title: Crucial Roles of Carotenoids as Bacterial Endogenous Defense System for Bacterial Radioresistance of Deinococcus radiodurans

abstract:
BackgroundThe central dogma of radiation biology is that the cytotoxic and mutagenic effects of radiation are principally the result of biological macromolecules (DNA, RNA, polysaccharides, proteins and lipids) damage caused during the course of irradiation. Previous studies on the radioresistance of the extremely radiation-resistant red-carotenoid-pigmented bacterium Deinococcus radiodurans have been focused on DNA repair system. However, relative little is known about the biochemical basis of the extraordinary recovery capacity of D. radiodurans for ionizing radiation and can include hundreds of DNA double-strand breaks.

Methodology/Principal FindingsHere we identified a novel carotenoid in D. radiodurans as ((all-E)-1-hydroxy-3,4-didehydro-1,2,2,3-quahydro-{beta},{psi}-carotene-4-one-1) and characterized the intracellular distribution of carotenoids. Following exposure to ionizing radiation, the wild-type parent of D. radiodurans survived much higher compared with radiation-sensitive colorless mutants screened by 60Co irradiation and by knockout of crtB or crtI genes for the carotenoid biosynthesis. Electron paramagnetic resonance spectroscopy analysis showed that two carotenoid can effectively scavenge radicals including superoxide anion (O2.-) and hydroxyl radical (.OH). Consistent results showing the substantial antioxidant abilities of the two carotenoids were obtained by UV-induction of bacteriophage {lambda} method in vivo, and the detectable reaction potentials of two carotenoids with nitric oxide (NO) evaluated by ultraviolet-visible near-infrared ray (UV/vis/NIR) spectra assay.

Conclusions/SignificanceOur findings support the idea that the degree of bacterial radioresistance of D. radiodurans is partly related to the levels of carotenoids in the cells. Carotenoids, as an important member of the structural and non-structural components of the cells, are endogenous scavengers of reactive oxygen species and can effectively quench radicals generated during irradiation. The current work suggests that carotenoids play a previously unrecognized protection role in the endogenous defense System (EDS) that facilitates the recovery from radiation injury. And we propose a complete model to elucidate the mechanisms of extreme radioresistance in this remarkable bacterium.


title: An altered metabolism in leukocytes showing in vitro igG memory from SARS-CoV-2-infected patients

abstract: Coronavirus disease 2019 (COVID 19) is a systemic infection that exerts a significant impact on cell metabolism. In this study we performed metabolomic profiling coupled with multivariate statistics analysis obtained from 43 in vitro cultures of peripheral blood mononuclear cells (PBMC), 19 of which displaying IgG memory for spike-S1 antigen 60-90 days after infection. By using mass spectrometry analysis, a significant up regulation of S-adenosyl-Homocysteine, Sarcosine and Arginine was found in leukocytes showing IgG memory. These metabolites are known to be involved in physiological recovering from viral infections and immune activities, and our findings might represent a novel and easy measure that could be of help in understanding SARS-Cov-2 effects on leukocytes.

category: cell biology


title: How effective are frogs in regulating crop pest population in a natural multi-trophic system??

abstract: Potential of frogs as important natural pest control agents has been highlighted earlier. But the effectiveness of frogs in regulating the pest load in intensive agricultural landscape in a multi-trophic system is not clear. We performed controlled field experiment in paddy field with a varying density (observed in high and low agricultural intensity (AI) areas) of a commonly found frog species and compared the pest and pest predator build-up. The consumption rate of the model amphibian was studied using enclosure experiment. The consequent trophic cascade effect of frogs on both crop pest and other arthropod pest predator was analyzed using mathematical population growth models. Although frogs consumed pests, they could not reduce crop pest abundance. although a lesser frog density found in high AI areas significantly affected the pest predator abundance. Based on the functional response result, mathematical growth models demonstrated that with a constant harvesting factor (Hollings Type II) frogs will always have a negative impact on the beneficial natural enemy population due to intraguild predation thereby limiting its potential as a pest regulator. Our study challenges the notion of frogs as an effective pest control agent and argues that increasing habitat diversity might improve overall biological pest suppression.

category: ecology


title: Predicting Bird Distributions Under Global Change

abstract: Global change creates an urgent need to predict spatial responses of biota to support the conservation of sufficient habitat to maintain biodiversity. We present species distribution model theory and a synthesis of avian literature on approaches to collecting occurrence data, selecting explanatory variables and analytical processes currently in use to predict future distributions. We find that interpreting the validity of current predictive distributions is hindered by variation in spatio-temporal resolution of data sets that force hypothesis testing under the Grinnellian niche concept. Broadly, the capacity of species to shift their geographic ranges under land use and climate change is expected to be limited by both large scale (i.e., the physiological or fundamental niche) and small scale (i.e., the realized or tolerance niche) factors. We highlight the strengths and weaknesses of widely used explanatory variables and analytical approaches tailored to macrohabitat characteristics and the Grinnellian niche concept. This synthesis addresses if and how current approaches align with theory and makes recommendations for future directions to improve the accuracy of predictive distribution modelling.


title: EXAMINING DISCRIMINATION PERFORMANCE AND LIKELIHOOD RATIO VALUES FOR TWO DIFFERENT LIKELIHOOD RATIO SYSTEMS USING THE PROVEDIT DATASET

abstract:
The conventional capillary electrophoresis (CE) genotyping workflow used in forensic DNA laboratories is composed of two processes: measurement and interpretation. The outcome of the measurement process is an electropherogram (EPG). The outcome of the interpretation process is a strength of evidence statement often reported in the form of a likelihood ratio (LR) which typically requires probabilistic genotyping software (PGS). An LR system is defined as the entire pipeline of the measurement and interpretation processes where PGS is a piece of the whole LR system. To gain understanding on how two LR systems perform, a total of 154 two-person mixture, 147 three-person mixture, and 127 four-person mixture profiles of varying DNA quality, DNA quantity, and mixture ratios were obtained from the filtered (.CSV) files of the GlobalFiler 29 cycles 15s PROVEDIt dataset and deconvolved in two independently developed fully continuous programs, STRmix v2.6 and EuroForMix v2.1.0. Various parameters were set in each software and LR computations obtained from the two software were based on same/fixed EPG features, same pair of propositions, number of contributors, theta, and population allele frequencies. The ability of each LR system to discriminate between contributor (H1-true) and non-contributor (H2-true) scenarios was evaluated qualitatively and quantitatively. Differences in the numeric LR values and their corresponding verbal classifications between the two LR systems were compared. The magnitude of the differences in the assigned LRs and the potential explanations for the observed differences greater than or equal to 3 on the log10 scale were described. Cases of LR < 1 for H1-true tests and LR > 1 for H2-true tests were also discussed. Our intent is to demonstrate the value of using a publicly available ground truth known mixture dataset to assess discrimination performance of any LR system and show the steps used to investigate and understand similarities and differences between different LR systems. We share our observations with the forensic community and describe how examining more than one PGS with similar discrimination power can be beneficial, help analysts compare interpretation especially with low-template profiles or minor contributor cases, and be a potential additional diagnostic check even if software in use does contain certain diagnostic statistics as part of the output.

HighlightsO_LIThe use of two different Likelihood Ratio (LR) systems to assign LRs is discussed.
C_LIO_LIH1-true and H2-true tests are performed using STRmix and EuroForMix and a large set of PROVEDIt mixture profiles.
C_LIO_LIAssessment of discrimination performance of two LR systems using ROC plots, scatter plots, and relative frequency histograms.
C_LIO_LIThe ability of the two LR systems to discriminate between contributors and non-contributors are statistically indistinguishable for the data that we considered.
C_LIO_LIPotential reasons for the differences in LR values between the two LR systems that are [≥] 3 on the log10 scale are investigated and discussed.
C_LIO_LIContributors with LRs < 1 and non-contributors with LRs > 1 generated from each LR system are discussed.
C_LI

category: genetics


title: The parasitic nematode Strongyloides ratti exists as populations of long-lived asexual lineages.

abstract: Nematodes are important parasites of people and animals and in natural ecosystems they are a major ecological force. Strongyloides ratti is a common parasitic nematode of wild rats and we have investigated its population genetics using single worm, whole genome sequencing. We find that S. ratti populations in the UK consist of mixtures of asexual lineages that are widely dispersed across a host population. These parasite lineages are likely very old and may have originated in Asia from where rats originated. Genes that underly the parasitic phase of the parasites life cycle are hyperdiverse, compared with the rest of the genome, and this may allow the parasites to maximise their fitness in a diverse host population. These patterns of parasitic nematode population genetics have not been found before and may also apply to Strongyloides spp. that infect people, which will affect how we should approach their control.


title: Loose ends in cancer genome structure

abstract: Recent pan-cancer studies have delineated patterns of structural genomic variation across thousands of tumor whole genome sequences. It is not known to what extent the shortcomings of short read ([≤] 150 bp) whole genome sequencing (WGS) used for structural variant analysis has limited our understanding of cancer genome structure. To formally address this, we introduce the concept of "loose ends" - copy number alterations that cannot be mapped to a rearrangement by WGS but can be indirectly detected through the analysis of junction-balanced genome graphs. Analyzing 2,319 pan-cancer WGS cases across 31 tumor types, we found loose ends were enriched in reference repeats and fusions of the mappable genome to repetitive or foreign sequences. Among these we found genomic footprints of neotelomeres, which were surprisingly enriched in cancers with low telomerase expression and alternate lengthening of telomeres phenotype. Our results also provide a rigorous upper bound on the role of non-allelic homologous recombination (NAHR) in large-scale cancer structural variation, while nominating INO80, FANCA, and ARID1A as positive modulators of somatic NAHR. Taken together, we estimate that short read WGS maps >97% of all large-scale (>10 kbp) cancer structural variation; the rest represent loose ends that require long molecule profiling to unambiguously resolve. Our results have broad relevance for future research and clinical applications of short read WGS and delineate precise directions where long molecule studies might provide transformative insight into cancer genome structure.

category: genomics


title: Crossing design shapes patterns of genetic variation in synthetic recombinant populations of Saccharomyces cerevisiae

abstract: "Synthetic recombinant" populations have emerged as a useful tool for dissecting the genetics of complex traits. They can be used to derive inbred lines for fine QTL mapping, or the populations themselves can be sampled for experimental evolution. In latter application, investigators generally value maximizing genetic variation in constructed populations. This is because in evolution experiments initiated from such populations, adaptation is primarily fueled by standing genetic variation. Despite this reality, little has been done to systematically evaluate how different methods of constructing synthetic populations shape initial patterns of variation. Here we seek to address this issue by comparing outcomes in synthetic recombinant Saccharomyces cerevisiae populations created using one of two strategies: pairwise crossing of isogenic strains or simple mixing of strains in equal proportion. We also explore the impact of the varying the number of parental strains. We find that more genetic variation is initially present and maintained when population construction includes a round of pairwise crossing. As perhaps expected, we also observe that increasing the number of parental strains typically increases genetic diversity. In summary, we suggest that when constructing populations for use in evolution experiments, simply mixing founder strains in equal proportion may limit the adaptive potential.


title: KIR gene content imputation from single-nucleotide polymorphisms in the Finnish population

abstract: The killer cell immunoglobulin-like receptor (KIR) gene cluster on chromosome 19 encodes cell surface glycoproteins that bind class I human leukocyte antigen (HLA) molecules as well as some other ligands. Through regulation of natural killer (NK) cell activity KIRs participate in tumour surveillance and clearing viral infections. KIR gene gene copy number variation associates with the outcome of transplantations and susceptibility to immune-mediated diseases. Inferring KIR gene content from genetic variant data is therefore desirable for immunogenetic analysis, particularly in the context of growing biobank genome data collections that rely on genotyping by microarray. Here we describe a stand-alone and freely available gene content imputation for 12 KIR genes. The models were trained using 818 Finnish biobank samples genotyped for 5774 KIR-region SNPs and analysed for KIR gene content with targeted sequencing. Cross-validation results demonstrate a high mean overall accuracy of 99.2% (95% CI: 97.8-99.7%) which compares favourably with previous methods including short-read sequencing based approaches.


title: The geometry of decision-making

abstract: Choosing among spatially-distributed options is a central challenge for animals, from deciding among alternative potential food sources or refuges, to choosing with whom to associate. Using an integrated theoretical and experimental approach (employing immersive virtual reality), we consider the interplay between movement and vectorial integration during decision-making regarding two, or more, options in space. In computational models of this process we reveal the occurrence of spontaneous and abrupt "critical" transitions (associated with specific geometrical relationships) whereby organisms spontaneously switch from averaging vectorial information among, to suddenly excluding one, among the remaining options. This bifurcation process repeats until only one option--the one ultimately selected--remains. Thus we predict that the brain repeatedly breaks multi-choice decisions into a series of binary decisions in space-time. Experiments with fruit flies, desert locusts, and larval zebrafish reveal that they exhibit these same bifurcations, demonstrating that across taxa and ecological context, there exist fundamental geometric principles that are essential to explain how, and why, animals move the way they do.

category: animal behavior and cognition


title: Microarrayed human bone marrow organoids for modeling blood stem cell dynamics

abstract: In many leukemia patients, a poor prognosis is attributed either to the development of chemotherapy resistance by leukemic stem cells (LSCs) or to the inefficient engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM). Here, we build a 3D in vitro model system of bone marrow organoids (BMOs) that recapitulate several structural and cellular components of native BM. These organoids are formed in a high-throughput manner from the aggregation of endothelial and mesenchymal cells within hydrogel microwells. Accordingly, the mesenchymal compartment shows partial maintenance of its self-renewal and multilineage potential, while endothelial cells self-organize into an interconnected vessel-like network. Intriguingly, such a vascular compartment enhances the recruitment of HSPCs in a chemokine ligand/receptor-dependent manner, reminiscent of HSPC homing behavior in vivo. Additionally, we also model LSC migration and nesting in BMOs, thus highlighting the potential of this system as a well accessible and scalable preclinical model for candidate drug screening and patient-specific assays.

category: bioengineering


title: Optimal dimensionality selection for independent component analysis of transcriptomic data

abstract: Independent Component Analysis (ICA) is an unsupervised machine learning algorithm that separates a set of mixed signals into a set of statistically independent source signals. Applied to high-quality gene expression datasets, ICA effectively reveals the source signals of the transcriptome as groups of co-regulated genes and their corresponding activities across diverse growth conditions. Two major variables that affect the output of ICA are the diversity and scope of the underlying data, and the user-defined number of independent components, or dimensionality, to compute. Availability of high-quality transcriptomic datasets has grown exponentially as high-throughput technologies have advanced; however, optimal dimensionality selection remains an open question. Here, we introduce a new method, called OptICA, for effectively finding the optimal dimensionality that consistently maximizes the number of biologically relevant components revealed while minimizing the potential for over-decomposition. We show that OptICA outperforms two previously proposed methods for selecting the number of independent components across four transcriptomic databases of varying sizes. OptICA avoids both over-decomposition and under-decomposition of transcriptomic datasets resulting in the best representation of the organisms underlying transcriptional regulatory network.

category: bioinformatics


title: The BH3-only protein NOXA serves as an independent predictor of breast cancer patient survival and defines susceptibility to microtubule targeting agents

abstract: Breast cancer (BC) treatment frequently involves microtubule-targeting agents (MTAs), such as paclitaxel, that arrest cells in mitosis. Sensitivity to MTAs is defined by a subset of pro- and anti-apoptotic BCL2 family proteins controlling mitochondrial apoptosis. Here, we aimed to determine their prognostic value in primary tumour samples from 92 BC patients. Our analysis identified high NOXA/PMAIP mRNA expression levels as an independent prognostic marker for improved relapse-free survival (RFS) and overall survival (OS) in multivariate analysis in BC patients, independent of their molecular subtype. Analysis of available TCGA datasets of 1060 BC patients confirmed our results and added a clear predictive value of NOXA mRNA levels for patients who received MTA-based therapy. In this TCGA cohort, 122 patients received MTA-treatment and high NOXA mRNA levels correlated with their progression-free interval (PFI) and OS. Our follow-up analyses in a panel of BC cell lines of different molecular subtypes identified NOXA protein expression as a key determinant of paclitaxel sensitivity in triple-negative breast cancer (TNBC) cells. Moreover, we noted highest additive effects between paclitaxel and chemical inhibition of BCLX, but not BCL2 or MCL1, documenting dependence of TNBC cells on BCLX for survival and paclitaxel sensitivity, defined by NOXA expression levels.

category: cancer biology


title: Translational control of fungal gene expression during the wheat-Fusarium graminearum interaction

abstract:
O_LITo date, translational regulation of key genes controlling infection-related processes in fungal pathogens during their interactions with plants has not been studied. Here, we employed ribosome profiling (ribo-seq) to study translational responses and how such responses are coordinated with transcriptional changes in the fungal pathogen Fusarium graminearum (Fg), which causes Fusarium head blight (FHB), a destructive disease of cereal crops worldwide.
C_LIO_LITranscription and translation were not always coordinated with approximately 22% of Fg genes showing a discordant relationship during wheat infection. Nitrite reductase, which we show here as an important component of fungal virulence, is only regulated at the translational level in Fg. In addition, more than 1000 new open reading frames (ORFs), many of which are short and highly conserved, were identified in the Fg genome.
C_LIO_LILike in higher eukaryotes, translation is controlled by upstream ORFs (uORFs) in Fg during infection. Similarly, miRNAs control both transcription and translation in Fg during wheat infection. However, Fgdicer2-dependent miRNAs do not have a significant effect on transcriptional gene expression at the global outset.
C_LIO_LIThe ribo-seq study undertaken here for the first time in any fungal pathogen discovered novel insights about the biology of an important plant pathogen.
C_LI

category: pathology


title: Predicting Composition of Genetic Circuits with Resource Competition: Demand and Sensitivity

abstract: The design of genetic circuits typically relies on characterization of constituent modules in isolation to predict the behavior of modules composition. However, it has been shown that the behavior of a genetic module changes when other modules are in the cell due to competition for shared resources. In order to engineer multi-module circuits that behave as intended, it is thus necessary to predict changes in the behavior of a genetic module when other modules load cellular resources. Here, we introduce two characteristics of circuit modules: the demand for cellular resources and the sensitivity to resource loading. When both are known for every genetic module in a circuit library, they can be used to predict any modules behavior upon addition of any other module to the cell. We develop an experimental approach to measure both characteristics for any circuit module using a resource sensor module. Using the measured resource demand and sensitivity for each module in a library, the outputs of the modules can be accurately predicted when they are inserted in the cell in arbitrary combinations. These resource competition characteristics may be used to inform the design of genetic circuits that perform as predicted despite resource competition.

category: synthetic biology


title: Female mice exhibit a more sensitive automated squint response to pain induced by CGRP and amylin

abstract: We developed an automated squint assay using both black C57BL/6J and white CD1 mice that measured the interpalpebral fissure area between the upper and lower eyelids as an objective quantification of pain. In C57BL/6J mice, we observed a squint response to increasing doses of a migraine trigger, the neuropeptide CGRP, including a significant response in female mice at a dose below detection by the manual grimace scale. Using the automated software, both C57BL/6J and CD1 mice lacked a detectable photic blink response. The CGRP-related peptide amylin induced squinting behavior in female mice, but not males. These data demonstrate that an automated squint assay can be used as an objective, real-time continuous-scale measure of pain that provides higher precision and real-time analysis compared to manual grimace assessments.

category: neuroscience


title: Cingulate-motor circuits update rule representations for sequential choice decisions

abstract: Anterior cingulate cortex mediates the flexible updating of an animals choice responses upon rule changes in the environment. However, how anterior cingulate cortex entrains motor cortex to reorganize rule representations and generate required motor outputs remains unclear. Here, we demonstrate that chemogenetic silencing of the projection terminals of cingulate cortical neurons in secondary motor cortex disrupted sequential choice performance in trials immediately following rule switches, suggesting that these inputs are necessary to update rule representations for choice decisions stored in the motor cortex. Indeed, the silencing of cingulate cortex decreased rule selectivity of secondary motor cortical neurons. Furthermore, optogenetic silencing of cingulate cortical neurons that was temporally targeted to error trials immediately after rule switches exacerbated errors in following trials. These results suggest that cingulate cortex monitors behavioral errors and update rule representations in motor cortex, revealing a critical role for cingulate-motor circuits in adaptive choice behaviors.


title: Dispersed Sleep Microstates and Associated Structural Changes in GBA1 Mouse: Relevance to Rapid Eye Movement Behavior Disorder

abstract: Rapid eye movement (REM) sleep behaviour disorder (RBD) is a rare parasomnia that may predict the later occurrence of alpha-synucleinopathies. Variants in the gene encoding for the lysosomal enzyme glucocerebrosidase, GBA, strongly increase the risk of RBD. In a GBA1-mouse model recently shown to mimic prodromal stages of -synucleinopathy, we now demonstrate striking REM and NREM sleep abnormalities accompanied by distinct structural changes in the more widespread sleep neurocircuitry.


title: Ketamine Produces a Long-Lasting Enhancement of CA1 Neuron Excitability

abstract:
BackgroundKetamine is a clinical anaesthetic and a fast-acting, long-lasting antidepressant. Ketamine is known for its antagonistic actions on N-methyl-D-aspartate (NMDA) receptors, but mechanisms leading to antidepression are not clear. The present study examined synaptic, neuronal and circuit-level loci, the duration of ketamines actions, and the involvement of NMDA receptors in these actions.

MethodsExtracellular evoked field potentials were recorded from the CA1 region of mouse hippocampal slices. Ketamine was tested at antidepressant (10 M) and anaesthetic (350 M) concentrations. Effects of NMDA receptor antagonists, DL-2-Amino-5-phosphonovaleric acid (APV) and MK-801, {gamma}-aminobutyric acid (GABA) receptor antagonist bicuculline, and a potassium channel blocker tetraethylammonium (TEA) were also studied.

ResultsKetamine decreased population spike (PS) amplitudes during application, but a long-lasting increase in PS amplitudes was seen during washout of ketamine. While the acute effects of ketamine were reversed by bicuculline, the washout increase was not altered. This long-term increase was statistically significant (p<0.01, Wilcoxon Rank Test), sustained for >2 hours, and involved postsynaptic mechanisms. A similar, long-lasting effect was produced by MK-801 but was only partially evident with APV, demonstrating the importance of channel block downstream of NMDA receptors. Furthermore, TEA produced a lasting excitability increase, indicating possible involvement of potassium channel block in ketamines long-term effects.

ConclusionsThis is this first report of a long-lasting increase in excitability following ketamine exposure. These results support a growing literature that increased GABA inhibition contributes to ketamine anaesthesia, while increased excitatory transmission contributes to its antidepressant effects.

Published AbstractsSociety for Neuroscience Annual Meeting 2019

Association of University Anesthesiologists Annual Meeting 2020

International Anesthesia Research Society Annual Meeting 2020

The Science of Consciousness Conference 2020

International Society for Anaesthetic Pharmacology Annual Meeting 2020

Western Anesthesia Residents Conference 2021

Association of University Anesthesiologists Annual Meeting 2021

International Anesthesia Research Society Annual Meeting 2021


title: Rational inattention in mice

abstract: Behavior exhibited by humans and other organisms is generally inconsistent and biased, and thus is often labeled irrational. However, the origins of this seemingly suboptimal behavior remain elusive. We developed a behavioral task and normative framework to reveal how organisms should allocate their limited processing resources such that there is an advantage to being imprecise and biased for a given metabolic investment that guarantees maximal utility. We found that mice act as rational-inattentive agents by adaptively allocating their sensory resources in a way that maximizes reward consumption in novel stimulus-reward association environments. Surprisingly, perception to commonly occurring stimuli was relatively imprecise, however this apparent statistical fallacy implies "awareness" and efficient adaptation to their neurocognitive limitations. Interestingly, distributional reinforcement learning mechanisms efficiently regulate sensory precision via top-down normalization. These findings establish a neurobehavioral foundation for how organisms efficiently perceive and adapt to environmental states of the world within the constraints imposed by neurobiology.


title: Whole genome phylogeny of Cyanobacteria documents a distinct evolutionary trajectory of marine picocyanobacteria

abstract: During their 2.7 Gyr long evolution cyanobacteria radiated into a large number of different lineages. To better understand the evolution of cyanobacteria we compared a whole genome phylogenetic tree using 1,047 concatenated single copy orthologues of Prochlorococcus marinus MIT9313 obtained from 93 reference prokaryotic species with traditional phylogenies inferred from 16S rRNA gene and 109 genes found in all used genomes. In contrast to the traditional phylogeny, our genome wide analysis shows a split between picocyanobacteria encompassing all marine Prochlorococcus species, marine "Synechococcus" species, and members of freshwater Cyanobium genus (altogether referred here as the "PSC" clade) and the remaining groups of extant cyanobacteria. To determine the influence of the horizontal gene transfer on the overall tree topology we removed the 374 genes identified as potentially transferred genes. A newly calculated tree utilizing the remaining 673 genes displayed the same topology as the former whole genome tree with the PSC clade as a basal group of all cyanobacteria. The picocyanobacteria also exhibited distinctly larger similarity to reference bacterias cell and genome size, carboxyzome architecture, various metabolic pathways, and chlorophyll synthesis than to the other cyanobacteria. Potentially horizontally transferred genes were found in connected chains throughout fundamental metabolic pathways suggesting that evolution of these genes was severely limited and or brought to a standstill. The environment related genes connected with metabolism of nitrogen, sulfur, and scarce seawater metals were more conserved in PSC group as they were already best tuned for its poor and stable environment. Other genes were found predominantly sequentially static as they were already accurately adapted with rare beneficial mutations. The PSC clade thus represents an isolated cyanobacterial lineage which followed a distinct evolutionary trajectory driven by its specific environment.

category: evolutionary biology


title: Fast and Accurate Estimation of Gas-Phase Entropy from the Molecular Surface Curvature

abstract:
Estimating entropy is crucial for understanding and modifying biological systems, such as protein-ligand binding. Current computational methods to estimate entropy require extensive, or at times prohibitively extensive, computational resources. This article presents SHAPE (SHape-based Accurate Predictor of Entropy), a new method that estimates the gas-phase entropy of small molecules purely from their surface geometry. The gas-phase entropy of small molecules can be computed in {approx}0.01 CPU hours with run time complexity of [Formula], where Na is the number of atoms. The accuracy of SHAPE is within 1 - 2% of computationally expensive quantum mechanical or molecular mechanical calculations. We further show that the inclusion of gas-phase entropy, estimated using SHAPE, improves the rank-order correlation between binding affinity and binding score from 0.18 to 0.40. The speed and accuracy of SHAPE make it well-suited for inclusion in molecular docking or QSAR (quantitative structure-activity relationships) methods.

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title: Coral: a web-based visual analysis tool for creating and characterizing cohorts

abstract:
SummaryA main task in computational cancer analysis is the identification of patient subgroups (i.e., cohorts) based on metadata attributes (patient stratification) or genomic markers of response (biomarkers). Coral is a web-based cohort analysis tool that is designed to support this task: Users can interactively create and refine cohorts, which can then be compared, characterized, and inspected down to the level of single items. Coral visualizes the evolution of cohorts and also provides intuitive access to prevalence information. Furthermore, findings can be stored, shared, and reproduced via the integrated session management. Coral is pre-loaded with data from over 128,000 samples from the AACR Project GENIE, The Cancer Genome Atlas, and the Cell Line Encyclopedia.

Availability and ImplementationCoral is publicly available at https://coral.caleydoapp.org. The source code is released at https://github.com/Caleydo/coral.

Contactthomas.zichner@boehringer-ingelheim.com

Supplementary informationSupplementary data are available at Bioinformatics online.


title: Automatic Consistency Assurance for Literature-based Gene Ontology Annotation

abstract:
BackgroundLiterature-based gene ontology (GO) annotation is a process where expert curators use uniform expressions to describe gene functions reported in research papers, creating computable representations of information about biological systems. Manual assurance of consistency between GO annotations and the associated evidence texts identified by expert curators is reliable but time-consuming, and is infeasible in the context of rapidly growing biological literature. A key challenge is maintaining consistency of existing GO annotations as new studies are published and the GO vocabulary is updated.

MethodIn this work, we introduce a formalisation of biological database annotation inconsistencies, identifying four distinct types of inconsistency. We propose a novel and efficient method using state-of-the-art text mining models to automatically distinguish between consistent GO annotation and the different types of inconsistent GO annotation. We evaluate this method using a synthetic dataset generated by directed manipulation of instances in an existing corpus, BC4GO.

Results and ConclusionTwo models built using our method for distinct annotation consistency identification tasks achieved high precision and were robust to updates in the GO vocabulary. We provide detailed error analysis for demonstrating that the method achieves high precision on more confident predictions. Our approach demonstrates clear value for human-in-the-loop curation scenarios.

Data availabilityThe synthetic dataset, and the code for generating it are available at https://github.com/jiyuc/BioConsistency.


title: Mechanical Forces during Lymph Node Expansion Govern Fibroblastic Reticular Network Remodeling

abstract: Emergent physical properties of tissues are not readily understood by reductionist studies of their constituent cells. Here, we show molecular signals controlling cellular physical properties, collectively determining tissue mechanics of lymph nodes, an immunologically-relevant, adult mammalian tissue. Lymph nodes paradoxically maintain robust tissue architecture in homeostasis yet are continually poised for extensive tissue expansion upon immune challenge. We find that following immune challenge, cytoskeletal mechanics of a cellular meshwork of fibroblasts determine tissue tension independently of extracellular matrix scaffolds. We determine that CLEC-2/podoplanin signalling regulates the cell surface mechanics of fibroblasts, permitting cell elongation and interdigitation through expedited access to plasma membrane reservoirs. Increased tissue tension through the stromal meshwork gates the initiation of fibroblast proliferation, restoring homeostatic cellular ratios and tissue structure through expansion.

category: biophysics


title: Tissue homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics

category: immunology


title: Single-Molecule Dynamics of SARS-CoV-2 5' Cap Recognition by Human eIF4F

abstract: Coronaviruses initiate translation through recognition of the viral RNA 5 m7GpppAm cap by translation factor eIF4F. eIF4F is a heterotrimeric protein complex with cap-binding, RNA-binding, and RNA helicase activities. Modulating eIF4F function through cellular regulation or small-molecule inhibition impacts coronavirus replication, including for SARS-CoV-2. Translation initiation involves highly coordinated dynamics of translation factors with messenger or viral RNA. However, how the eIF4F subunits coordinate on the initiation timescale to define cap-binding efficiency remains incompletely understood. Here we report that translation supported by the SARS-CoV-2 5-UTR is highly sensitive to eIF4A inhibition by rocaglamide. Through a single-molecule fluorescence approach that reports on eIF4E-cap interaction, we dissect how eIF4F subunits contribute to cap-recognition efficiency on the SARS-CoV-2 5 UTR. We find that free eIF4A enhances cap accessibility for eIF4E binding, but eIF4G alone does not change the kinetics of eIF4E-RNA interaction. Conversely, formation of the full eIF4F complex significantly alters eIF4E-cap interaction, suggesting that coordinated eIF4E and eIF4A activities establish the net eIF4F-cap recognition efficiency. Moreover, the eIF4F complex formed with phosphomimetic eIF4E(S209D) binds the viral UTR more efficiently than with wild-type eIF4E. These results highlight a dynamic interplay of eIF4F subunits and mRNA that determines cap-recognition efficiency.


title: Rapid propagation of membrane tension at a presynaptic terminal

abstract: Many cellular activities, such as cell migration1, cell division2,3, signaling4,5, infection6, phagocytosis7 and exo-endocytosis8-11, generate membrane tension gradients that in turn regulate them12. Moreover, membrane flows, which are driven by tension gradients, can limit exo-endocytosis coupling in space and time, as net membrane flow from exocytic to endocytic sites is required to maintain membrane homeostasis13. However, there is controversy over how rapidly plasma membrane flows can relax tension gradients; contrary to the common view12,14,15, recent work showed membrane tension does not equilibrate in several cell types16. Here we show membrane tension can propagate rapidly or slowly, spanning orders of magnitude in speed, depending on cell type. In a neuronal terminal specialized for rapid synaptic vesicle turnover and where exo-endocytosis events occur at distinct loci, membrane tension equilibrates within seconds. By contrast, membrane tension does not propagate in neuroendocrine adrenal chromaffin cells secreting catecholamines. Thus, slow membrane flow and tension equilibration may confine exo- and exocytosis to the same loci17. Stimulation of exocytosis causes a rapid, global decrease in the synaptic terminal membrane tension, which recovers slowly due to endocytosis. Our results demonstrate membrane tension propagates rapidly at neuronal terminals and varies during synaptic activity, likely contributing to exo-endocytosis coupling.


title: Somatic reversion impacts MDS/AML evolution in the short telomere syndromes

abstract: Inherited mutations in telomerase and other telomere maintenance genes manifest in the premature aging short telomere syndromes. Myelodysplastic syndromes and acute myeloid leukemia (MDS/AML) account for 75% of associated malignancies, but how these cancers overcome the germline telomere defect is unknown. We used targeted ultra-deep sequencing to detect candidate somatic reversion mutations hypothesizing they may promote MDS/AML evolution. While no controls carried somatic mutations in telomere maintenance genes (0 of 28), 29% of adults with germline telomere maintenance defects carried at least one (16 of 56, P<0.001). In addition to TERT promoter mutations which were present in 19%, we identified POT1 and TERF2IP mutations in 13%. POT1 mutations impaired telomere binding and in some cases were identical to those found in familial melanoma associated with longer telomere length. Exclusively in patients with germline defects in telomerase RNA (TR), we identified somatic mutations in nuclear RNA exosome genes, RBM7, SKIV2L2, and DIS3, where loss-of-function upregulates mature TR levels. Paradoxically, somatic reversion events were more prevalent in patients who were MDS/AML-free (P=0.01, RR 5.0, 95% CI 1.4-18.9), and no MDS/AML patient had more than one mutant clone (P=0.048). Our data identify diverse somatic adaptive mechanisms in the short telomere syndromes, and raise the possibility that their presence alleviates the telomere crisis that promotes transformation to MDS/AML.


title: Machine learning-enabled cancer diagnostics with widefield polarimetric second-harmonic generation microscopy

abstract: The extracellular matrix (ECM) collagen undergoes major remodeling during tumorigenesis. However, alterations to the ECM are not widely considered in cancer diagnostics, due to mostly uniform appearance of collagen fibers in white light images of hematoxylin and eosin-stained tissue sections. Polarimetric second-harmonic generation (P-SHG) microscopy enables label-free visualization and ultrastructural investigation of non-centrosymmetric molecules, which, when combined with texture analysis, provides multiparameter characterization of tissue collagen. This paper demonstrates whole slide imaging of breast tissue microarrays using high-throughput widefield P-SHG microscopy. The resulting P-SHG parameters are used in classification to differentiate tumor tissue from normal with 94.2% accuracy and F1-score, and 6.3% false discovery rate. Subsequently, the trained classifier is employed to predict tumor tissue with 91.3% accuracy, 90.7% F1-score, and 13.8% false omission rate. As such, we show that widefield P-SHG microscopy reveals collagen ultrastructure over large tissue regions and can be utilized as a sensitive biomarker for cancer diagnostics and prognostics studies.


title: Prenatal Alcohol and THC E-Cigarette Exposure Effects on Motor Development

abstract: It has been well established that prenatal alcohol exposure can lead to a wide range of neurological and behavioral deficits, including alterations in motor domains. However, much less is known about the effects of prenatal cannabis exposure on motor development, despite the fact that cannabis is the most commonly consumed illicit drug among women. Cannabis use among pregnant women has become increasingly popular given the wide-spread perception that consumption is safe during pregnancy. Moreover, alcohol and cannabis are commonly used together, even among pregnant women. Yet, few studies have explored the potential consequences of combined prenatal exposure on behavioral domains. Using our previously established model, pregnant Sprague-Dawley rats were exposed to vaporized alcohol, THC via e-cigarettes, the combination, or a vehicle from gestational days 5-20. Following birth, offspring were tested on early sensorimotor development, adolescent motor coordination, and adolescent activity levels. Prenatal e-cigarette THC exposure delayed sensorimotor development early in life and impaired motor coordination later in adolescence. However, combined prenatal alcohol and THC exposure produced hyperactivity among male offspring. These data suggest that prenatal cannabis exposure may lead to impaired motor skills throughout early development, and that combined exposure with alcohol during gestation may also lead to hyperactivity in adolescence. These findings have important implications for pregnant women and public policy.

category: pharmacology and toxicology


title: Lecture-based, problem-based, digital problem-based and distance learning on knowledge improvement in medical education: a meta-analysis

abstract: Problem-based learning (PBL), an educational approach well applied in education, was believed as a deep method that can promote problem solving, and critical thinking. Varies implementation of PBL across different settings were introduced. How to objectively evaluate knowledge effectiveness of PBLs remains a challenge. The present study is aiming to systematically investigate the knowledge improvement between types of PBL in medical education. Our meta-analysis showed that distance learning using digital PBL could be a good alternative to traditional learning in medical education.

category: scientific communication and education


title: Inhibitory Gating of Coincidence-Dependent Sensory Binding in Secondary Auditory Cortex

abstract: Integration of multi-frequency sounds into a unified perceptual object is critical for recognizing syllables in speech. This "feature binding" relies on the precise synchrony of each components onset timing, but little is known regarding its neural correlates. We find that multi-frequency sounds prevalent in vocalizations, specifically harmonics, preferentially activate the mouse secondary auditory cortex (A2), whose response deteriorates with shifts in component onset timings. The temporal window for harmonics integration in A2 was broadened by inactivation of somatostatin-expressing interneurons (SOM cells), but not parvalbumin-expressing interneurons (PV cells). Importantly, A2 has functionally connected subnetworks of neurons encoding harmonic, but not inharmonic sounds. These subnetworks are stable across days and exist prior to experimental harmonics exposure, suggesting their formation during development. Furthermore, A2 inactivation impairs performance in a discrimination task for coincident harmonics. Together, we propose A2 as a locus for harmonic integration, which may form the circuit basis for vocal processing.


title: Effect of straw incorporation and nitrification inhibitor on nitrous oxide emission in various cropland soils and its microbial mechanism

abstract: Nitrification inhibitor and straw incorporation are widely used to improve crop nitrogen use efficiency in agricultural soil, but their effects on nitrous oxide (N2O) emission across different soil types and the microbial mechanisms remain less understood. In this study, we used controlled experiment and DNA-based molecular analysis to study how nitrification inhibitor (dicyandiamide, DCD) and straw incorporation affect soil nitrogen balance, N2O emission and microbial nitrifiers/denitrifers in three distinct agricultural soils (the black, fluvo-aquic and red soils) across China. Both DCD and straw incorporation improved nitrogen balance by increasing NH4+ and decreasing NO3- in all soils. DCD tended to decrease N2O emission from all soils especially the fluvo-aquic one, while straw incorporation reduced N2O emission only in the fluvo-aquic soil but increased N2O emission in the other two especially the red soil (by [~]600%). T-RFLP analysis revealed that the denitriers community structure are distinct among the three soils but was not strongly affected by DCD or straw incorporation. qPCR analysis revealed that DCD or straw incorporation had no significant effect on nitrifier abundance but increased nitrous oxide reductase nosZ gene abundance in the black/fluvo-aquic soil rather than the red soil. Structural equational modelling further confirmed that, when accounting for treatments and soil properties, nosZ gene abundance is the only biological factor significantly determined N2O emission in different soil types. Taken together, our work advanced the knowledge on the agricultural practices and N2O emission in cropland soils, suggesting that straw incorporation may not be a good choice for the red and black soil areas; management practices should be used as per soil type to balance between nitrogen use efficiency and N2O emission.


title: Feasibility of an Inhalable Ultrasound Contrast Agent to Enhance Airway Imaging

abstract:
IntroductionUltrasound is a relatively inexpensive and non-ionizing imaging modality, but is under-utilized in large airway assessments due to poor image quality. No commercially available contrast agents currently exist for sonographic evaluation of the respiratory system, nor has a respiratory route of microbubble contrast agent (MCA) administration been previously described for the enhancement of airway imaging.

MethodsWe conducted a feasibility study to assess proof-of-concept for an inhalable ultrasound MCA composed of lipid-encapsulated decaflourobutane gas. The MCA was nebulized and administered as an aerosol through the lumen of an ex vivo porcine trachea, with image enhancement evaluated by comparing images pre- and post-exposure. Additionally, primary human bronchial epithelial (hBE) cells from three donors were differentiated at an air-liquid interface and exposed apically to 25 L of undiluted MCA or vehicle control to assess contrast agent-induced cytotoxicity and inflammation. Basolateral medium was collected 24-hours post-exposure and lactate dehydrogenase (LDH) and interleukin-8 (IL-8) concentrations were measured as biomarkers of cytotoxicity and inflammation, respectively.

ResultsContrast microbubbles remained intact following nebulization and enhanced sonographic delineation of ex vivo porcine tracheal walls, indicating adherence of the nebulized MCA to the lumenal mucosa. No significant cytotoxic or inflammatory effects were observed in cultured hBE cells following exposure to MCA.

ConclusionsWe present proof-of-concept for an inhaled MCA for the enhancement of sonographic evaluations of the large airways. Pending further evaluations for safety and effectiveness, inhaled MCA may be feasible for clinical ultrasound applications, such as enhancing ultrasound-guided tracheal intubation, detecting airway bleeds, or monitoring large airway diseases in pediatric populations.


title: Microscopic characterization of local strain field in healing tissue in the central third defect of mouse patellar tendon at early-phase of healing

abstract: Tendons exhibit a hierarchical collagen structure, wherein higher-level components, such as collagen fibres and fascicles, are elongated, slid, and rotated during macroscopic stretching. These mechanical behaviours of collagen fibres play important roles in stimulating tenocytes, imposing stretching, compression, and shear deformation. It was hypothesised that a lack of local fibre behaviours in healing tendon tissue may result in a limited application of mechanical stimuli to cells within the tissue, leading to incomplete recovery of tissue structure and functions in regenerated tendons. Therefore, the present study aimed to measure the microscopic strain field in the healing tendon tissue. A central third defect was created in the patellar tendon of mice, and the regenerated tissue in the defect was examined by tensile testing, collagen fibre analysis, and local strain measurement using confocal microscopy at 3 and 6 weeks after surgery. Healing tissue at 3 weeks exhibited a significantly lower strength and disorganised collagen fibre structure compared with the normal tendon. These characteristics at 6 weeks remained significantly different from those of the normal tendon. Moreover, the magnitude of local shear strain in the healing tissue under 4% tissue strain was significantly smaller than that in the normal tendon. Differences in the local strain field may be reflected in the cell nuclear shape and possibly the amount of mechanical stimuli applied to the cells during tendon deformation. Accordingly, restoration of a normal local mechanical environment in the healing tissue may be key to a better healing outcome of tendon injury.


title: Mitochondrial One-Carbon Flux has a Growth-Independent Role in Promoting Breast Cancer Metastasis

abstract:
Progression of primary cancer to metastatic disease is the most common cause of death in cancer patients with minimal treatment options available. Canonical drugs target mainly the proliferative capacity of cancer cells, which often leaves slow-proliferating, persistent cancer cells unaffected. Metabolic determinants that contribute to growth-independent functions supporting resistance and metastatic dissemination are still poorly understood.

In the present study, we revealed that antifolate treatment results in an uncoupled and autarkic mitochondrial one-carbon (1C) metabolism allowing sustained serine catabolism and formate overflow when cytosolic 1C metabolism is impaired. Interestingly, antifolate dependent growth-arrest did not correlate with decreased migration capacity. Therefore, using the antifolate Methotrexate as a tool compound allowed us to disentangle proliferation and migration to profile the metabolic phenotype of migrating (growth-arrested) cells. Supported by an increased NAD/NADH ratio, we observed increased serine de novo synthesis and increased serine catabolism to formate.

Consequently, inhibition of serine de novo synthesis using the competitive PHGDH-inhibitor BI-4916 or direct inhibition of mitochondrial 1C metabolism reduced cancer cell migration. Using an orthotopic breast cancer model, we show that sole inhibition of mitochondrial serine catabolism does not affect primary tumor growth but strongly inhibits pulmonary metastasis.

We conclude that mitochondrial 1C metabolism, despite being dispensable for proliferative capacities, confers an advantage to cancer cells by supporting their motility potential.

Our results improve our understanding of 1C metabolism and of metabolic determinants that support the process of cancer cell migration and metastasis.


title: Mitochondria preserve an autarkic one-carbon cycle to confer growth-independent cancer cell migration and metastasis


title: Protein sensors of bacterial kinase activity reveal antibiotic-dependent kinase activation in single cells

abstract: A lack of direct single-cell readouts for bacterial kinase activity remains a major barrier to our understanding of most signaling systems. At the single-cell-level, protein kinase activity is typically inferred by the activity of downstream transcriptional reporters. Complicating this approach in vivo, promoters are often co-regulated by several pathways, making the activity of a specific kinase difficult to deconvolve. Here, we have designed and constructed new, direct and specific sensors of bacterial kinase activity, including FRET-based sensors, as well as a synthetic transcription factor that responds to phosphorylation. We demonstrate the utility of these reporters in measuring kinase activity in population-based and single-cell assays during various growth phases and antibiotic treatments. These sensors respond to a highly conserved bacterial Ser/Thr kinase, PrkC that has no known dedicated transcription factor and whose regulon is known to be convolved with an essential signaling system. We used these new sensors to measure PrkC activity in colonies, bulk culture, and single cells. Together these new sensors provide evidence for considerable heterogeneity in PrkC activity in actively growing populations. We further demonstrate that PrkC activity increases in response to a cell-wall active antibiotic that blocks the late steps in peptidoglycan synthesis (cefotaxime), but not the early steps (fosfomycin). This is consistent with a model where PrkC senses and responds to blocks in the extracellular steps in cell wall synthesis. As the design of these phosphorylation sensors is modular, we anticipate that this work may have broad applications to other bacterial signaling systems in the future.


title: Dynamics of repair and regeneration of adult zebrafish respiratory gill tissue after cryoinjury.

abstract: The study of respiratory tissue damage and repair is critical to understand not only the consequences of respiratory tissue exposure to infectious agents, irritants and toxic chemicals, but also to comprehend the pathogenesis of chronic inflammatory lung diseases. To gain further insights into these processes, we developed a gill cryoinjury model in the adult zebrafish. Time course analysis showed that cryoinjury of the gills triggered an inflammatory response, extensive cell death and collagen deposition at the site of injury. However, the inflammation was rapidly resolved, collagen accumulation dissipated and by 3 weeks after injury the affected gill tissue had begun to regenerate. RNA seq analysis of cryoinjured gills, combined with a comparison of zebrafish heart cryoinjury and caudal fin resection datasets, highlighted the differences and similarities of the transcriptional programmes deployed in response to injury in these three zebrafish models. Comparative RNA seq analysis of cryoinjured zebrafish gills with mouse pulmonary fibrosis datasets also identified target genes, including the understudied FIBIN, as differentially expressed in the two species. Further mining, including of human datasets, suggests that FIBIN may contribute to the successful resolution of tissue damage without fibrosis.


title: Spatial profiling of microbial communities by sequential FISH with error-robust encoding

abstract: Spatial analysis of microbiomes at single cell resolution with high multiplexity and accuracy has remained challenging. Here we present spatial profiling of a microbiome using sequential error-robust fluorescence in situ hybridization (SEER-FISH), a highly multiplexed and accurate imaging method that allows mapping of microbial communities at micron-scale. We show that multiplexity of RNA profiling in microbiomes can be increased significantly by sequential rounds of probe hybridization and dissociation. Combined with error-correction strategies, we demonstrate that SEER-FISH enables accurate taxonomic identification in complex microbial communities. Using microbial communities composed of diverse bacterial taxa isolated from plant rhizospheres, we show that SEER-FISH can quantify the abundance of each taxon and map microbial biogeography on roots. SEER-FISH should enable accurate spatial profiling of the ecology and function of complex microbial communities.

category: microbiology


title: The 3-phosphoinositide-dependent protein kinase 1 is an essential upstream activator of protein kinase A in malaria parasites

abstract: Cyclic AMP (cAMP) signalling is crucial for the propagation of asexual malaria blood stage parasites. Recent work on Plasmodium falciparum demonstrated that phosphorylation of the invasion ligand AMA1 by the catalytic subunit of cAMP-dependent protein kinase A (PfPKAc) is an essential step during parasite invasion into red blood cells. However, the exact mechanisms regulating PfPKAc activity are only partially understood and PfPKAc function has not been extensively studied in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown mutant, we confirm the essential role for PfPKAc in erythrocyte invasion and demonstrate that PfPKAc is involved in regulating gametocyte deformability. Interestingly, we observed that the conditional overexpression of PfPKAc also caused a profound lethal phenotype by preventing intra-erythrocytic parasite multiplication. Whole genome sequencing of parasites selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the putative parasite orthologue of 3-phosphoinositide-dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we show that PfPDK1 is essential for PfPKAc activation, most likely by phosphorylating T189 in the PfPKAc activation loop. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and identify PfPDK1 as a crucial upstream regulator in this pathway and potential new drug target.


title: The type 3 secretion system requires actin polymerization to open translocon pores

abstract:
Many bacterial pathogens require a type 3 secretion system (T3SS) to establish a niche. Host contact activates bacterial T3SS assembly of a translocon pore in the host plasma membrane. Following pore formation, the T3SS docks onto the translocon pore. Docking establishes a continuous passage that enables the translocation of virulence proteins, effectors, into the host cytosol. Here we investigate the contribution of actin polymerization to T3SS-mediated translocation. Using the T3SS model organism Shigella flexneri, we show that actin polymerization is required for assembling the translocon pore in an open conformation, thereby enabling effector translocation. Opening of the pore channel is associated with a conformational change to the pore, which is dependent upon actin polymerization and a coiled-coil domain in the pore protein IpaC. An IpaC mutant is identified that shows actin polymerization-dependent pore opening is distinct from the previously described actin polymerization-dependent ruffles that are required for bacterial internalization. Moreover, actin polymerization is not required for other pore functions, including docking or pore protein insertion into the plasma membrane. Thus, activation of the T3SS is a multilayered process in which host signals are sensed by the translocon pore leading to the activation of effector translocation.

Significance StatementThe type 3 secretion system (T3SS) is required for the virulence of a variety of bacteria that infect humans. The T3SS forms in the host cell membrane a pore that is the conduit for delivering virulence proteins into the cell. Here, we demonstrate that actin polymerization is necessary to convert pores into an open conformation that is competent for virulence protein delivery. We find that activation of type 3 secretion proceeds in a multistep process whereby bacteria dock onto the translocon pore and then activate secretion and delivery of virulence proteins.


title: Abiotic factors modulate interspecies competition mediated by the type VI secretion system effectors in Vibrio cholerae

abstract: Vibrio cholerae, the etiological pathogen of cholera, relies on its type VI secretion system (T6SS) as an effective weapon to survive in highly competitive communities. The anti-bacterial and anti-eukaryotic functions of T6SS depend on its secreted effectors that target multiple essential cellular processes. However, the mechanisms that account for effector diversity and different effectiveness during interspecies competition remain elusive. Here, we report that environmental cations and temperature play a key role in dictating effector-mediated competition of Vibrio cholerae. We found that V. cholerae could employ its cell-wall-targeting effector TseH to outcompete the otherwise resistant Escherichia coli and the V. cholerae immunity deletion mutant{Delta} tsiH when Ca2+ and Mg2+ were supplemented. The E. coli {Delta}phoQ mutant was more sensitive to TseH-mediated killing during competition, suggesting the metal-sensing PhoPQ two-component system is protective to E. coli from TseH activity. Using transcriptome analysis, we found multiple stress response systems, including acid stress response, oxidative stress response, and osmotic stress response, were activated in E. coli expressing TseH in comparison with E. coli expressing the inactive mutant TseHH64A. The membrane-targeting lipase effector TseL also exhibited reduced killing against E. coli when divalent cations were removed. In addition, competition analysis of E. coli with V. cholerae single-effector active strains reveals a temperature-dependent susceptibility of E. coli to effectors, VasX, VgrG3, and TseL. These findings suggest that abiotic factors, that V. cholerae frequently encounters in natural habitats, play a crucial role in dictating the competitive fitness conferred by the type VI secretion system in complex multispecies communities.


title: Expression of putative effectors of different Xylella fastidiosa subspecies/strains reveals recognition and defense activation in various model plants

abstract: The re-emergence of Gram-negative bacterium Xylella fastidiosa in Europe in 2013 impelled the scientific community to discover novel strategies for crop protection. The wide host range of Xylella indicates the existence of yet not characterized pathogenic mechanisms to overcome plant defenses. The recent uprising accuracy of a variety of bioinformatics tools, with the ability to predict the function of putative microbial protein represent a useful approach for understanding which of these proteins are associated with pathogens virulence. In this study we collected a number of putative effectors from two X. fastidiosa strains: Temecula1 and CoDiRo and the subspecies (ssp.) Sandyi Ann-1. We designed an in-planta Agrobacterium based expression system that drives the expressed proteins to the cell apoplast, in order to investigate their ability to activate defense in various model plants. Furthermore, we organized the resulted proteins according to their sequential and structural similarities via the I-TASSER online tool. We identified that various X. fastidiosa proteins were able to differentially elicit cell death-like phenotypes in Nicotiana tabacum, N. sylvestris and N. benthamiana. These proteins are members of different enzymatic groups: a) hydrolases/hydrolases inhibitors, b) serine proteases and c) metal transferases. Collectively, we identified structurally similar proteins that were able to differentially elicit cell death-like phenotypes in different cultivars of the same species. Our findings provide the bases for further studies on the mechanisms that underlie host-defense activation by X. fastidiosa putative effectors, as well as, pathogens adaptation in susceptible hosts.


title: Genetic tools for the stable overexpression of circular RNAs

abstract: Circular RNAs (circRNAs) are a class of non-coding RNAs that feature a covalently closed ring structure formed through backsplicing. circRNAs are broadly expressed and contribute to biological processes through a variety of functions. Standard gain-of-function and loss-of-function approaches to study gene functions have significant limitations when studying circRNAs. Overexpression studies in particular suffer from the lack of efficient genetic tools. While mammalian expression plasmids enable transient overexpression of circRNAs in cultured cells, most cell biological studies require long-term ectopic expression. Here we report the development and characterization of genetic tools enabling stable circRNA overexpression in vitro and in vivo. We demonstrated that circRNA expression constructs can be delivered to cultured cells via transposons, whereas lentiviral vectors have limited utility for the delivery of circRNA constructs. We further showed that circRNA transposons can be supplied to mouse livers via hydrodynamic tail vein injection, resulting in ectopic circRNA expression in a hepatocellular carcinoma mouse model. Furthermore, we generated genetically engineered mice harboring circRNA expression constructs. We demonstrate that this approach enables constitutive, global circRNA overexpression as well as inducible circRNA expression directed specifically to melanocytes in a melanoma mouse model. Overall, these tools expand the genetic toolkit available for the functional characterization of circRNAs of interest.

category: molecular biology


title: Human HELB is a processive motor protein which catalyses RPA clearance from single-stranded DNA

abstract: Human HELB is a poorly-characterised helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single molecule approaches to characterise the biochemical activities of HELB protein with a particular focus on its interactions with RPA and RPA-ssDNA filaments. HELB is a monomeric protein which binds tightly to ssDNA with a site size of [~]20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5'-to-3' direction accompanied by the formation of DNA loops and with an efficiency of 1 ATP per base. HELB also displays classical helicase activity but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from single-stranded DNA.


title: Lack of Cas13a inhibition by anti-CRISPR proteins from Leptotrichia prophages

abstract: CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements, like bacteriophages and plasmids. To overcome CRISPR immunity, phages have evolved diverse families of anti-CRISPR proteins (Acrs), each of which inhibits the nucleic acid binding or cleavage activities of specific Cas protein families. Recently, Lin et al. (2020) described the discovery and characterization of 7 different Acr families (AcrVIA1-7) that inhibit type VI-A CRISPR systems, which use the nuclease Cas13a to perform RNA-guided RNA cleavage. In this Matters Arising article, we detail several inconsistencies that question the results reported in the Lin et al. (2020) study. These include inaccurate bioinformatics analyses, as well as reported experiments involving bacterial strains that are impossible to construct. The authors were unable to provide their published strains with which we might reproduce their experiments. We independently tested the Acr sequences described in Lin et al. (2020) in two different Cas13 inhibition assays, but could not detect anti-CRISPR activity. Taken together, our data and analyses prompt us to question the claim that AcrVIA1-7 reported in Lin et al. are bona fide type VI anti-CRISPR proteins.


title: AURKB/Ipl1 restarts replication forks to recover from replication stress.

abstract: Aurora kinase B (AURKB in human, Ipl1 in S. cerevisiae) is a master regulator of mitosis and its dysregulation has been implicated in chromosome instability. AURKB accumulates in the nucleus in S-phase and is regulated by CHK1 but has not been implicated in in the DNA Damage Response (DDR). Here we show that AURKB has a conserved role to recover from replication stress and restart replication forks. Active AURKB is localized to replication forks after a prolonged arrest. CHK1 phosphorylation of AURKB induces activating phosphorylation of PLK1 and both Aurora and Plk1 are required to deactivate the DDR. Clinical trials with AURKB inhibitors are designed to target established roles for AURKB in mitosis. Our data suggest combinations of AURKB inhibitors and DNA damaging agents could be of therapeutic importance.

category: cell biology


title: DeepNull: Modeling non-linear covariate effects improves phenotype prediction and association power

abstract: Genome-wide association studies (GWAS) examine the association between genotype and phenotype while adjusting for a set of covariates. Although the covariates may have non-linear or interactive effects, due to the challenge of specifying the model, GWAS often neglect such terms. Here we introduce DeepNull, a method that identifies and adjusts for non-linear and interactive covariate effects using a deep neural network. In analyses of simulated and real data, we demonstrate that DeepNull maintains tight control of the type I error while increasing statistical power by up to 20% in the presence of non-linear and interactive effects. Moreover, in the absence of such effects, DeepNull incurs no loss of power. When applied to 10 phenotypes from the UK Biobank (n=370K), DeepNull discovered more hits (+6%) and loci (+7%), on average, than conventional association analyses, many of which are biologically plausible or have previously been reported. Finally, DeepNull improves upon linear modeling for phenotypic prediction (+23% on average).

category: genetics


title: The Interplay of Electrostatics and Chemical Positioning in the Evolution of Antibiotic Resistance in TEM β-Lactamases

abstract: The interplay of enzyme active site electrostatics and chemical positioning are important for understanding the origin(s) of enzyme catalysis and the design of novel catalysts. We reconstruct the evolutionary trajectory of TEM-1 {beta}-lactamase to TEM-52 towards extended-spectrum activity to better understand the emergence of antibiotic resistance and to provide insights into the structure-function paradigm and non-covalent interactions involved in catalysis. Utilizing a detailed kinetic analysis and the vibrational Stark effect, we quantify the changes in rates and electric fields in the Michaelis and acyl-enzyme complexes for penicillin G and cefotaxime to ascertain the evolutionary role of electric fields to modulate function. These data are combined with MD simulations to interpret and quantify the substrate-dependent structural changes during evolution. We observe that this evolutionary trajectory utilizes a large preorganized electric field and substrate-dependent chemical positioning to facilitate catalysis. This governs the evolvability, substrate promiscuity, and protein fitness landscape in TEM {beta}-lactamase antibiotic resistance.


title: Spatial-transcriptomics reveals unique defining molecular features of fluorescence-sorted 5-aminolevulinic acid+ infiltrative tumor cells associated with glioblastoma recurrence and poor survival

abstract: Spatiotemporal-heterogeneity of glioblastoma (GBM) originating from the genomic and transcriptional variation in spatially distinct intra-tumor sites, may contribute to subtype switching in GBM prior to and upon recurrence. Fluorescence-guided neurosurgical resection utilizing 5-aminolevulinic acid (5ALA) has enabled the isolation of infiltrative margin tumor cells (5ALA+ cells) from a background of non-neoplastic cells. We have explored the spatial-transcriptomic (ST) landscape to interrogate molecular signatures unique to infiltrating 5ALA+ cells in comparison to GBM core, rim, and invasive margin non-neoplastic cells. ST analysis reveals that GBM molecular subtype plasticity is not restricted to recurrence, but manifests regionally in a cell-type-specific manner. Whilst GBM core and rim are highly enriched with Classical and Proneural subtypes, the unique enrichment of the Mesenchymal subtype (MES) in 5ALA+ cells supports the hypothesis that MES 5ALA+ cells may drive GBM recurrence. Upregulation of the wound response pathway in 5ALA+ cells signifies the possibility of hijacking the wound healing pathway of neural cells to promote tumor growth. Exon-intron split analysis revealed an upregulation of exonic counts for MES and wound-response genes in 5ALA+ cells, implying that these genes are under active post-transcriptional control. Network analysis suggests that wound response genes, including chemokine CCL2 that recruits regulatory T-cells and monocytic myeloid-derived suppressor cells, are controlled by an IRF8-mediated transcriptional program in 5ALA+ cells. A higher stemness signature both in 5ALA+ cells and non-neoplastic cells of the invasive margin emphasizes the role of this microenvironment in stemness acquisition and defines 5ALA+ cells as a rare sub-population of GBM stem cells. Finally, we establish a link between the unique molecular signatures of 5ALA+ cells and poor survival and GBM recurrence. Characterization of the 5ALA+ infiltrative sub-population offers an opportunity to develop more effective GBM treatments and urges focus away from the GBM proliferative core, upon which failed targeted therapies have been predicated.


title: Comprehensive analysis of clustered mutations in cancer reveals recurrent APOBEC3 mutagenesis of ecDNA

abstract: Clustered somatic mutations are common in cancer genomes with prior analyses revealing several types of clustered single-base substitutions, including doublet- and multi-base substitutions, diffuse hypermutation termed omikli, and longer strand-coordinated events termed kataegis. Here, we provide a comprehensive characterization of clustered substitutions and clustered small insertions and deletions (indels) across 2,583 whole-genome sequenced cancers from 30 cancer types. While only 3.7% of substitutions and 0.9% of indels were found to be clustered, they contributed 8.4% and 6.9% of substitution and indel drivers, respectively. Multiple distinct mutational processes gave rise to clustered indels including signatures enriched in tobacco smokers and homologous-recombination deficient cancers. Doublet-base substitutions were caused by at least 12 mutational processes, while the majority of multi-base substitutions were generated by either tobacco smoking or exposure to ultraviolet light. Omikli events, previously attributed to the activity of APOBEC3 deaminases, accounted for a large proportion of clustered substitutions. However, only 16.2% of omikli matched APOBEC3 patterns with experimental validation confirming additional mutational processes giving rise to omikli. Kataegis was generated by multiple mutational processes with 76.1% of all kataegic events exhibiting AID/APOBEC3-associated mutational patterns. Co-occurrence of APOBEC3 kataegis and extrachromosomal-DNA (ecDNA) was observed in 31% of samples with ecDNA. Multiple distinct APOBEC3 kataegic events were observed on most mutated ecDNA. ecDNA containing known cancer genes exhibited both positive selection and kataegic hypermutation. Our results reveal the diversity of clustered mutational processes in human cancer and the role of APOBEC3 in recurrently mutating and fueling the evolution of ecDNA.


title: Effect of Suppression of Rotational Joint Instability on Cartilage and Meniscus Degeneration in Mouse Osteoarthritis Model

abstract:
ObjectiveJoint instability and meniscal dysfunction contribute to the onset and progression of knee osteoarthritis (OA). In the destabilization of the medial meniscus (DMM) model, secondary OA occurs due to the rotational instability and increases compressive stress resulting from the meniscal dysfunction. We created a new controlled abnormal tibial rotation (CATR) model that reduces the rotational instability that occurs in the DMM model. So, we aimed to investigate whether rotational instability affects articular cartilage degeneration using the DMM and CATR models, as confirmed using histology and immunohistochemistry.

DesignTwelve-week-old male mice were randomized into 3 groups: DMM group, CATR group, and INTACT group (right knee of the DMM group). After 8 and 12 weeks, we performed the tibial rotational test, safranin-O/fast green staining, and immunohistochemical staining for TNF- and MMP-13.

ResultsThe rotational instability in the DMM group was significantly higher than that of the other groups. And articular cartilage degeneration was higher in the DMM group than in the other groups. However, meniscal degeneration was observed in both DMM and CATR groups. The TNF- and MMP-13 positive cell rates in the articular cartilage of the CATR group were lower than those in the DMM group.

ConclusionsWe found that the articular cartilage degeneration was effectively suppressed by controlling the rotational instability caused by meniscal dysfunction. These findings suggest that suppression of rotational instability in the knee joint is an effective therapeutic measure for preventing OA progression.


title: Cell type- and region-specific modulation of cocaine seeking by micro-RNA-1 in striatal projection neurons

abstract: The persistent and experience-dependent nature of drug addiction may result in part from epigenetic alterations, including non-coding micro-RNAs (miRNAs), which are both critical for neuronal function and modulated by cocaine in the striatum. Two major striatal cell populations, the striato-nigral and striato-pallidal projection neurons, express respectively the D1 (D1-SPNs) and D2 (D2-SPNs) dopamine receptor, and display distinct but complementary functions in drug-evoked responses. However, a cell-type-specific role for miRNAs action has yet to be clarified. Here, we evaluated the expression of a subset of miRNAs proposed to modulate cocaine effects in the nucleus accumbens (NAc) and dorsal striatum (DS) upon sustained cocaine exposure in mice and showed that these selected miRNAs were preferentially up-regulated in the NAc. We then focused on miR-1 considering the important role of some of its predicted mRNA targets, such as fosb and npas4, in the effects of cocaine. We validated these targets in vitro and in vivo and further showed that overexpression of miR-1 in D1-SPNs of the DS reduced cocaine-induced CPP reinstatement, whereas it increased cue-induced reinstatement of cocaine-SA, without affecting other cocaine-mediated adaptive behavior. In addition, miR-1 overexpression in D2-SPNs of the DS reduced the motivation to self-administer cocaine but did not modify other measured behaviors. Together, our results highlight a precise cell-type- and region-specific control of relapse to cocaine-seeking behaviors by miR-1, and illustrate the importance of cell-specific investigations.


title: Early Th2 cytokine production in Heligmosomoides polygyrus and Toxoplasma gondii co-infected mice is associated with reduced IFNγ production, increased parasite loads and increased mortality

abstract: Co-infections are a common reality but understanding how the immune system responds in this context is complex and can be unpredictable. Despite this, it is key to develop models that will provide better translatability to real world situations. Heligmosomoides polygyrus (parasitic roundworm) and Toxoplasma gondii (protozoan parasite) are well studied organisms that stimulate a characteristic Th2 and Th1 response respectively. IFN{gamma}-producing T cells, NK and {gamma}{delta} T cells contribute to early protective immunity during T. gondii infection. To minimise immunopathology, IL-10 is also key to a successful response. Previous research has found H. polygyrus to improve survival during co-infection with both parasites. IFN{gamma}-producing CD4+ and CD8+ T cells were implicated in this protection. Using a similar approach, we have found the opposite. Our co-infected animals displayed greater mortality and intestinal pathology than either single infection. This was associated with an early increase in Th2 cytokines in the Peyers patches, mesenteric lymph nodes and spleen. Co-infected animals also had reduced IFN{gamma}-producing cells at day 5 post T. gondii infection in the Peyers patches (CD8 T cells only) and in the MLN (NK, NKT, {gamma}{delta} T, CD4+ T and CD8+ T cells). This correlated with increased parasite loads in the MLN at 10 days post T. gondii infection. Our results demonstrate that co-infection dynamics can vary dramatically and that careful consideration needs to be taken when interpreting data in each situation.


title: Novel aspects of iron homeostasis in pathogenic bloodstream form Trypanosoma brucei

abstract:
Iron is an essential regulatory signal for virulence factors in many pathogens. Mammals and bloodstream form (BSF) Trypanosoma brucei obtain iron by receptor-mediated endocytosis of transferrin bound to receptors (TfR) but the mechanisms by which T. brucei subsequently handles iron remains enigmatic. Here, we analyse the transcriptome of T. brucei cultured in iron-rich and iron-poor conditions. We show that adaptation to iron-deprivation induces upregulation of TfR, a cohort of parasite-specific genes (ESAG3, PAGS), genes involved in glucose uptake and glycolysis (THT1 and hexokinase), endocytosis (Phosphatidic Acid Phosphatase, PAP2), and most notably a divergent RNA binding protein RBP5, indicative of a non-canonical mechanism for regulating intracellular iron levels. We show that cells depleted of TfR by RNA silencing import free iron as a compensatory survival strategy. The TfR and RBP5 iron response are reversible by genetic complementation, the response kinetics are similar, but the regulatory mechanisms are distinct. Increased TfR protein is due to increased mRNA. Increased RBP5 expression, however, occurs by a post-transcriptional feedback mechanism whereby RBP5 interacts with its own, and with PAP2 mRNAs. Further observations suggest that increased RBP5 expression in iron-deprived cells has a maximum threshold as ectopic overexpression above this threshold disrupts normal cell cycle progression resulting in an accumulation of anucleate cells and cells in G2/M phase. This phenotype is not observed with overexpression of RPB5 containing a point mutation (F61A) in its single RNA Recognition Motif. Our experiments shed new light on how T. brucei BSFs reorganise their transcriptome to deal with iron stress revealing the first iron responsive RNA binding protein that is co-regulated with TfR, is important for cell viability and iron homeostasis; two essential processes for successful proliferation.

Author SummaryAfrican trypanosomes are single-celled extracellular parasites of humans and animals relying on essential host nutrients for survival. They satisfy their iron needs by capturing host transferrin-bound iron using a surface-localised transferrin receptor (TfR) that is structurally distinct from its host counterpart. Little is known about the trypanosome response to fluctuations in host iron availability, with the exception of modulated TfR expression. We show that unlike other eukaryotes, at the transcriptome level, trypanosomes do not regulate iron-dependent enzymes as a mechanism to cope with iron deprivation.

Instead, we identify a group of novel iron responsive trypanosome-specific genes, particularly an RNA Binding Protein RBP5 that is responsive to iron levels, albeit mediated by a distinct mechanism from TfR. We show that although RBP5 expression is elevated at the mRNA and protein levels, increased abundance above a maximum threshold is toxic. The trypanosome TfR has been suggested as a therapeutic target, but whether it is essential for optimal host colonisation is unclear. Our data demonstrate that trypanosomes efficiently import free iron from their environment independent of TfR suggesting that alternative iron uptake pathways exist, and that any therapeutic interventions targeting TfR must be evaluated with caution.


title: ColFeatures: Automated data extraction and classification of bacterial colonies

abstract: Examining microbial colonies on agar plates have been at the core of microbiology for many decades. It is usually done manually, and therefore subject to bias besides requiring a considerable amount of time and effort. In order to optimize and standardize the identification of bacterial colonies growing on agar plates, we have developed an open access tool available on GitHub: ColFeatures. The software allows automated identification of bacterial colonies, extracts key morphological data and generate labels that ensure tracking of temporal development. We included machine learning algorithms that provide sorting of environmental isolates by using cluster methodologies. Furthermore, we show how cluster performance is evaluated using index scores (Silhouette, Calinski-Harabasz, Davies-Bouldin) to ensure the outcome of colony classification. As automation becomes more prominent in microbiology, tools as ColFeatures will assist identification of bacterial colonies on agar plates, bypassing human bias and complementing sequencing or mass spectrometry information that often comes attached with a considerable price tag.


title: Electrolysis of Bacterial Biofilm

abstract: Biofilms are a significant global health problem and are of most important clinical impediments of the century. Biofilms play a crucial role in antimicrobial resistance and chronic infections by acting as reservoir for microorganisms. Therefore greater understanding of biofilm and novel methods to eliminate them are needed. We considered electrolysis as a contrivance to decompose and disperse the Pseudomonas aeruginosa biofilm in microtiter dishes. We showed electrolysis effectively eliminated the Pseudomonas aeruginosa biofilm with the potential to change our approach in tackling biofilms.


title: Deciphering microbiome impacts on fungal-microalgal interaction in a marine environment using metabolomics

abstract: The comprehension of microbial interactions is one of the key challenges in microbial ecology. The present study focuses on studying the chemical interaction between the toxic dinoflagellate Prorocentrum lima PL4V strain and associated fungal strains (two Penicillium sp. strains and three Aspergillus sp) among which the Aspergillus pseudoglaucus strain MMS1589 was selected for further co-culture experiment. Such rarely studied interaction (fungal-microalgal) was explored in axenic and non-axenic conditions, in a dedicated microscale marine environment (hybrid solid/liquid conditions), to delineate specialized metabolome alteration in relation to the P. lima and A. pseudoglaucus co-culture in regard to the presence of their associated bacteria. Such alteration was monitored by high-performance liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). In-depth analysis of the resulting data highlighted (1) the chemical modification associated to fungal-microalgal co-culture, and (2) the impact of associated bacteria in microalgal resilience to fungal interaction. Even if only a very low number of highlighted metabolites were fully characterised due to the poor chemical investigation of the studied species, a clear co-culture induction of the dinoflagellate toxins okadaic acid and dinophysistoxin 1 was observed. Such results highlight the importance to consider microalgal microbiome to study parameters regulating toxin production. Finally, a microscopic observation showed an unusual physical interaction between the fungal mycelium and the dinoflagellates.


title: Production of human translation-competent lysates using dual centrifugation

abstract: Protein synthesis is a central process in gene expression and the development of efficient in vitro translation systems has been the focus of scientific efforts for decades. The production of translation-competent lysates originating from human cells or tissues remains challenging, mainly due to the variability of cell lysis conditions. Here we present a robust and fast method based on dual centrifugation that allows for detergent-free cell lysis under controlled mechanical forces. We optimized the lysate preparation to yield cytoplasm-enriched extracts from human cells that efficiently translate mRNAs in a cap-dependent as well as in an IRES-mediated way. Reduction of the phosphorylation state of eIF2 using recombinant GADD34 and 2-aminopurine considerably boosts the protein output, reinforcing the potential of this method to produce recombinant proteins from human lysates.


title: Unleashing a Novel Function of Endonuclease G in Mitochondrial Genome Instability

abstract: Having its own genome makes mitochondria a unique and semiautonomous organelle within cells. Mammalian mitochondrial DNA (mtDNA) is double-stranded closed circular molecule of about 16 kb coding 37 genes. Mutations, including deletions in the mitochondrial genome can culminate in different human diseases. Mapping of the deletion junctions suggests that the breakpoints are generally seen at hotspots.  9-bp deletion (8271-8281), seen in the intergenic region of cytochrome c oxidase II/tRNALys, is the most common mitochondrial deletion. While it is associated with several diseases like myopathy, dystonia, and hepatocellular carcinoma, it has also been used as an evolutionary marker. However, the mechanism responsible for its fragility is unclear. In the current study, we show that Endonuclease G, a mitochondrial nuclease responsible for nonspecific cleavage of nuclear DNA during apoptosis, can induce breaks at sequences associated with  9-bp deletion, when it is present on a plasmid or in the mitochondrial genome. Through a series of in vitro and intracellular studies, we show that Endonuclease G binds to G-quadruplex structures formed at the hotspot and induces DNA breaks. Besides, we reconstitute the whole process of  9-bp deletion using purified Endonuclease G to induce breaks in mtDNA, followed by mitochondrial extract-mediated DSB repair and establish that microhomology-mediated end joining is responsible for the generation of mtDNA deletion. Finally, we show that the whole process is regulated by different stress conditions, which may modulate release of Endonuclease G to the mitochondrial matrix. Therefore, we uncover a new role for Endonuclease G in generating deletions, which is dependent on the formation of G4 DNA within the mitochondrial genome. Thus, in this study we identify a novel property of Endonuclease G, besides its role in apoptosis, and the recently described  elimination of paternal mitochondria during fertilization.


title: Inferring Mycobacterium bovis transmission between cattle and badgers using isolates from the Randomised Badger Culling Trial

abstract: Mycobacterium bovis (M. bovis) is a causative agent of bovine tuberculosis, a significant source of morbidity and mortality in the global cattle industry. The Randomised Badger Culling Trial was a field experiment carried out between 1998 and 2005 in the South West of England. As part of this trial, M. bovis isolates were collected from contemporaneous and overlapping populations of badgers and cattle within ten defined trial areas. We combined whole genome sequences from 1,442 isolates with location and cattle movement data, identifying transmission clusters and inferred rates and routes of transmission of M. bovis. Most trial areas contained a single transmission cluster that had been established shortly before sampling, often contemporaneous with the expansion of bovine tuberculosis in the 1980s. The estimated rate of transmission from badger to cattle was approximately two times higher than from cattle to badger, and the rate of within-species transmission considerably exceeded these for both species. We identified long distance transmission events linked to cattle movement, recurrence of herd breakdown by infection within the same transmission clusters and superspreader events driven by cattle but not badgers. Overall, our data suggests that the transmission clusters in different parts of South West England that are still evident today were established by long-distance seeding events involving cattle movement, not by recrudescence from a long-established wildlife reservoir. Clusters are maintained primarily by within-species transmission, with less frequent spill-over both from badger to cattle and cattle to badger.

category: genomics


title: de novo variant calling identifies cancer mutation profiles in the 1000 Genomes Project

abstract: Detection of de novo variants (DNVs) is critical for studies of disease-related variation and mutation rates. We developed a GPU-based workflow to rapidly call DNVs (HAT) and demonstrated its effectiveness by applying it to 4,216 Simons Simplex Collection (SSC) whole-genome sequenced parent-child trios from DNA derived from blood. In our SSC DNV data, we identified 78 {+/-} 15 DNVs per individual, 18% {+/-} 5% at CpG sites, 75% {+/-} 9% phased to the paternal chromosome of origin, and an average allele balance of 0.49. These calculations are all in line with DNV expectations. We sought to build a control DNV dataset by running HAT on 602 whole-genome sequenced parent-child trios from DNA derived from lymphoblastoid cell lines (LCLs) from the publicly available 1000 Genomes Project (1000G). In our 1000G DNV data, we identified 740 {+/-} 967 DNVs per individual, 14% {+/-} 4% at CpG sites, 61% {+/-} 11% phased to the paternal chromosome of origin, and an average allele balance of 0.41. Of the 602 trios, 80% had > 100 DNVs and we hypothesized the excess DNVs were cell line artifacts. Several lines of evidence in our data suggest that this is true and that 1000G does not appear to be a static reference. By mutation profile analysis, we tested whether these cell line artifacts were random and found that 40% of individuals in 1000G did not have random DNV profiles; rather they had DNV profiles matching B-cell lymphoma. Furthermore, we saw significant excess of protein-coding DNVs in 1000G in the gene IGLL5 that has already been implicated in this cancer. As a result of cell line artifacts, 1000G has variants present in DNA repair genes and at Clinvar pathogenic or likely-pathogenic sites. Our study elucidates important implications of the use of sequencing data from LCLs for both reference building projects as well as disease-related projects whereby these data are used in variant filtering steps.