradeoff between stability and energy efficiency.


title: Porous yet Dense matrices: using ice to shape collagen 3D cell culture systems with increased physiological relevance

abstract: Standard in vitro cell culture is one of the pillars of biomedical science. However, there is increasing evidence that 2D systems provide biological responses that are often in disagreement with in vivo observations, partially due to limitations in reproducing the native cellular microenvironment. 3D materials that are able to mimic the native cellular microenvironment to a greater extent tackle these limitations. Here, we report Porous yet Dense (PyD) type I collagen materials obtained by ice-templating followed by topotactic fibrillogenesis. These materials combine extensive macroporosity, favouring the cell migration and nutrients exchange, as well as dense collagen walls, which mimic locally the Extracellular Matrix. When seeded with Normal Human Dermal Fibroblasts (NHDFs), PyD matrices allow for a faster and more extensive colonisation when compared with equivalent Non-Porous matrices. The textural properties of the PyD materials also impact cytoskeletal and nuclear 3D morphometric parameters. Due to the effectiveness in creating a biomimetic 3D environment for NHDFs and the ability to promote cell culture for more than 28 days without subculture, we anticipate that PyD materials could configure an important step towards in vitro systems applicable to other cell types and with higher physiological relevance.


title: kb_DRAM: Annotating and functional profiling of genomes with DRAM in KBase

abstract:
SummaryAnnotation is predicting the location of and assigning function to genes in a genome. DRAM is a tool developed to annotate bacterial, archaeal and viral genomes derived from pure cultures or metagenomes. DRAM distills multiple gene annotations to summaries of functional potential. Despite these benefits, a downside of DRAM is processing requires large computation resources, which limits its accessibility, and it did not integrate with downstream metabolic modelling tools. To alleviate these constraints, DRAM and the viral counterpart, DRAM-v, are now available and integrated in the freely accessible KBase cyberinfrastructure. With kb_DRAM users can generate DRAM annotations and functional summaries from microbial or viral genomes in a point and click interface, as well as generate genome scale metabolic models from these DRAM annotations.

Availability and ImplementationThe kb_DRAM software is available at https://github.com/shafferm/kb_DRAM. The kb_DRAM apps on KBase can be found in the catalog at https://narrative.kbase.us/#catalog/modules/kb_DRAM. A narrative with examples of running all KBase apps is available at https://kbase.us/n/88325/84/.

ContactMichael Shaffer, michael.t.shaffer@colostate.edu; Kelly Wrighton, kelly.wrighton@colostate.edu

Supplementary InformationSupplementary data are available at Bioinformatics online.

category: bioinformatics


title: Palo: Spatially-aware color palette optimization for single-cell and spatial data

abstract:
SummaryIn the exploratory data analysis of single-cell or spatial genomic data, single cells or spatial spots are often visualized using a two-dimensional plot where cell clusters or spot clusters are marked with different colors. With tens of clusters, current visualization methods often assigns visually similar colors to spatially neighboring clusters, making it hard to identify the distinction between clusters. To address this issue, we developed Palo that optimizes the color palette assignment for single-cell and spatial data in a spatially-aware manner. Palo identifies pairs of clusters that are spatially neighboring to each other and assigns visually distinct colors to those neighboring pairs. We demonstrate that Palo leads to improved visualization in real single-cell and spatial genomic datasets.

AvailabilityPalo R package is freely available at https://github.com/Winnie09/Palo.

Contactzhicheng.ji@duke.edu


title: Planemo: a command-line toolkit for developing, deploying, and executing scientific data analyses

abstract: There are thousands of well-maintained high-quality open-source software utilities for all aspects of scientific data analysis. For over a decade, the Galaxy Project has been providing computational infrastructure and a unified user interface for these tools to make them accessible to a wide range of researchers. In order to streamline the process of integrating tools and constructing workflows as much as possible, we have developed Planemo, a software development kit for tool and workflow developers and Galaxy power users. Here we outline Planemos implementation and describe its broad range of functionality for designing, testing and executing Galaxy tools, workflows and training material. In addition, we discuss the philosophy underlying Galaxy tool and workflow development, and how Planemo encourages the use of development best practices, such as test-driven development, by its users, including those who are not professional software developers. Planemo is a mature project widely used within the Galaxy community which has been downloaded over 80,000 times.


title: Blood-based transcriptomic signature panel identification for cancer diagnosis: Benchmarking of feature extraction methods

abstract: Liquid biopsy has shown promise for cancer diagnosis due to its minimally invasive nature and the potential for novel biomarker discovery. However, the low concentration of relevant blood-based biosources and the heterogeneity of samples (i.e. the variability of relative abundance of molecules identified), pose major challenges to biomarker discovery. Moreover, the number of molecular measurements or features (e.g., transcript read counts) per sample could be in the order of several thousand, whereas the number of samples is often substantially lower, leading to the curse of dimensionality. These challenges, among others, elucidate the importance of a robust biomarker panel identification or feature extraction step wherein relevant molecular measurements are identified prior to classification for cancer detection. In this work, we performed a benchmarking study on eleven feature extraction methods using transcriptomic profiles derived from different blood-based biosources. The methods were assessed both in terms of their predictive performance and the robustness of the biomarker panels in diagnosing cancer or stratifying cancer subtypes. While performing the comparison, the feature extraction methods are categorised into feature subset selection methods and transformation methods. A transformation feature extraction method, namely PLS-DA, was found to perform consistently superior in terms of classification performance, while a feature subset selection method, namely Ranger, selected feature sets that were the most robust to sub-sampling in terms of consistently selecting the same features. As part of the benchmarking study, a generic pipeline has been created and made available as an R package to ensure reproducibility of the results and allow for easy extension of this study to other datasets.


title: Novel tuberculosis combination regimens of two and three-months therapy duration

abstract: The tuberculosis treatment duration is 4-6 months, but can be 18-20 months with multidrug resistance. Ultrashort regimens that cure regardless of resistance status ["pan-tuberculosis regimens"] would be a major step towards global tuberculosis control. Starting with seven drugs [bedaquiline, delamanid, pretomanid, OPC-167832, sutezolid, moxifloxacin, and pyrazinamide] in clinical testing, we calculated that there were thousands of possible novel combinations to evaluate. We used mathematical and pharmacokinetics/pharmacodynamic-based hollow fiber modeling to reduce this complexity to nine combinations, which we tested. Each regimens fast and slow growth bacteria kill-slope trajectories and times-to-extinction were estimated and translated to minimum required therapy duration in patients. Sputum microbial kill trajectories of two combination regimens that have undergone clinical testing were correctly predicted by this approach. Four pan-tuberculosis regimens were predicted to achieve relapse-free cure after 2-3 months therapy in patients. The kill-slopes were used to design expedited clinical trials that minimize patients risks.

category: pharmacology and toxicology


title: Effect of GDF15 on acetaminophen (APAP)-induced liver injury in mice

abstract: Acetaminophen (APAP)-induced liver injury (AILI) has been recognized as a pivotal contributor to drug-induced liver failure in western countries, but its molecular mechanism remains poorly understood. After data mining using the Gene Expression Omnibus (GEO) database, we found that the gene expression of growth differentiation factor 15 (GDF15) increased significantly after APAP-induced hepatotoxicity in mouse liver. However, the precise role of GDF15 in AILI has yet to be clarified. To deal with this question, we first examined the temporal gene expression pattern of GDF15 after APAP treatment. Next, overexpression of hepatic GDF15 using adeno-associated virus serotype 8 (AAV8) was employed to investigate the role of GDF15 in AILI. In addition, therapeutic administration of recombinant human GDF15 (rhGDF15) was conducted to probe its curative effect after APAP overdose. We observed that protein expression of GDF15 was significantly increased after APAP overdose. However, there were no significant differences in AILI-related outcomes after overexpression or supplementation of GDF15. The contents of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), morphological feature of liver, the level of reduced form of glutathione (GSH), the activation of c-Jun-N-terminal kinase (JNK), the expression of genes relevant to oxidative stress and cell proliferation were not remarkably changed between AAV8-GFP and AAV8-Gdf15 groups. Although the liver injury was not improved by rhGDF15 administration, the gene expression levels of glutamate-cysteine ligase modifier subunit (Gclm) and glutamate-cysteine ligase catalytic subunit (Gclc) which function downstream of Nrf2 signaling pathway were significantly increased. Altogether, despite a noticeable elevated expression after APAP overdose, GDF15 overexpression or supplementation exerted no significant ameliorative effect on AILI. Further mechanism and consequence of Gclm and Gclc elevation by GDF15 remains to be elucidated.


title: Hippocampal theta and episodic memory

abstract: Computational models of rodent physiology implicate hippocampal theta as a key modulator of learning and memory (Buzsaki & Moser, 2013; J. E. Lisman & Jensen, 2013), yet human hippocampal recordings have shown divergent theta correlates of memory formation. Herweg et al. (2020) suggest that decreases in memory-related broadband power mask narrowband theta increases. Their survey also suggests that thetas role in memory appears strongest in contrasts that isolate associative retrieval processes and when aggregating signals across large brain regions. We evaluate these hypotheses by analyzing human hippocampal recordings captured as 161 neurosurgical patients performed a free recall task. Using the irregular-resampling auto-spectral analysis to separate broad and narrow-band components of the field potential we show: 1) Broadband and narrowband components of theta exhibit opposite effects, with broadband signals decreasing and narrow-band theta increasing during successful encoding; 2) Whereas low-frequency theta oscillations increase prior to successful recall, higher-frequency theta and alpha oscillations decrease, masking thetas positive effect when aggregating across the full band; 3) Thetas effects on memory encoding and retrieval do not differ between reference schemes that accentuate local signals (bipolar) and those that aggregate across large reference (whole brain average). In line with computational models that ascribe a fundamental role for hippocampal theta in memory our large-scale study of human hippocampal recordings shows that 3-4 Hz theta oscillations reliably increase during successful memory encoding and prior to spontaneous recall of previously studied items.

category: neuroscience


title: Astrocyte mitochondria produce nitric oxide from nitrite to modulate cerebral blood flow during brain hypoxia

abstract: In low oxygen conditions, increases in cerebral blood flow maintain brain oxygen delivery but the mechanisms underlying hypoxia-induced dilations of cerebral vasculature are incompletely understood. Here we show that astrocytes accumulate nitrite and in response to hypoxia produce nitric oxide via mitochondrial reduction of nitrite by a molybdenum-containing enzyme sulfite oxidase. These data suggest that astrocytes can modulate cerebral blood flow in accord with brain tissue oxygenation via mitochondrial production of nitric oxide.


title: Virgin Birth: A genetic basis for facultative parthenogenesis

abstract: Sexual reproduction evolved 1-2 billion years ago and underlies the biodiversity of our planet. Nevertheless, devolution of sexual into asexual reproduction can occur across all phyla of the animal kingdom. The genetic basis for how parthenogenesis can arise is completely unknown. To understand the mechanism and benefits of parthenogenesis, we have sequenced the genome of the facultative parthenogen, Drosophila mercatorum, and compared its organisation and expression pattern during parthenogenetic or sexual reproduction. We identified three genes, desat2, Myc, and polo in parthenogenetic D. mercatorum that when mis-regulated in a non-parthenogenetic species, D. melanogaster, enable facultative parthenogenetic reproduction. This simple genetic switch leads us to propose that sporadic facultative parthenogenesis could evolve as an  escape route preserving the genetic lineage in the face of sexual isolation.

category: evolutionary biology


title: Neutrophil Peptidylarginine Deiminase 4 is Essential for Detrimental Age-related Cardiac Remodeling and Dysfunction in Mice

abstract:
AimsWe aimed to study the long-term effect of neutrophils on cardiac health during the process of natural aging. We hypothesized that neutrophil PAD4, via its role in neutrophil extracellular trap (NET) formation, is involved in myocardial remodeling and cardiac fibrosis development, resulting in turn in impaired cardiac function.

Methods and resultsWe generated mice with deletion of Padi4, a NET-essential gene, under the neutrophil-specific promoter S100A8 (PAD4fl/flMRP8Cre+). These mice and their littermate controls were aged for two years (coinciding with approximately 70 years of age in humans; the age at which HF is the number one cause of hospitalization), after which cardiac function and remodeling were evaluated. We performed a comprehensive echocardiography analysis including both structural and functional parameter measurements. Deletion of PAD4 in neutrophils resulted in a protection against both systolic, and diastolic dysfunction. Interestingly, these mice showed protection against age induced fibrosis, detected as through the absence of cardiac collagen deposition. To explore this further, cardiac gene expression and plasma cytokine levels were evaluated. Here we saw a clear impact of PAD4-deficiency on cardiac neutrophil recruitment, with both cardiac genes as well as plasma cytokines involved in neutrophil recruitment being downregulated in aged PAD4fl/flMRP8Cre+ animals in comparison to littermate PAD4fl/fl controls, including decreased plasma levels of C-X-C ligand 1 (CXCL1).

ConclusionOur data confirms neutrophil PAD4 involvement in heart failure progression by promoting cardiac remodeling, leading to cardiac dysfunction with old age. We saw that the deletion of PAD4 specifically in neutrophils had an influence on the CXCL1-CXCR2 axis, which is known to be involved in HF development.

Translational perspectiveIn the developed world, an estimated 2% of the population lives with heart failure (HF). HF can be viewed as an upcoming pandemic, which is only expected to increase due to the aging of the global population. Therefore, research in HF development and progression are needed to establish new avenues for treatment and improved therapies. In our study, we were able to show the contribution of neutrophil PAD4 to HF pathogenesis, providing new supporting evidence for the involvement of NETs in detrimental cardiac remodeling.

category: physiology


title: The sunflower WRINKLED1 transcription factor regulates fatty acid biosynthesis genes through an AW box binding sequence with a particular base bias

abstract: Sunflower (Helianthus annuus L.) is an important oilseed crop in which the biochemical pathways leading to seed oil synthesis and accumulation have been widely studied. However, how these pathways are regulated is less well understood. The WRINKLED1 (WRI1) transcription factor is considered a master regulator in the transcriptional control of triacylglycerol biosynthesis, acting through the AW box binding element (CNTNG(N)7CG) that resides in the promoter of target genes. Here, we identified the sunflower WRI1 gene and characterized its activity in electrophoretic mobility shift assays. We studied its role as a co-regulator of sunflower genes involved in plastidial fatty acid synthesis, identifying genes bound by this transcription factor. Sunflower WRI1-targets included genes encoding all subunits of the pyruvate dehydrogenase complex, the -CT and BCCP genes of the acetyl-CoA carboxylase complex, genes encoding acyl carrier proteins and key genes of the fatty acid synthase complex (KASIII, KASI and KAR), together with the FATA1 gene. As such, sunflower WRI1 regulates seed plastid fatty acid biosynthesis in a coordinated manner, establishing a WRI1 push and pull strategy that drives oleic acid synthesis for its export into the cytosol. We also analyzed the sequence of the functional sunflower AW box, determining the base bias at the N positions in the active sunflower AW box motif. Accordingly, we conclude that the sunflower AW box is sequence-sensitive at the non-conserved positions, enabling WRI1-binding. Moreover, we found that sunflower WRI1 could bind to a non-canonical AW-box motif, opening the possibility of searching for new target genes.

category: plant biology


title: Interferon-induced lysosomal membrane permeabilization causes cDC1-deserts in tumors.

abstract: The landscape of migratory conventional dendritic cells (cDCs) comprises XCR1+CD11b- cDC1s, XCR1-CD11b+ cDC2s and intermediate XCR1low/negCD11b- states. cDC1 are unique in their ability to cross-prime lymph node CD8 T cells in a CD4 T cell-dependent manner. In perturbed cancer states cDC1s become particularly scarce in tumors and tumor draining lymph nodes, which decreases T cell infiltration, immunotherapy responses and patient survival. The causes of cDC1 paucity are not fully understood and no specific therapy currently exists. Here, we find that cDC1s undergo apoptosis in tumor microenvironments. Gene expression analysis of independent murine and human RNA sequencing datasets point to a shared cDC1 lysosomal stress response state across various tumors. Modeling primary cDC1 behavior in lung tumors in vivo and ex vivo, we show that two distinct yet interconnected pathways converge to cause apoptosis of cDC1: mTOR inhibition leads to an increase in the proteolytic activity of lysosomes, while lysosomal membrane permeabilization (LMP) allows the release of proteolytic enzymes to the cytosol and apoptotic death. Pathway and regulon analysis of the cDC1 transcriptome suggest that mTOR inhibition and lysosomal stress happen downstream of type I IFNs. Accordingly, exposure of the Mutu cDC1 line to type I IFNs inhibits mTOR, stresses lysosomes and triggers LMP and death. Further supporting this finding, in mixed bone-marrow chimeras IFNRA deletion rescues primary cDC1s from mTOR inhibition, lysosomal stress, LMP and death. We have therefore elucidated IFN-induced lysosomal death as a key mechanism of cDC1s paucity in tumors that should be prevented to increase tumor immunity through reinvigoration of the cDC1 pool.

category: immunology


title: Arctic phytoplankton spring bloom diversity across the marginal ice zone in Baffin Bay

abstract: Phytoplankton under-ice blooms have been recently recognized as an important Arctic phenomenon for global primary production and biogeochemical cycling. Drastic sea-ice decline in both extension and thickness enables the development of early blooms, sometimes hundreds of kilometers beneath the pack ice. Baffin Bay is a semi-enclosed sea where Arctic and North Atlantic water masses interact. It is totally covered by sea-ice by March and ice-free by August/September. In the present work, we investigated the phytoplankton community structure across the marginal ice zone between the ice-free, Atlantic-influenced, east and the ice-covered, Arctic-influenced, west Baffin Bay using 18S rRNA high-throughput amplicon sequencing, flow cytometry cell counting and numerous environmental and biological data collected and compiled in the scope of the Green Edge project. Sampling was performed during June-July 2016 in a total of 16 stations with around 6 depths each. Stations were clustered into "Under Ice" (UI), "Marginal Ice Zone" (MIZ) and "Open Water" (OW) on the basis of its sea ice cover upon sampling. Phytoplankton community structure was analyzed by 18S rRNA metabarcoding with the microdiversity approach. The UI sector was characterized by a shallow nitracline, high pico-phytoplankton abundance and a shared dominance between Micromonas and Phaeocystis in the 0.2-3 {micro}m size fraction, as well as an increased contribution of Cryptophyceae and non-diatom Ochrophyta in the 3-20 {micro}m size fraction. Several amplicon sequence variants (ASVs) were flagged as indicator for the UI+MIZ sector group, including known ice-associated taxa such as the diatoms Melosira arctica and Pseudo-nitzschia seriata, but also specific ASVs assigned to the green alga Micromonas polaris and the cryptophyte Falcomonas daucoides, the silicoflagellate Dictyocha speculum, one member of the uncultivated MOCH-2 group, and a Pterosperma sp. (green algae) rarely seen in other metabarcoding datasets, including from the Arctic. The OW sector harbored a community adapted to a nutrient-depleted/high light environment, with a significant contribution of the haptophytePhaeocystis pouchetii and big centric diatoms, including several Thalassiosira species.

category: microbiology


title: Expression and Purification of BsaXI Restriction Endonuclease and Engineering New Specificity from BsaXI Specificity (S) Subunit

abstract: BsaXI is a Type IIB restriction endonuclease (REase) that cleaves both sides of its recognition sequence 5 {downarrow}N9 AC N5 CTCC N10{downarrow} 3 (complement strand 5 {downarrow}N7 GGAG N5 GT N12{downarrow} 3), creating 3-base 3 overhangs. Here we report the cloning and expression of bsaXIS and bsaXIRM genes in E. coli. BsaXI activity was successfully reconstituted by mixing the BsaXI RM fusion subunit with the BsaXI S subunit and the enzyme complex further purified by chromatography over 6 columns. As expected, the S subunit consisted of two subdomains encoding TRD1-CR1 (TRD, target recognition domain, CR, conserved region) for 5 AC 3, and TRD2-CR2 presumably specifying 5 CTCC 3. TRD1-CR1 (TRD2-CR2 deletion) or duplication of TRD1 (TRD1-CR1-TRD1-CR2) both generated a new specificity 5 AC N5 GT 3 when the S variants were complexed with the RM subunits. Circular permutation of TRD1 and TRD2, i.e. relocation of TRD2-CR2 to the N-terminus and TRD1-CR1 to the C-terminus generated the same specificity with the RM subunits, although some wobble cleavage was detected. The TRD2 domain in the BsaXI S subunit can be substituted by a close homolog ([~]59% sequence identity) and generated the same specificity. However, TRD2-CR2 domain alone failed to express in E. coli, but CR1-TRD2-CR2 protein could be expressed and purified which showed partial nicking activity with the RM subunits. This work demonstrated that like Type I restriction systems, the S subunit of a Type IIB system could also be manipulated to create new specificities. Genome mining of BsaXI TRD2 homologs in GenBank found more than 36 orphan TRD2 homologs, implying that quite a few orphan TRD2s are present in microbial genomes that may be potentially paired with other TRDs to create new restriction specificities.


title: Parsing the role of NSP1 in SARS-CoV-2 infection

abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 19 (COVID-19) pandemic. Despite its urgency, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis and its ability to antagonize innate immune responses. SARS-CoV-2 leads to shutoff of cellular protein synthesis and over-expression of nsp1, a central shutoff factor in coronaviruses, inhibits cellular gene translation. However, the diverse molecular mechanisms nsp1 employs as well as its functional importance in infection are still unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant in which nsp1 does not bind ribosomes, we untangle the effects of nsp1. We uncover that nsp1, through inhibition of translation and induction of mRNA degradation, is the main driver of host shutoff during SARS-CoV-2 infection. Furthermore, we find the propagation of nsp1 mutant virus is inhibited specifically in cells with intact interferon (IFN) response as well as in-vivo, in infected hamsters, and this attenuation is associated with stronger induction of type I IFN response. This illustrates that nsp1 shutoff activity has an essential role mainly in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover the central role it plays in SARS-CoV-2 pathogenesis, explicitly through blockage of the IFN response.


title: STORM-seq Reveals Differentiation Trajectories of Primary Human Fallopian Tube Epithelium

abstract: We present Single-cell TOtal RNA Miniaturized sequencing (STORM-seq), a full-length single-cell ribo-reduced RNA sequencing protocol, optimized to profile thousands of cells per run. Using off-the-shelf reagents and random hexamer priming, STORM-seq recovers comprehensive RNA profiles from single cells with library complexity approaching that of bulk RNA-seq. STORM-seq identifies thousands of additional coding and non-coding transcripts not detected by existing methods, and recovers clinically relevant structural variants at the single-cell level. We apply STORM-seq to primary human fallopian tube epithelium (FTE), a complex solid tissue key to both human reproductive biology and ovarian carcinogenesis. In differentiation trajectory analyses, the improved resolution from STORM-seq reveals intermediate/transitional cell states, and a putative progenitor cell population. The results support a trajectory from a bipotent progenitor population to ciliated and secretory cell types in normal FTE. These findings are consistent across human subjects, sequencing depths, and platforms. Long intergenic non-coding RNAs (lincRNAs) appear as key driver genes in both ciliated and secretory lineage trajectories, underscoring the importance of both coding and non-coding RNA in this tissue. By capturing essentially complete individual cellular transcriptomes, STORM-seq sheds new light on the transcriptional programs that establish cellular state and fate in complex tissues.

category: molecular biology


title: Oxidative phosphorylation pathway disruption is an alternative pathological mechanism leading to Diamond-Blackfan anemia

abstract:
Diamond-Blackfan anemia (DBA) is a rare congenital disorder characterized by the failure of erythroid progenitor differentiation; however, the molecular mechanisms leading to erythroid defects remain unclear. By analyzing the transcriptomic profiles of bone marrow from patients with DBA (n = 10), we identified the dysfunction of the oxidative phosphorylation (OXPHOS) pathway as a possible cause of DBA. We established a DBA cell model using differentiating hematopoietic stem progenitor cells in which the OXPHOS pathway was suppressed to completely recapitulate the defects in erythroid progenitor differentiation, ribosome biogenesis, and heme biosynthesis, which are representative characteristics of patients with DBA. Disruption of the OXPHOS pathway led to ribosomal defects and associated erythroid defects via abolishment of the Ran GTPase activating protein RanGAP1, which is pivotal in the RNA transport pathway. The composition of the ribosomal proteins in the established DBA cells was unchanged, but an overall reduction in ribosomal protein levels was observed, leading to an alteration in the translation of a subset of transcripts specific to erythropoiesis. We revealed that the OXPHOS pathway participates in erythropoiesis, particularly at an early stage, and reinforced the relationship between the OXPHOS pathway and erythropoiesis. Coenzyme Q10, an activator of OXPHOS, largely rescued the erythroid defects in DBA cells. Our results reveal that OXPHOS repression is an alternative pathological mechanism leading to DBA, demonstrating its potential as a therapeutic pathway.

Key PointsO_LIOxidative phosphorylation (OXPHOS) pathway disruption is an alternative pathological mechanism underlying Diamond-Blackfan anemia (DBA).
C_LIO_LISuppression of OXPHOS leads to defects in erythropoiesis and ribosomal biogenesis via the RanGAP1 protein.
C_LI

category: cell biology


title: Imaging tools generated by CRISPR/Cas9 tagging reveal cytokinetic diversity in mammalian cells

abstract: Cytokinesis is required to physically separate the daughter cells at the end of mitosis. This process occurs via the ingression of an actomyosin ring that assembles in anaphase and pulls in the overlying plasma membrane as it constricts. Mechanistic studies have uncovered different pathways that regulate the assembly and position of the ring in mammalian cells, but the majority of these studies were done using HeLa cells with overexpressed transgenes, and the relative requirement for these mechanisms among the majority of cell types is not known. Here, we used CRISPR/Cas9 gene editing to endogenously tag cytokinesis proteins, anillin, Ect2 and RhoA, as well as other cellular components, with fluorescent proteins. These tools enabled the visualization of cytokinesis by live imaging to quantitatively study these proteins at endogenous levels. As a proof-of-concept, tagging anillin in multiple mammalian cell lines revealed cytokinetic diversity, which will be useful for studies of how mechanisms controlling cytokinesis vary among cell types. We also successfully tagged multiple cellular components in the same cell line, demonstrating the versatility of these tagging tools.


title: Endosomes deliver ceramide phosphoethanolamine with unique acyl chain anchors to the cleavage furrow during male meiotic cytokinesis

abstract: Cell division, wherein one cell divides into two daughter cells, is fundamental to all living organisms. Cytokinesis, the final step in cell division, begins with the formation of an actomyosin contractile ring, positioned midway between the segregated chromosomes. Constriction of the ring with concomitant membrane deposition in a spatiotemporal manner generates a cleavage furrow that physically separates the cytoplasm. Unique lipids with specific biophysical properties have been shown to localize to intercellular bridges (also called midbody) connecting the two dividing cells; however, their biological roles and delivery mechanisms remain largely unknown. In this study, we show that Ceramide phosphoethanolamine (CPE), the structural analog of sphingomyelin, has unique acyl chain anchors in spermatocytes and is essential for meiotic cytokinesis. The head group of CPE is also important for spermatogenesis. We find that aberrant central spindle and contractile ring behavior but not mislocalization of phosphatidylinositol phosphates (PIPs) at the plasma membrane is responsible for the male meiotic cytokinesis defect in CPE deficient animals. Further, we demonstrate the enrichment of CPE in multivesicular bodies marked by Rab7, which in turn localize to cleavage furrow. Volume electron microscopy analysis using correlative light and focused ion beam scanning electron microscopy shows that CPE enriched Rab7 positive endosomes are juxtaposed on contractile ring material. Correlative light and transmission electron microscopy reveal Rab7 positive endosomes as a multivesicular body-like organelle that releases its intraluminal vesicles in the vicinity of ingressing furrows. Genetic ablation of Rab7 or expression of dominant negative Rab11 results in significant meiotic cytokinesis defects. Our results imply that endosomal delivery of CPE to ingressing membranes is crucial for meiotic cytokinesis.


title: TNIP1 inhibits Mitophagy via interaction with FIP200 and TAX1BP1

abstract: Mitophagy is a form of selective autophagy that disposes of superfluous and potentially damage-inducing organelles in a tightly controlled manner. While the machinery involved in mitophagy induction is well known, the regulation of the components is less clear. For example, it is still unknown how the ULK1 complex is dissociated prior to closing of the autophagosome. Here, using Chemically Inducible Dimerization (CID) and mitophagy assays with the fluorescent probe mtKeima, we reveal that the ubiquitin binding domain-containing protein TNIP1 is able to induce mitophagy when ectopically targeted to mitochondria. Conversely, we demonstrate that TNIP1 knock down accelerates mitophagy rates, and that ectopic TNIP1 negatively regulates the rate of mitophagy. These functions of TNIP1 depend on a previously unrecognized, evolutionarily conserved LIR motif as well as its AHD3 domain, which are required for binding to the ULK1 complex member FIP200 and the autophagy receptor TAX1BP1, respectively. Taken together, our findings identify a novel negative regulator of mitophagy that acts at the early steps of autophagosome biogenesis and provide a molecular rationale for how the ULK1 complex might be dissociated from the closing autophagosome.


title: Genetic mapping of multiple metabolic traits identifies novel genes for adiposity, lipids and insulin secretory capacity in outbred rats

abstract: Despite the successes of human genome-wide association studies, the causal genes underlying most metabolic traits remain unclear. We used outbred heterogeneous stock (HS) rats, coupled with expression data and mediation analysis, to identify quantitative trait loci (QTLs) and candidate gene mediators for adiposity, glucose tolerance, serum lipids and other metabolic traits. Physiological traits were measured in 1519 male HS rats, with liver and adipose transcriptomes measured in over 410 rats. Genotypes were imputed from low coverage whole genome sequence. Linear mixed models were used to detect physiological and expression QTLs (pQTLs and eQTLs, respectively), employing both SNP- and haplotype-based models for pQTL mapping. Genes with cis-eQTLs that overlapped pQTLs were assessed as causal candidates through mediation analysis. We identified 15 SNP-based pQTLs and 19 haplotype-based pQTLs, of which 11 were in common. Using mediation, we identified the following genes as candidate mediators of pQTLs: Grk5 for a fat pad weight pQTL on Chr1, Krtcap3 for fat pad weight and serum lipids pQTLs on Chr6, Ilrun for a fat pad weight pQTL on Chr20 and Rfx6 for a whole pancreatic insulin content pQTL on Chr20. Furthermore, we verified Grk5 and Ktrcap3 using gene knock-down/out models, thereby shedding light on novel regulators of obesity.

category: genetics


title: Deterioration of the human transcriptome with age due to increasing intron retention and spurious splicing

abstract: Adult aging is characterized by a progressive deterioration of biological functions at physiological, cellular and molecular levels, but its damaging effects on the transcriptome are not well characterized. Here, by analyzing splicing patterns in [~]1,000 human subjects sampled across multiple tissues, we found that splicing fidelity declines with age. Most prominently, genuine introns fail to be spliced out, manifesting as a broad surge in intron retention, and this is exacerbated by the increase in diverse spurious exon-exon junctions with age. Both of these effects are prominently detected in the majority of human tissues. Collectively, they result in the progressive deterioration of the active transcriptome, wherein functional mRNAs are increasingly diluted with non-functional splicing isoforms. We discuss the concept of "splicing damage" and formulate methods to quantify it. Using these tools, we show that splicing damage increases both with age and with the incidence of diseases. Altogether, this work uncovers transcriptome damage as a critical molecular indicator of human aging and healthspan.


title: Molecular docking between human TMPRSS2 and the serine protease Kunitz-type inhibitor rBmTI-A

abstract: SARS-CoV-2 entrance into host cells is dependent of ACE2 receptor and viral protein S initiation by serine protease TMPRSS2. Cleavage of coronavirus protein S at the junctions Arg685/Ser686 and Arg815/Ser816 leads to the production of the S1/S2 and S2 fragments needed for the fusion of viral and cell membranes. Studying and identifying serine protease inhibitors is an important step towards the development of candidate drugs to prevent SARS-CoV-2 infection. It has already been stablished that camostat mesylate, a serine protease inhibitor, is capable of blocking TMPRSS2 activity and prevent SARS-CoV-2 entrance into host cells. In this work, the interaction between the two domains of Kunitz-type serine protease inhibitor rBmTI-A and TMPRSS2 was studied through molecular docking. rBmTI-A domain 2 (P1 site Leu84) had the best complex results with predicted binding affinity of -12 Kcal.mol-1 and predicted dissociation constant at 25{degrees}C of 1.6 nM. The results suggest that rBmTI-A is capable of binding TMPRSS2 cleavage site at the junction Arg815/Ser816 using essentially the same residues that camostat mesylate.

category: biochemistry


title: Hypoxia-inducible factor 1 signaling drives placental aging and can elicit inflammatory changes in uterine myocytes

abstract: Most cases of pre-term labor have unknown cause, and the burden of preterm birth is immense. Placental aging has been proposed to promote labor onset, but specific mechanisms remain elusive. We report findings stemming from unbiased transcriptomic analysis of mouse placenta, which revealed that hypoxia-inducible factor 1 (HIF-1) stabilization is a hallmark of advanced gestational timepoints, accompanied by mitochondrial dysfunction and cellular senescence; we validated these findings in human placenta. In parallel in primary mouse trophoblasts and human choriocarcinoma cells, we modeled HIF-1 induction using prolyl hydroxylase inhibitors, and demonstrated resultant mitochondrial dysfunction and cellular senescence. Transcriptomic analysis revealed that HIF-1 stabilization recapitulated gene signatures observed in aged placenta. Further, conditioned media from trophoblasts following HIF-1 induction was sufficient to induce a contractile phenotype in immortalized uterine myocytes, suggesting a mechanism by which the aging placenta may drive the transition from uterine quiescence to contractility at the onset of labor. These results provide clear evidence for placental aging in normal pregnancy. Mechanistic dissection of this phenomenon demonstrates how hypoxia in late gestation may be a causal determinant of the mitochondrial dysfunction and senescence observed within the trophoblast as well as a trigger for uterine contraction.


title: Peritoneal M2 macrophage-derived extracellular vesicles as natural multi-target nanotherapeutics to attenuate cytokine storm after severe infections

abstract: Cytokine storm is a primary cause for multiple organ damage and death after severe infections, such as SARS-CoV-2. However, current single cytokine-targeted strategies display limited therapeutic efficacy. Here, we report that peritoneal M2 macrophages-derived extracellular vesicles (M2-EVs) are multi-target nanotherapeutics to resolve cytokine storm. In detail, primary peritoneal M2 macrophages exhibited superior anti-inflammatory potential than immobilized cell lines. Systemically administrated M2-EVs entered major organs and were taken up by phagocytes (e.g., macrophages). M2-EVs treatment effectively reduced excessive cytokine (e.g., TNF- and IL-6) release in vitro and in vivo, thereby attenuated oxidative stress and multiple organ (lung, liver, spleen and kidney) damage in endotoxin-induced cytokine storm. Moreover, M2-EVs simultaneously inhibited multiple key proinflammatory pathways (e.g., NF-{kappa}B, JAK-STAT and p38 MAPK) by regulating complex miRNA-gene and gene-gene networks, and this effect was collectively mediated by many functional cargos (miRNAs and proteins) in EVs. In addition to the direct anti-inflammatory role, human peritoneal M2-EVs expressed angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2 spike protein, and thus could serve as nanodecoys to prevent SARS-CoV-2 pseudovirus infection in vitro. As cell-derived nanomaterials, the therapeutic index of M2-EVs can be further improved by genetic/chemical modification or loading with specific drugs. This study highlights that peritoneal M2-EVs are promising multifunctional nanotherapeutics to attenuate infectious diseases-related cytokine storm.


title: Intratumoral Recall of Childhood Vaccine-Specific CD4+ T cells Coordinates Type I and II Antitumor Immunity

abstract: CD4+ T cells are key contributors to cancer immune surveillance. Here we report that childhood vaccine associated antigens engage simultaneous antitumor functions of CD8+ T cells and eosinophils via intratumor vaccine-specific CD4+ T cell recall. Prior vaccination against poliovirus potentiates antitumor efficacy of polio virotherapy in mice, and intratumor recall of poliovirus or tetanus immunity delayed tumor growth. Antitumor effects of recall antigens were mediated by CD4+ T cells, independent of CD40L signaling, and were dependent on eosinophils and CD8+ T cells. Recall antigen therapy caused marked tumor infiltration of type 2 innate lymphoid cells (ILC2s) expressing granzyme B and PD1 and eosinophils, coinciding with decreased proportions of intratumor Tregs. A pan-cancer analysis revealed an inverse relationship between intratumor eosinophils and Tregs, but not CD4+ or CD8+ T cells. This work defines cancer immunotherapy potential of childhood vaccines and implicates type II immunity in CD4+ T cell cancer immune surveillance.


title: Modulation of the extracellular matrix by Streptococcus gallolyticus subsp. gallolyticus and importance in cell proliferation

abstract:
Streptococcus gallolyticus subspecies gallolyticus (Sgg) has a strong clinical association with colorectal cancer (CRC) and actively promotes the development of colon tumors. Previous work showed that this organism stimulates CRC cells proliferation and tumor growth. However, the molecular mechanisms underlying these activities are not well understood. Here, we found that Sgg upregulates the expression of several types of collagens in HT29 and HCT116 cells, with type VI collagen (ColVI) being the highest upregulated collagen type. Knockdown of ColVI abolished the ability of Sgg to induce cell proliferation and reduced the adherence of Sgg to CRC cells. The extracellular matrix (ECM) is an important regulator of cell proliferation. Therefore, we further examined the role of decellularized matrix (dc-matrix), which is free of live bacteria or cells, in Sgg-induced cell proliferation. Dc-matrix prepared from Sgg-treated cells showed a significantly higher pro-proliferative activity than that from untreated cells or cells treated with the control bacteria. On the other hand, dc-matrix from Sgg-treated ColVI knockdown cells showed no difference in the capacity to support cell proliferation compared to that from untreated ColVI knockdown cells, suggesting that the ECM by itself is a mediator of Sgg-induced cell proliferation. Furthermore, Sgg-treated CRC cells formed significantly larger tumors in vivo, whereas Sgg treatment had no effect on ColVI knockdown cells, suggesting that ColVI is important for Sgg to promote tumor growth in vivo. These results highlight a dynamic bidirectional interplay between Sgg and the ECM, where Sgg upregulates collagen expression. The Sgg-modified ECM in turn affects the ability of Sgg to adhere to host cells and more importantly, acts as a mediator for Sgg-induced CRC cell proliferation. Taken together, our results reveal a novel mechanism in which Sgg stimulates CRC proliferation through modulation of the ECM.

Author SummaryColorectal cancer (CRC) is a leading cause of cancer-related death. The development of CRC can be strongly influenced by specific gut microbes. Understanding how gut microbes modulate CRC is critical to developing novel strategies to improve clinical diagnosis and treatment of this disease. S. gallolyticus subsp. gallolyticus (Sgg) has a strong clinical association with CRC and actively promotes the development of colon tumors. However, the mechanisms Sgg utilizes to promote tumors are not well understood. Our results showed for the first time a dynamic interplay between Sgg and the extracellular matrix. We found that Sgg upregulates the expression of collagens which in turn affects the interaction between Sgg and CRC cells and mediates CRC cell proliferation. These findings draw attention to a previously unrecognized dynamic bidirectional interplay between a CRC-associated microbe and the extracellular matrix (ECM). Given the importance of the ECM in normal homeostasis and in tumor microenvironment, these findings have important implications in the context of microbial contribution to cancer.


title: Revealing the microbial heritage of traditional Brazilian cheeses through metagenomics

abstract: Brazilian artisanal cheeses date from the first Portuguese settlers and evolved via local factors, resulting in unique products that are now part of the patrimony and identity of different Brazilian regions. In this study, we combined several culture-independent approaches, including 16S/ITS metagenetics, assembly- and deep profiling-metagenomics to characterize the originality of the microbiota of five varieties of Brazilian artisanal cheeses from the South and Southeast regions of Brazil. Their core microbiota contained mainly lactic acid bacteria (LAB), of which Lactococcus lactis subsp. lactis was the most frequent, followed by Streptococcus thermophilus in the South region. Moreover, several samples from the Southeast region contained, as dominant LAB, two other food Streptococci belonging to a new species of the salivarius group and S. infantarius. Rinds of samples from the Southeast region were dominated by the halotolerant bacterium Corynebacterium variabile and the yeasts Diutina catenulata and, to a lesser extent, by Debaryomyces hansenii and Kodamaea ohmeri. Rinds from the South region contained mainly LAB due to their short ripening time, and the predominant yeast was D. hansenii. Phylogenomic analysis based on L. lactis metagenome-assembled genomes (MAGs) showed that most Brazilian strains are closely related and form a different clade from those whose genomes are available at this time, indicating that they belong to a specific group. Lastly, functional analysis showed that S. infantarius acquired a [~]26 kb DNA fragment from S. thermophilus starter strains that carry the LacSZ system, allowing fast lactose assimilation, an adaptation advantage for growth in milk. Finally, our study identified several areas of concern, such as the presence of somatic cell DNA and high levels of antibiotic resistance genes in several cheese microbiota, implying that the milk used was from diseased herds. Overall, the data from this study highlight the potential value of the traditional and artisanal cheese production network in Brazil, and provide a metagenomic-based scheme to help manage this resource safely.


title: Contribution of the Golgi apparatus in morphogenesis of a virus induced cytopathic vacuolar system

abstract: The Golgi apparatus (GA) in mammalian cells is pericentrosomally anchored and exhibits a stacked architecture. During infections by members of the alphavirus genus, the host cell GA is thought to give rise to distinct mobile pleomorphic vacuoles known as CPV-II (cytopathic vesicle-II) via unknown morphological steps. To dissect this, we adopted a phased electron tomography approach to image multiple overlapping volumes of a cell infected with Venezuelan equine encephalitis virus (VEEV) and complemented it with localization of peroxidase tagged Golgi marker. Analysis of the tomograms revealed a pattern of progressive cisternal bending into double-lamellar vesicles as a central process underpinning the biogenesis and the morphological complexity of this vacuolar system. Here we propose a model for the conversion of GA to CPV-II that reveals a unique pathway of intracellular virus envelopment. Our result has implications for alphavirus virus induced displacement of Golgi cisternae to the plasma membrane to aid viral egress operating late in the infection cycle.


title: Group II Intron-Like Reverse TranscriptasesFunction in Double-Strand Break Repair by Microhomology-Mediated End Joining

abstract: Bacteria encode free-standing reverse transcriptases (RTs) of unknown function that are closely related to group II intron-encoded RTs. Here, we found that a Pseudomonas aeruginosa group II intron-like RT (G2L4 RT) with YIDD instead of YADD at its active site functions in DNA repair in its native host and when transferred into Escherichia coli. G2L4 RT has biochemical activities strikingly similar to those of human DNA repair polymerase{theta} and uses them for translesion DNA synthesis and double-strand break repair (DSBR) via microhomology-mediated end-joining (MMEJ) in vitro and in vivo. We also found that a group II intron RT can function similarly to G2L4 RT in DNA repair, with reciprocal substitutions at the active site showing an I residue favors MMEJ and an A residue favors primer extension in both enzymes. The DNA repair functions of these enzymes utilize conserved structural features of non-LTR-retroelement RTs, including human LINE-1 and other eukaryotic non-LTR-retrotransposon RTs, suggesting such enzymes may have an inherent ability to function in DSBR in a wide range of organisms.


title: Opposing effects of calcium stress in human muscle: defective glucose utilization leading to diabetes vs. stress-adaptive effects of a junctophilin fragment

abstract:
Contraction of striated muscle, which is controlled by movements of calcium ions between cellular stores (SR) and cytosol, is the function consuming the most energy in mammals, which also makes muscle the main user of circulating glucose and regulator of its level. Accordingly, inadequate removal of glucose by muscle is the main proximate cause of hyperglycemia, leading to insulin resistance and diabetes. Multiple diseases of muscle course with chronically elevated cytosolic calcium. Here we show in a sample of malignant hyperthermia-susceptible (MHS) patients, a significant decrease in content of junctophilin1, an essential protein of the couplon --the supramolecular device that effects calcium release from the SR-- accompanied by an increase in a 44 kDa C- terminal junctophilin fragment, JPh44, and the activating cleavage of GSK3{beta}, a specialized kinase that inhibits glycogen synthase. We trace these changes to activation, by the excess calcium, of calpain1, a serine/threonine kinase. By high-resolution imaging of antibody markers or fluorescent probes expressed in patients and murine muscle and patient-derived and C2C12 myotubes, we demonstrate detailed colocalization of junctophilin1 with the calcium channel RyR1, marking the couplon, and of calpain1 with both. We also show that the JPh44 fragment moves away from the couplon and into nuclei, a movement greatly increased in MHS. Cultured myotubes expressing a JPh44-like construct had altered gene transcription in multiple pathways, including changes that promote glucose utilization. Together with less transcription, the expression of GSK3{beta} was reduced, which predicts increased glucose utilization. In summary, the changes observed in these patients add to the wide-ranging alterations in metabolism that eventually lead to hyperglycemia and diabetes, assign the changes to proteolysis by calcium-activated calpain1, and reveal the production and beneficial, stress-adaptive function of a novel regulator of transcription, JPh44. Similar stress-adaptive roles have been described for a junctophilin2 fragment in heart failure.

Alternative AbstractCalcium ion movements between cellular stores and the cytosol govern muscle contraction, the most energy- consuming function in mammals, which confers striated myocytes a pivotal role in glycemia regulation. Chronic myoplasmic calcium elevation, found in malignant hyperthermia-susceptible (MHS) patients and in other myopathies, has been suggested to underlie the progression from hyperglycemia to insulin resistance. However, what drives such progression remains elusive. We find that muscle cells derived from MHS patients have decreased content of junctophilin1, a protein that colocalizes in the couplon with both the calcium channel RyR1 and calpain1, accompanied by an increase in a 44 kDa junctophilin1 fragment (JPh44) that localizes in the nuclei. We also find increased an activated fragment of GSK3{beta} -- a specialized kinase that inhibits glycogen synthase, which favors glycogenolysis. We trace these changes to activated proteolysis by calpain1, secondary to increased myoplasmic calcium. We demonstrate that a JPh44-like construct induces transcriptional changes predictive of increased glucose utilization in myotubes, including decreased translation of GSK3{beta}. These effects reveal a stress-adaptive response, similar to that of a junctophilin 2 fragment described in heart failure, mediated by the novel regulator of transcription JPh44.


title: Lnc956-TRIM28-HSP90B1 complex on replication forks promotes CMG helicase retention to ensure stem cell genomic stability and embryogenesis

abstract: Replication stress is a major source of endogenous DNA damage. Despite that numerous proteins have been identified on replication forks to modulate fork or replication machinery activities, it remains unexplored whether non-coding RNAs can localize on stalled forks and play critical regulatory roles. Here we identify an uncharacterized lncRNA NONMMUT028956 (Lnc956 for short) predominantly expressed in mouse embryonic stem cells. Lnc956 is recruited to stalled replication forks to prevent fork collapse and preserve genomic stability, and is essential for mouse embryogenesis. Mechanistically, it drives assembly of the Lnc956-TRIM28-HSP90B1 ribonucleoprotein (RNP) complex on stalled forks in an inter-dependent manner downstream of ATR signaling. This RNP complex physically associates with MCM2-7 hexamer via TRIM28 and directly regulates the CMG helicase retention on chromatin. The regulation of RNP on CMG retention is mediated by HSP90B1s chaperoning function. These findings reveal a novel pathway which actively regulates replisome retention to prevent fork collapse.


title: Base editing derived models of human WDR34 and WDR60 disease alleles replicate retrograde IFT and hedgehog signaling defects and suggest disturbed Golgi protein transport

abstract:
Cytoplasmic Dynein-2 or IFT-dynein is the only known retrograde motor for intraflagellar transport, enabling protein trafficking from the ciliary tip to the base. Dysfunction of WDR34 and WDR60, the two intermediate chains of this complex, causes Short Rib Thoracic Dystrophy (SRTD), human skeletal chondrodysplasias with high lethality. Complete loss of function of WDR34 or WDR60 is lethal in vertebrates and individuals with SRTD carry at least one putative hypomorphic missense allele. Gene knockout is therefore not suitable to study the effect of these human missense disease alleles.

Using CRISPR single base editors, we recreated three different patient missense alleles in cilia-APEX-IMCD3 cells. Consistent with previous findings in dynein-2 full loss of function models and patient fibroblasts, mutant cell lines showed hedgehog signaling defects as well as disturbed retrograde IFT. Transcriptomics analysis revealed differentially regulated expression of genes associated with various biological processes, including G-protein-coupled receptor signaling as well extracellular matrix composition, endochondral bone growth and chondrocyte development. Further, we also observed differential regulation of genes associated with Golgi intracellular transport, including downregulation of Rab6b, a GTPase involved in Golgi-ER retrograde protein trafficking and interacting with components of cytoplasmic dynein-1, in mutant ciliated and non-ciliated clones compared to controls. In addition to providing cellular model systems enabling investigations of the effect of human SRTD disease alleles, our findings indicate non-ciliary functions for WDR34 and WDR60 in addition to the established roles as components of the retrograde IFT motor complex in cilia.


title: Discovery of a novel jingmenvirus in Australian sugarcane Soldier fly (Inopus flavus) larvae

abstract: In Australia, soldier flies are major pests of sugarcane, and they can cause significant yield losses in some areas, possibly due to virus transmission to the plants. We sequenced fly larvae salivary glands and identified a novel jingmenvirus, putatively named Inopus flavus jingmenvirus 1 (IFJV1). Phylogenetic trees confirmed that IFJV1 groups with insect-associated jingmenviruses, newly identified flavivirus-like viruses with a segmented genome. After the design and validation of molecular detection systems for IFJV1, larval homogenates were passaged on insect and vertebrate cells but IFJV1 could only be detected in the first two passages in insect cells and not at all in vertebrate cells. Despite this lack of consistent replication in laboratory models, this virus does replicate in its host Inopus flavus, as sequenced small RNA from larvae match the IFJV1 sequences, are predominantly 21 nucleotides-long and map to the whole sequences on both strands, which is typical of an actively replicating virus. This discovery confirms the worldwide presence of jingmenviruses, which until now had only been detected on four continents. However, the study of IFJV1 tropism and of the possible pathogenicity to its host or the sugarcane it parasitizes requires the development of a stable replication model.


title: Hemin availability induces coordinated DNA methylation and gene expression changes in Porphyromonas gingivalis

abstract:
Periodontal disease is a common chronic inflammatory disease driven by the sub-gingival microbiota. Porphyromonas gingivalis is an important bacterium in the development of the disease that expresses a variety of virulence determinants. The environmental concentration of hemin, an essential nutrient of this organism, is a global regulator of virulence in P. gingivalis: high levels of hemin lead to increased expression of several virulence determinants. However, the mechanism through which hemin influences bacterial gene expression is poorly understood. Bacterial epigenetic processes, primarily DNA methylation, have the potential to fulfil this mechanistic role. Here, we characterised the methylome of P. gingivalis, and compared its variation to transcriptomic changes in response to changes in hemin concentration.

Gene expression and DNA methylation profiling of P. gingivalis W50 was performed, following continuous culture in chemostats with excess or limited hemin, using Illumina RNA-Seq and Nanopore DNA sequencing. DNA methylation quantification was carried out for N6-methyladenine (6mA) and 5-methylcytosine (5mC) base pair modifications. Differential expression and differential methylation in response to excess hemin availability are presented after multiple testing correction (FDR 5%).

In excess hemin there were 161 over- and 268 under-expressed genes, compared to limited hemin culture. Genes under-expressed in excess hemin were involved in iron recruitment (the hemophore HmuY) and transport (TonB-dependent receptors), and those over-expressed were involved in Fe-S and 4FE-4S cluster binding, and metal cluster binding. Hemin-dependent differentially methylated signals were observed for both 6mA and 5mC, with 49 and 47 signals, respectively. Coordinated genome-wide differential expression and methylation effects were observed in 6 genes, a Ppx/GppA family phosphatase, a lactate utilization protein, 4-alpha-glucanotransferase, two ABC transporter proteins, and a hypothetical protein HMPREF1322_RS00730. The findings indicate that altered genome methylation occurs in response to the availability of hemin and give insights into the molecular mechanisms of regulation of virulence in this bacterium.

Author SummaryDNA methylation has important roles in bacteria, including in the regulation of transcription. Porphyromonas gingivalis, an oral pathogen in periodontitis, exhibits well-established gene expression changes in response to hemin availability. However, the gene regulatory processes underlying these effects remain unknown. To this end, we profiled the novel P. gingivalis epigenome, and assessed epigenetic and transcriptome variation under limited and excess hemin conditions. As expected, multiple gene expression changes were detected in response to limited and excess hemin conditions. Notably, we also detected differential DNA methylation signatures for both N6-methyladenine (6mA) and 5-methylcytosine (5mC) in response to hemin availability. Joint analyses identified a subset of coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify novel regulatory processes underlying the mechanism of hemin regulated gene expression in P. gingivalis, with phenotypic impacts on its virulence in periodontal disease.


title: Structural insights into the regulation of acetylation-dependent genome condensation

abstract: Histone modifications are deposited by chromatin modifying enzymes and read out by proteins that recognize the modified state. BRD4-NUT is an oncogenic fusion protein of the acetyl lysine reader BRD4 that binds to the acetylase p300 and enables formation of long-range intra- and interchromosomal interactions. We here examine how acetylation reading and writing enable formation of such interactions. We show that NUT contains an acidic transcriptional activation domain that binds to the TAZ2 domain of p300. We use NMR to investigate the structure of the complex and found that the TAZ2 domain has an autoinhibitory role for p300. NUT-TAZ2 interaction or mutations found in cancer that interfere with autoinhibition by TAZ2 allosterically activate p300. p300 activation results in a self-organizing, acetylation-dependent feed-forward reaction that enables long-range interactions by bromodomain multivalent acetyl-lysine binding. We discuss the implications for chromatin organisation, gene regulation and dysregulation in disease.


title: Structural insights into p300 regulation and acetylation-dependent genome organisation


title: Mitochondrial Redox Adaptations Enable Aspartate Synthesis in SDH-deficient Cells

abstract: The oxidative tricarboxylic acid (TCA) cycle is a central mitochondrial pathway integrating catabolic conversions of NAD+ to NADH and anabolic production of aspartate, a key amino acid for cell proliferation. Several TCA cycle components are implicated in tumorigenesis, including loss of function mutations in subunits of succinate dehydrogenase (SDH), also known as complex II of the electron transport chain (ETC). Mechanistic understanding of how proliferating cells tolerate the metabolic defects of SDH loss is still lacking. Here, we identify that SDH supports cell proliferation through aspartate synthesis but, unlike other ETC impairments, is not restored by electron acceptor supplementation. Interestingly, we find aspartate production and cell proliferation are restored to SDH impaired cells by concomitant inhibition of ETC complex I (CI). We determine that the benefits of CI inhibition in this context are dependent on decreasing mitochondrial NAD+/NADH, which drives SDH-independent aspartate production. We also find that genetic loss or restoration of SDH selects for cells with concordant CI activity, establishing distinct modalities of mitochondrial metabolism for maintaining aspartate synthesis. Collectively, these data identify a metabolically beneficial mechanism for CI loss in proliferating cells and reveal that compartmentalized redox changes can impact cellular fitness.


title: Validation of a small molecule inhibitor of PDE6D-RAS interaction with potent anti-leukemic effects

abstract: RAS mutations prevalent in high-risk leukemia have been linked to relapse and chemotherapy resistance. Efforts to directly target RAS proteins have been largely unsuccessful. However, since RAS-mediated transformation is dependent on signaling through the RAS-related C3 botulinum toxin substrate (RAC) small GTPase, we hypothesized that targeting RAC may be an effective therapeutic approach in RAS mutated tumors. Here we describe multiple small molecules capable of inhibiting RAC activation in acute lymphoblastic leukemia cell lines. One of these, DW0254, also demonstrates promising anti-leukemic activity in RAS-mutated cells. Using chemical proteomics and biophysical methods, we identified the hydrophobic pocket of phosphodiester 6 subunit delta (PDE6D), a known RAS chaperone, as a target for this compound. Inhibition of RAS localization to the plasma membrane upon DW0254 treatment is associated with RAC inhibition through a phosphatidylinositol-3-kinase/AKT-dependent mechanism. Our findings provide new insights on the importance of PDE6D-mediated transport for RAS-dependent RAC activation and leukemic cell survival.

category: cancer biology


title: EPI-589, a redox-active neuroprotectant, potently protects cultured cells from oxidative stress and alleviates symptomatic and pathological progression of motor neuron disease in the wobbler mouse

abstract: Oxidative stress is believed to play a significant role in the pathophysiology of amyotrophic lateral sclerosis (ALS), the most common form of motor neuron disease. The present study aims to firstly investigate the antioxidant activities of EPI-589, a small-molecule quinone derivative, under cell-free or cell culture conditions, and explore the in vivo efficacy of EPI-589 in the wobbler mouse model of human motor neuron disease. The reduced form of EPI-589 showed hydroxyl radical scavenging activities, whereas the oxidized form i.e. EPI-589 did not. In cellular models utilizing ALS patient-derived fibroblasts carrying mutations in the fused in sarcoma (FUS) gene or superoxide dismutase 1 (SOD1) gene, EPI-589 potently protected cells from oxidative stress induced by buthionine sulfoximine and ferric citrate. Protective effect of EPI-589 was also observed in culture of mouse immortalized striatal STHdHQ7/Q7 cells with cystine deprivation. In wobbler mice, oral administration of dietary EPI-589 provided long-lasting amelioration of both of deterioration of the rotarod walking performance and progression of forelimb deformity in wobbler mice throughout the treatment. In separate studies, we found that EPI-589 significantly suppressed changes of pathophysiological markers such as plasma phosphorylated neurofilament heavy chain, urinary 8-hydroxy-2-deoxyguanosine, and cervical N-acetylaspartate in untreated wobbler mice. Thus, the present study firstly demonstrates that EPI-589 is a highly potent, redox-active neuroprotectant and robustly delays the symptomatic and pathophysiological progression of motor neuron disease in the wobbler mouse, and these findings strongly encourage further exploration of the therapeutic potential of EPI-589 for the treatment of ALS.


title: Loss of Stathmin-2, a hallmark of TDP-43-associated ALS, causes motor neuropathy

abstract: TDP-43 mediates proper Stathmin-2 (STMN2) mRNA splicing, and STMN2 protein is reduced in the spinal cord of most ALS patients. To test the hypothesis that STMN2 loss contributes to ALS pathogenesis, we generated constitutive and conditional STMN2 knockout mice. Constitutive STMN2 loss results in early-onset sensory and motor neuropathy featuring impaired motor behavior and dramatic distal neuromuscular junction (NMJ) denervation of fast-fatigable motor units, which are selectively vulnerable in ALS, without axon or motoneuron degeneration. Selective excision of STMN2 in motoneurons leads to similar NMJ pathology. STMN2 KO heterozygous mice, which better model the partial loss of STMN2 protein found in ALS patients, display a slowly progressive, motor-selective neuropathy with functional deficits and NMJ denervation. Thus, our findings strongly support the hypothesis that STMN2 reduction due to TDP-43 pathology contributes to ALS pathogenesis.


title: Structural basis for ultrapotent neutralization of human metapneumovirus

abstract: Human metapneumovirus (hMPV) is a leading cause of morbidity and hospitalization among children worldwide, however, no vaccines or therapeutics are currently available for hMPV disease prevention and treatment. The hMPV fusion (F) protein is the sole target of neutralizing antibodies. To map the immunodominant epitopes on the hMPV F protein, we isolated a panel of human monoclonal antibodies (mAbs), and the mAbs were assessed for binding avidity, neutralization potency, and epitope specificity. We found the majority of the mAbs target diverse epitopes on the hMPV F protein, and we discovered multiple mAb binding approaches for antigenic site III. The most potent mAb, MPV467, which had picomolar potency, was examined in prophylactic and therapeutic mouse challenge studies, and MPV467 limited virus replication in mouse lungs when administered 24 hrs before or 72 hrs after viral infection. We determined the structure of MPV467 in complex with the hMPV F protein using cryo-electron microscopy to a resolution of 3.3 [A], which revealed a complex novel prefusion-specific epitope overlapping antigenic sites II and V on a single protomer. Overall, our data reveal new insights into the immunodominant antigenic epitopes on the hMPV F protein, identify a new mAb therapy for hMPV F disease prevention and treatment, and provide the discovery of a unique pre-fusion-specific epitope on the hMPV F protein.


title: Transcriptome profiling of barley and tomato shoot and root meristems unravels physiological variations underlying photoperiodic sensitivity

abstract: The average sowing date of crops in temperate climate zones has been shifted forwards by several days, resulting in a changed photoperiod regime at the emergence stage. In the present study, we performed a global transcriptome profiling of plant development genes in the seedling stage of root and shoot apical meristems of a photoperiod-sensitive species (barley) and a photoperiod insensitive species (tomato) in short-day conditions (8h). Variant expression indicated differences in physiological development under this short day-length regime between species and tissues. The barley tissue transcriptome revealed reduced differentiation compared to tomato. In addition, decreased photosynthetic activity was observed in barley, indicating a slower physiological development of shoot meristems than in tomatoes. The photomorphogenesis controlling cryptochrome gene cry1, with an effect on physiological differentiation, showed an underexpression in barley compared to tomato shoot meristems. This might lead to a cascade of suspended sink-source activities, which ultimately delay organ development and differentiation in barley shoot meristems under short photoperiods.

category: developmental biology


title: First assessment of Iranian pomegranate germplasm using targeted metabolites and morphological traits to develop the core collection and modeling of the current and future spatial distribution under climate change conditions

abstract: Pomegranate has been considered a medicinal plant due to its rich nutrients and bioactive compounds. Since environmental conditions affect the amount and composition of metabolites, selecting suitable locations for cultivation would be vital to achieve optimal production. In this study, the diversity of targeted metabolites and morphological traits among 152 Iranian pomegranate genotypes was evaluated to establish a core collection. The multivariate analyses were conducted including principal component analysis (PCA), and cluster analysis. In addition, the current and future geographical distribution of pomegranate in Iran was predicted to identify suitable locations using the MaxEnt model. The results showed high diversity in the studied morphological and metabolic traits. The PCA results indicated that FFS, NFT, JA, and AA are the most important traits in discriminating the studied genotypes. A constructed core collection using maximization strategy consisted of 20 genotypes and accounted for 13.16% of the entire collection. Shannon-Weaver diversity index of a core collection was similar or greater than the entire collection. Evaluation of the core collection using four parameters of MD, VD, CR, and VR also indicated the maintenance of the genetic diversity of the original set. According to the MaxEnt model, altitude, average temperature of coldest quarter, and isothertmality were the key factors for the distribution of pomegranate. The most suitable areas for pomegranate cultivation were also determined which were located in arid and semi-arid regions of Iran. The geographic distribution of pomegranate in the future showed that the main provinces of pomegranate cultivation would be less affected by climatic conditions by the middle of the century. The results of this study provide valuable information for selection of elite genotypes to develop the breeding programs to obtain the cultivars with the highest levels of metabolic compounds for pharmaceutical purposes, as well as identification of the most suitable agro-ecological zones for orchard establishment.


title: Formulation of substrates with agricultural and forestry wastes for Camellia oleifera Abel seedling cultivation

abstract: AbstractsFive compound substrates for the cultivation of Camellia oleifera Abel seedlings were obtained by substituting the peats in conventional substrate formulas with the composts of Camellia oleifera shell, pine chips, palm fiber residues, chicken manure, and sheep manure. The changes in the physical and chemical properties of the substrate after seedling cultivation were determined and the effects of these substrates on the development of Camellia oleifera seedling were analyzed. It was found that the survival rates of the one-year-old seedlings produced from stem cuttings in all groups were greater than 97.5% after 6 months cultivation. The seedlings exhibited greater seedling heights, larger ground diameters, longer root systems and larger total root volume than those obtained with conventional substrates. The compound substrate formulated with the compost of Camellia oleifera shell+ palm fiber residue+ chicken manure (A3), vermiculite and perlite (6:3:1) is the most optimal, which gives the 100% survival rate of cultivated seedlings, the greatest seedling heights, and the largest ground diameters. In particular, the ground diameters and 26.67% of the seedling heights reach the grade 1 standard for the two-year-old seedlings.


title: Altered integration of excitatory inputs onto the basal dendrites of layer 5 pyramidal neurons in a mouse model of Fragile X Syndrome

abstract: To uncover how synaptic integration of feedforward sensory inputs is affected in autism spectrum disorders (ASD), we used two-photon uncaging of caged glutamate to activate two clustered spines in the basal dendrites of layer 5 (L5) pyramidal neurons from a mouse model of Fragile X syndrome (FXS), the most common genetic cause of ASD. While subthreshold excitatory inputs integrate linearly in wild-type animals, surprisingly those with FXS summate sublinearly, contradicting what would be expected of a hyperexcitable cortex typically associated with ASD. Knockdown of the regulatory {beta}4 subunit of BK channels, rescues the synaptic integration, a result that was corroborated with numerical simulations. Taken together, these findings suggest that there is a differential impairment in the integration of feedforward sensory and feedback predictive inputs in L5 pyramidal neurons in FXS and potentially other forms of ASD. These results challenge the traditional view that FXS and other ASD are characterized by sensory hypersensitivity, but instead by hyposensitivity of sensory inputs and hypersensitivity of predictive inputs onto cortical neurons.


title: A Genetic History of Continuity and Mobility in the Iron Age Central Mediterranean

abstract: The Iron Age saw the expansion of Phoenician and Greek colonies across the Mediterranean and the rise of Carthage as the major maritime power of the region. These events were facilitated by the ease of long-distance travel following major advances in seafaring. We know from the archaeological record that trade goods and materials were moving across great distances in unprecedented quantities, but it is unclear how these patterns correlate with human mobility. To investigate population mobility and interactions directly, we sequenced the genomes of 30 ancient individuals from Carthaginian and Etruscan port cities around the central Mediterranean, in Tunisia, Sardinia, and central Italy. At all three locations, there is a meaningful contribution of autochthonous populations (from Bronze Age North Africa, Sardinia, and Italy, respectively), as well as highly heterogeneous ancestry including many individuals with ancestry from other parts of the Mediterranean region. These results highlight both the role of autochthonous populations and the extreme interconnectedness of populations in the Iron Age Mediterranean. By studying these trans-Mediterranean neighbors together, we explore the complex interplay between local continuity and mobility that shaped the Iron Age societies of the central Mediterranean.

category: genomics


title: An integrated single-cell atlas of the skeleton from development through adulthood

abstract: AO_SCPLOWBSTRACTC_SCPLOWThe recent growth of single-cell transcriptomics has turned single-cell RNA sequencing (scRNA-seq) into a near-routine experiment. Breakthroughs in improving scalability have led to the creation of organism-wide transcriptomic datasets, aiming to comprehensively profile the cell types and states within an organism throughout its lifecycle. To date however, the skeleton remains a majorly underrepresented organ system in organism-wide atlases. Considering how the skeleton not only serves as the central framework of the vertebrate body but is also the home of the hematopoietic niche and a central player in major metabolic and homeostatic processes, this presents a major deficit in current reference atlas projects. To address this issue, we integrated seven separate scRNA-seq datasets containing skeletal cells and their developmental precursors, generating an atlas of over 800,000 cells. This skeletal cell atlas describes cells across the mesenchymal lineage from the induction of the limb field to adult bone, encompassing 50 different cell states. In addition, the original datasets were reannotated, enabling the discovery of novel, highly specific marker genes, some of which we have validated in vivo by whole-mount in situ hybridization. Furthermore, expanding the repertoire of available time points and cell types within a single dataset allowed for more complete analyses of cell-cell communication or in silico perturbation studies. Taken together, we present a missing piece in the current atlas mapping efforts, which will be of value to researchers in the fields of skeletal biology, hematopoiesis, metabolism and regenerative medicine.


title: Ontogeny of oscillatory slow-wave and neuronal population activity in human iPSC-3D cortical circuits

abstract:
Oscillatory slow-wave activity (0.5--100 Hz) emerges during fetal human cortex development reflecting functional consequences of cellular brain ontogeny. Human induced pluripotent stem cell-derived (iPSC) neural in vitro models recapitulate aspects of in vivo cellular brain ontogeny, while neuronal mesoscale functional ontogeny is largely uncharacterized. We utilized a human iPSC-derived 3D cortical aggregate model to assess properties of emerging oscillatory slow-wave activity and its relation to synchronous neuronal population activity in cortical circuits. We reveal that oscillatory slow-wave activity (< 1 Hz), phased locked to synchronous population bursting, emerges within 14 days in vitro followed by consecutive stages of emerging delta (1--4 Hz), theta (4--11 Hz), beta (11--30 Hz), and gamma (30--55 Hz) oscillatory activity, accompanied by stage-specific changes in neuronal population burst pattern characteristics.

We provide a classification of neuronal mesoscale functional ontogeny stages of developing human iPSC-cortical circuits, where each stage is defined by specific oscillatory slow-wave activity and characteristic synchronous neuronal bursting patterns.


title: Dissecting neural computations of the human auditory pathway using deep neural networks for speech

abstract: The human auditory system extracts rich linguistic abstractions from the speech signal. Traditional approaches to understand this complex process have used classical linear feature encoding models, with limited success. Artificial neural networks have recently achieved remarkable speech recognition performance and offer potential alternative computational models of speech processing. We used the speech representations learned by state-of-the-art deep neural network (DNN) models to investigate neural coding across the ascending auditory pathway from the peripheral auditory nerve to auditory speech cortex. We found that representations in hierarchical layers of the DNN correlated well to neural activity throughout the ascending auditory system. Unsupervised speech models achieve the optimal neural correlations among all models evaluated. Deeper DNN layers with context-dependent computations were essential for populations of high order auditory cortex encoding, and the computations were aligned to phonemic and syllabic context structures in speech. Accordingly, DNN models trained on a specific language (English or Mandarin) predicted cortical responses in native speakers of each language. These results reveal convergence between representations learned in DNN models and the biological auditory pathway and provide new approaches to modeling neural coding in the auditory cortex.


title: Individual neurophysiological signatures of spontaneous rhythm processing

abstract:
When sensory input conveys rhythmic regularity, we are able to form predictions about the timing of upcoming events. Although rhythm processing capacities can differ considerably between individuals, these differences are often obscured by subject- and trial-level data averaging procedures in M/EEG research. Here, instead, we systematically assessed the neurophysiological variability exhibited by individuals listening to isochronous equitone sequences interspersed with unexpected deviant tones. First, we focused on rhythm tracking and tested the phase-relationship between neural activity in the delta-band and pre-tone onsets. We observed clear between- and within-subject variability, confirming that individuals employ variable, flexible, but consistent rhythm tracking at the single-trial level. Next, we modelled single-trial time-locked neural responses in the beta-band to investigate individual tendencies to employ binary or triplet grouping ("tic-toc effect"). Beta-band activity showed binary (strong-weak), triplet (strong-weak-weak) and mixed accentuation patterns, resembling the superimposition of a beat. Thus, we identified individual preferences for grouping patterns and tendencies to switch between binary, triplet, or combined patterns over trials. Notably, we demonstrated that the processing of standard and deviant tones was modulated by the employed grouping.

This approach to neurophysiological profiling offers a sensitive strategy to identify individual and dynamically evolving neural signatures of rhythm and beat processing. We suggest that the observed neurophysiological variability reflects flexible windows of opportunity for evaluating and adapting to (un)predictable environmental rhythms.

Significance statementFor decades, music, speech and rhythm research has investigated how humans process, predict, and adapt to environmental rhythms. By taking an individual-trial and -subject approach, we avert the common pooling of EEG data in favor of individual and time-varying neural signatures of rhythm tracking and beat processing. Notably, the superimposition of a beat induces pattern accentuations that affect how individuals respond to expected (standard) and unexpected (deviant) auditory events at the single-trial level. This subject- and trial-based approach supports individual neural profiling and potentially aids the identification of altered neural activity and its consequences for natural listening.


title: The Regulatory Mendelian Mutation score for GRCh38

abstract:
MotivationVarious genome sequencing efforts for individuals with rare Mendelian disease have increased the research focus on the non-coding genome and the clinical need for methods that prioritize potentially disease causal non-coding variants. Some methods and annotations are not available for the current human genome build (GRCh38), for which the adoption in databases, software and pipelines was slow.

ResultsHere, we present an updated version of the Regulatory Mendelian Mutation (ReMM) score, re-trained on features and variants derived from the GRCh38 genome build. Like its GRCh37 version, it achieves good performance on its highly imbalanced data. To improve accessibility and provide users with a toolbox to score their variant files and lookup scores in the genome, we developed a website and API for easy score lookup.

Availability and ImplementationPre-scored whole genome files of GRCh37 and GRCh38 genome builds are available on Zenodo https://doi.org/10.5281/zenodo.6138102. The website and API are available at https://remm.bihealth.org.


title: Mutational footprint of platinum chemotherapy in a secondary thyroid cancer

abstract: Although papillary thyroid carcinoma (PTC) is the most frequent endocrine tumor with a generally excellent prognosis, a patient developed a clinically aggressive PTC eleven years after receiving platinum chemotherapy for ovarian endometrioid adenocarcinoma. Germline and somatic analyses of multi-temporal and multi-regional molecular profiles indicated that ovarian and thyroid tumors did not share common genetic alterations. PTC tumors had driver events associated with aggressive PTC behavior, an RBPMS-NTRK3 fusion and a TERT promoter mutation. Spatial and temporal genomic heterogeneity analysis indicated a close link between anatomical locations and molecular patterns of PTC. Mutational signature analyses demonstrated a molecular footprint of platinum exposure, and that aggressive molecular drivers of PTC were linked to prior platinum-associated mutagenesis. This case provides a direct association between platinum chemotherapy exposure and secondary solid tumor evolution, in specific aggressive thyroid carcinoma, and suggests that uniform clinical assessments for secondary PTC after platinum chemotherapy may warrant further evaluation.


title: Connectivity in Large-Scale Resting State Brain Networks is Related to Motor Learning: a High-Density EEG Study

abstract: Previous research has shown that resting-state functional connectivity (rsFC) between different brain regions (seeds) is related to motor learning and motor memory consolidation. Using high-density electroencephalography (hdEEG) we addressed this question from a brain network perspective. Specifically, we examined frequency-dependent functional connectivity in resting-state networks from twenty-nine young healthy participants before and after they were trained on a motor sequence learning task. Consolidation was assessed with an overnight retest on the motor task. We showed training-related decreases in gamma band connectivity within the motor network, and between the motor and functionally distinct resting-state networks including the attentional network. Brain-behavior correlation analyses revealed that baseline beta, delta and theta rsFC was related to subsequent motor learning and memory consolidation such that lower connectivity within the motor network and between the motor and several distinct resting-state networks was correlated with better learning and overnight consolidation. Last, training-related increases in beta-band connectivity between the motor and the visual networks were related to greater consolidation. Altogether our results indicate that connectivity in large-scale resting-state brain networks is related to - and modulated by - motor learning and memory consolidation processes. These finding corroborate previous seed-based connectivity research and provide evidence that frequency-dependent functional connectivity in resting state networks is critically linked to motor learning and memory consolidation.