title: Metacommunity dynamics and the spurious detection of species associations in co-occurrence analyses: patch disturbance matters

abstract:
O_LI1: Many statistical methods attempt to detect species associations - and so infer inter-specific interactions - from species co-occurrence patterns. Habitat heterogeneity and out-of-equilibrium colonization histories are well recognized as potentially causing species associations, even when interactions are absent. The potential for patch disturbance, a classical component of metacommunity dynamics, to also drive spurious species associations has however been over-looked.
C_LIO_LI2: Using a new general metacommunity model, we derive mathematical predictions regarding how patch disturbance would affect the patterns of species associations detected in "null" co-occurrence matrices. We also conduct numerical simulations to test our predictions and to compare the performance of several widespread statistical methods, including direct tests of pairwise independence, matrix permutation approaches and joint species distribution modelling.
C_LIO_LI3: We show how classical metacommunity dynamics can produce statistical associations, both positive and negative, even when species do not interact, when there is no habitat heterogeneity, and at equilibrium. This occurs as soon as there is some rate of patch disturbance (i.e. simultaneous extinction of several species in a patch) and/or a finite life-span of patches, a common feature of a broad range of plant, animal or microbial systems.
C_LIO_LI4: Patch disturbance can compromise species co-occurrence analyses and cause the artefactual detection of species associations if not taken into account. Including patch age (i.e. the time since the last patch disturbance event) as a covariate in a joint species distribution model can resolve the artefact. However, this requires additional data that often are not available in practice. We argue that the consequences of patch disturbance should not be underestimated when analysing species distribution patterns in metacommunity-like systems.
C_LI

category: ecology


title: Metacommunity dynamics and the detection of species associations in co-occurrence analyses: why patch disturbance matters


title: Drivers of genetic differentiation and recent evolutionary history of an Eurasian wild pea

abstract: Genetic diversity a major determinant for the capacity of species to persist and adapt to their environments. Unraveling the factors affecting genetic differentiation is crucial to understand how genetic diversity is shaped and species may react to changing environments. We employed genotyping by sequencing to test the influence of climate, space, latitude, altitude and land cover on genetic differentiation in a collection of 81 wild pea samples (Pisum sativum ssp. elatius) from across its distribution range from western Europe to central Asia. We also attempted to elucidate the species recent evolutionary history and its effect on the current distribution of genetic diversity. Association of single SNPs with climate variables were analyses to test for signatures of local adaptation. Genetic variation was geographically structured into six distinct genetic cluster. Two of which were associated with a taxonomic group (Pisum sativum ssp. humile) that according to some researchers does not qualify for a sub-species rank due to its alleged lack of genetic distinctness from other conspecific groups. The effect of the tested factors influencing genetic differentiation were rather variable among genetic clusters. The climate predictors were most important in all clusters. Land use was more important in clusters from areas strongly influenced by human land use, especially by agriculture. We found a statistically significant association of 3,623 SNPs (2.4 % of all SNPs) with one of the environmental predictors. Most of them were correlated with latitude followed by temperature, precipitation and altitude. Estimation of SNP effects of the candidates resulted in a missense to silent ratio of 0.45, suggesting many of the observed candidates SNPs may alter the encoded amino acid sequence. Wild peas went through a genetic bottleneck during the last glacial period followed by population recovery. Probably associated with this population recovery, we detected a range expansion, which may have led to an eastward range expansion of the European cluster to Turkey and thereof southwards and eastwards. Overall, the interplay of several environmental factors and the recent evolutionary history affected the distribution of genetic diversity in wild peas where each subpopulations were differently affected by those factors and processes.

category: evolutionary biology


title: Altered DNA methylation in ion transport and immune signalling genes is associated with severity in Pancreatic Ductal Adenocarcinoma

abstract:
BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the leading cancers worldwide and has a poor survival, with a relative five-year survival rate of only 8.5%. In this study we investigated epigenetic marksassociated with PDAC severity and prognosis, through studying alterations in DNA methylation patterns.

MethodsDNA methylome for tumor and adjacent normal tissue samples from PDAC patients (n=7) were generated using Illumina 450K bead chips. Differentially methylated positions (DMPs) were identified with |delta beta| > 0.2 and p-value<0.01 by comparing tumors with the adjacent normal tissues. Validation of differential methylation and associated gene expression at selected genes was carried out in an independent cohort PDAC patient.

ResultsWe identified 76 DMPs in PDAC patients that mapped to 43 genes. Among them, 44.7% (n=34) were hypo-methylated and 55.3% (n=42) were hyper-methylated DMPs in cancer samples. The trends of change in methylation at these 76 DMPs from well to moderate were like that from moderate to poorly differentiated cancer samples. The gradual trend in differential methylation was observed both in our cohort and the TCGA-PAAD cohort, suggesting methylation marks can serve as early indicators of disease pathology. Altered promoter methylation, which may affect gene expression, was observed for transcription regulators (BHLHE23, GSC2, FOXE1 and TWIST1), gated ion channels (KCNA6, and CACNB2), tumor suppressors (RASSF1, SPRED2, and NPY) and genes functioning in interferon signalling (SIGIRR, MX2, and OAS2). We also have compared the TCGA-PAAD dataset with normal pancreatic tissue data from GTEx V8 dataset leading to a confluent observation.

ConclusionsWe reported the first study on methylome in PDAC tumors from patients in India. We identified altered DNA methylation associated with increasing severity in PDAC among some genes like SIGIRR, MX2 along with other previously reported loci. We also concluded a confluence in our observation when comparing the TCGA-PAAD dataset with GTEx V8 dataset.

category: cancer biology


title: Preterm birth is associated with xenobiotics and predicted by the vaginal metabolome

abstract: Spontaneous preterm birth (sPTB) is a leading cause of maternal and neonatal morbidity and mortality, yet both its prevention and early risk stratification are limited. The vaginal microbiome has been associated with PTB risk, possibly via metabolic or other interactions with its host. Here, we performed untargeted metabolomics on 232 vaginal samples, in which we have previously profiled the microbiota using 16S rRNA gene sequencing. Samples were collected at 20-24 weeks of gestation from women with singleton pregnancies, of which 80 delivered spontaneously before 37 weeks of gestation. We find that the vaginal metabolome correlates with the microbiome and separates into six clusters, three of which are associated with spontaneous preterm birth (sPTB) in Black women. Furthermore, while we identify five metabolites that associate with sPTB, another five associate with sPTB only when stratifying by race. We identify multiple microbial correlations with metabolites associated with sPTB, including intriguing correlations between vaginal bacteria that are considered sub-optimal and metabolites that were enriched in women who delivered at term. We propose that several sPTB-associated metabolites may be exogenous, and investigate another using metabolic models. Notably, we use machine learning models to predict sPTB risk using metabolite levels, weeks to months in advance, with high accuracy. We show that these predictions are more accurate than microbiome-based and maternal covariates-based models. Altogether, our results demonstrate the potential of vaginal metabolites as early biomarkers of sPTB and highlight exogenous exposures as potential risk factors for prematurity.

category: systems biology


title: COVID-eVax, an electroporated plasmid DNA vaccine candidate encoding the SARS-CoV-2 Receptor Binding Domain, elicits protective immune responses in animal models of COVID-19

abstract: The COVID-19 pandemic caused by the {beta}-coronavirus SARS-CoV-2 has made the development of safe and effective vaccines a critical global priority. To date, four vaccines have already been approved by European and American authorities for preventing COVID-19 but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle, a technology previously utilized for cancer vaccines. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 Spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax - a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein RBD - induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function and significantly lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started in Italy.

category: immunology


title: Regulatory dissection of the severe COVID-19 risk locus introgressed by Neanderthals

abstract: Individuals infected with the SARS-CoV-2 virus present with a wide variety of phenotypes ranging from asymptomatic to severe and even lethal outcomes. Past research has revealed a genetic haplotype on chromosome 3 that entered the human population via introgression from Neanderthals as the strongest genetic risk factor for the severe COVID-19 phenotype. However, the specific variants along this introgressed haplotype that contribute to this risk and the biological mechanisms that are involved remain unclear. Here, we assess the variants present on the risk haplotype for their likelihood of driving the severe COVID-19 phenotype. We do this by first exploring their impact on the regulation of genes involved in COVID-19 infection using a variety of population genetics and functional genomics tools. We then perform an locus-specific massively parallel reporter assay to individually assess the regulatory potential of each allele on the haplotype in a multipotent immune-related cell line. We ultimately reduce the set of over 600 linked genetic variants to identify 4 introgressed alleles that are strong functional candidates for driving the association between this locus and severe COVID-19. These variants likely drive the locus impact on severity by putatively modulating the regulation of two critical chemokine receptor genes: CCR1 and CCR5. These alleles are ideal targets for future functional investigations into the interaction between host genomics and COVID-19 outcomes.

category: genetics


title: NMPylation and de-NMPylation of SARS-CoV-2 Nsp9 by the NiRAN domain

abstract: Nsp12, the catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), contains two active sites that catalyze nucleotidyl-monophosphate (NMP) transfer (NMPylation). RNA synthesis is mediated by the RdRp active site that is conserved among all RNA viruses and has been a focus of mechanistic studies and drug discovery. The second active site resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. Both catalytic reactions are essential for viral replication, but the mechanism and targets of NiRAN are poorly characterized. One recent study showed that NiRAN transfers NMP to the first residue of RNA-binding protein Nsp9. Another study reported a structure of SARS-CoV-2 replicase with an extended Nsp9 in the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 Nsp12 efficiently and reversibly NMPylates the native but not the extended Nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas a substitution of a catalytic RdRp Asp residue does not. NMPylation is inhibited by nucleotide analogs, pyrophosphate, and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We hypothesize that Nsp9 remodels both active sites of Nsp12 to support initiation of RNA synthesis by RdRp and subsequent capping of the product RNA by the NiRAN domain.

category: biochemistry


title: A large disordered region confers a wide spanning volume to vertebrate Suppressor of Fused as shown in a trans-species solution study.

abstract:
Hedgehog (Hh) pathway inhibition by the conserved protein Suppressor of Fused (SuFu) is crucial to vertebrate development. By constrast, SuFu removal has little effect in drosophila.

Previous publications showed that the crystal structures of human and drosophila SuFu consist of two ordered domains that are capable of breathing motions upon ligand binding. However, the crystal structure of human SuFu does not give information about 20 N-terminal residues (IDR1) and an eighty-residue-long disordered region (IDR2) in the C-terminus, whose function is important for the pathway repression. These two IDRs are species-dependent.

We studied SuFus structure in solution, both with circular dichroism and small angle X-ray scattering, comparing drosophila, zebrafish and human species, to better understand this considerable difference. Our studies show that, in spite of similar crystal structures restricted to ordered domains, drosophila and vertebrate SuFu have very different structures in solution. The IDR2 of vertebrates spans a large area, thus enabling it to reach for partners and be accessible for post-translational modifications. Furthermore, we show that the IDR2 region is highly conserved within phyla but varies in length and sequence, with insects having a shorter disordered region while that of vertebrates is broad and mobile. This major variation may explain the different phenotypes observed upon SuFu removal.

category: biophysics


title: Comparing the Accuracy and Precision of Multi-echo Combination Methods for Quantitative Susceptibility Mapping Using Laplacian-Based Methods at 3 T

abstract:
PurposeTo compare different multi-echo combination methods for MRI quantitative susceptibility mapping (QSM). Given the current lack of consensus, we aimed to elucidate how to optimally combine multi-echo gradient-recalled echo (GRE) signal phase information, either before or after applying Laplacian-base methods (LBMs) for phase unwrapping or background field removal.

MethodsMulti-echo GRE data were simulated in a numerical head phantom, and multiecho GRE images were acquired at 3 T in ten healthy volunteers. To enable image-based estimation of GRE signal noise, five volunteers were scanned twice in the same session without repositioning. Five QSM processing pipelines were designed: one applied nonlinear phase fitting over echo times (TEs) before LBMs; two applied LBMs to the TE-dependent phase and then combined multiple TEs via either TE-weighted or signal-to-noise ratio (SNR)-weighted averaging; two calculated TE-dependent susceptibility maps via either multi-step or single-step QSM and then combined multiple TEs via magnitude-weighted averaging. Results from different pipelines were compared using visual inspection; summary statistics of susceptibility in deep gray matter, white matter, and venous regions; phase noise maps (error propagation theory); and, in the healthy volunteers, regional fixed bias analysis (Bland-Altman) and regional differences between the means (nonparametric tests).

ResultsNonlinearly fitting the multi-echo phase over TEs before applying LBMs provided the highest regional accuracy of{chi} and the lowest phase noise propagation compared to averaging the LBM-processed TE-dependent phase. This result was especially pertinent in high-susceptibility venous regions.

ConclusionFor multi-echo QSM, we recommend combining the signal phase by nonlinear fitting before applying LBMs.

category: neuroscience


title: Multi-echo Quantitative Susceptibility Mapping: How to Combine Echoes for Accuracy and Precision


title: Multi-echo Quantitative Susceptibility Mapping: How to Combine Echoes for Accuracy and Precision at 3 T


title: Normative modeling of neuroimaging data using generalized additive models of location scale and shape

abstract: Normative modeling aims to quantify the degree to which an individuals brain deviates from a reference sample with respect to one or more variables, which can be used as a potential biomarker of a healthy brain and as a tool to study heterogeneity of psychiatric disorders. The application of normative models is hindered by methodological challenges and lacks standards for the usage and evaluation of normative models. In this paper, we present generalized additive models for location scale and shape (GAMLSS) for normative modeling of neuroimaging data, a flexible modeling framework that can model heteroskedasticity, non-linear effects of variables, and hierarchical structure of the data. It can model non-Gaussian distributions, and it allows for an automatic model order selection, thus improving the accuracy of normative models while mitigating problems of overfitting. Furthermore, we describe measures and diagnostic tools suitable for evaluating normative models and step-by-step examples of normative modeling, including fitting several candidate models, selecting the best models, and transferring them to new scan sites.


title: Willingness to Pay in the Human Brain: A fMRI Activation Likelihood Estimation Meta-analysis

abstract: The neural substrate of willingness to pay (WTP) ultimately supports human economic exchange activities and plays a crucial role in daily life. This paper aimed to identify the neural basis of WTP for food and nonfood, as well as the brain regions related to real and hypothetical WTP choices. We found the human brain centers of WTP by performing an activation likelihood estimation (ALE) meta-analysis (27 experiments, 796 subjects) on the existing neuroimaging studies. The conjunction analysis revealed that WTP for food and nonfood engaged a common cluster in the paracingulate and cingulate gyrus, revealing a common reward circuit in the brain. The frontal medial cortex and paracingulate gyrus were particularly activated by WTP for nonfood. Furthermore, the left caudate, left thalamus, angular gyrus and supramarginal gyrus (subregions of inferior parietal lobule) were more convergently activated by hypothetical WTP choice. Our findings support the idea that a common currency representation in the brain and reward-specific neural basis. Results also provide evidence of neural representations of the hypothetical bias.


title: A palmitoylation code controls PI4KIIIα complex formation and PI(4,5)P2 homeostasis at the plasma membrane

abstract:
PI4KIII is the major enzyme responsible for generating the phosphoinositide PI(4)P at the plasma membrane. This lipid kinase forms two multicomponent complexes, both including a palmitoylated anchor, EFR3. Whereas both PI4KIII complexes support production of PI(4)P, the distinct functions of each complex and mechanisms underlying the interplay between them remain unknown. Here, we present roles for differential palmitoylation patterns within a tri-Cys motif in EFR3B (Cys5/Cys7/Cys8) in controlling the distribution of PI4KIII between these two complexes at the plasma membrane and corresponding functions in phosphoinositide homeostasis. Spacing of palmitoyl groups within three doubly palmitoylated EFR3B "lipoforms" affects both its interactions with TMEM150A, a transmembrane protein governing formation of a PI4KIII complex functioning in rapid PI(4,5)P2 resynthesis following PLC signaling, and its partitioning within liquid-ordered and -disordered regions of the plasma membrane. This work identifies a palmitoylation code in controlling protein-protein and protein-lipid interactions affecting a plasma membrane-resident lipid biosynthetic pathway.

SUMMARY STATEMENTDifferent palmitoylation patterns on a lipid kinase adaptor protein control partitioning of the kinase between two spatiotemporally and functionally distinct complexes within the plasma membrane.

category: cell biology


title: Negative modulation of macroautophagy by HERPUD1 is counteracted by an increased ER-lysosomal network with impact in drug-induced stress cell survival

abstract:
Macroautophagy and the ubiquitin proteasome system work as an interconnected network in the maintenance of cellular homeostasis. Indeed, efficient activation of macroautophagy upon nutritional deprivation is sustained by degradation of preexisting proteins by the proteasome. However, the specific substrates that are degraded by the proteasome in order to activate macroautophagy are currently unknown. By quantitative proteomic analysis we identified several proteins downregulated in response to starvation but independently of ATG5 expression. Among them, the most significant was HERPUD1, an ER protein of short-half life and a well-known substrate of the proteasome. We found that increased HERPUD1 stability by deletion of its ubiquitin-like domain (UBL) plays a negative role on basal and induced macroautophagy. Moreover, we found it triggers ER expansion by reordering the ER in crystalloid structures, but in the absence of unfolded protein response activation. Surprisingly, we found ER expansion led to an increase in the number and function of lysosomes establishing a tight network with the presence of membrane-contact sites. Importantly, a phosphomimetic S59D mutation within the UBL mimics UBL deletion on its stability and the ER-lysosomal network expansion revealing an increase of cell survival under stress conditions. Altogether, we propose stabilized HERPUD1 downregulates macroautophagy favoring instead a closed interplay between the ER and lysosomes with consequences in drug-cell stress survival.

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title: Accurate viral genome reconstruction and host assignment with proximity-ligation sequencing

abstract: Viruses play crucial roles in the ecology of microbial communities, yet they remain relatively understudied in their native environments. Despite many advancements in high-throughput whole-genome sequencing (WGS), sequence assembly, and annotation of viruses, the reconstruction of full-length viral genomes directly from metagenomic sequencing is possible only for the most abundant phages and requires long-read sequencing technologies. Additionally, the prediction of their cellular hosts remains difficult from conventional metagenomic sequencing alone. To address these gaps in the field and to accelerate the study of viruses directly in their native microbiomes, we developed an end-to-end bioinformatics platform for viral genome reconstruction and host attribution from metagenomic data using proximity-ligation sequencing (i.e., Hi-C). We demonstrate the capabilities of the platform by recovering and characterizing the metavirome of a variety of metagenomes, including a fecal microbiome that has also been sequenced with accurate long reads, allowing for the assessment and benchmarking of the new methods. The platform can accurately extract numerous near-complete viral genomes even from highly fragmented short-read assemblies and can reliably predict their cellular hosts with minimal false positives. To our knowledge, this is the first software for performing these tasks. Being significantly cheaper than long-read sequencing of comparable depth, the incorporation of proximity-ligation sequencing in microbiome research shows promise to greatly accelerate future advancements in the field.

category: genomics


title: Differential repression of Otx2 underlies the capacity of NANOG and ESRRB to induce germline entry

abstract: Primordial germ cells (PGCs) are induced in the embryo by signals, including BMP emanating from extra-embryonic ectoderm, that act on cells in the post-implantation epiblast. PGC development can be recapitulated in vitro through the exposure of epiblast-like cells (EpiLCs) to appropriate cytokines, resulting in differentiation into PGC-like cells (PGCLCs). Interestingly, the requirement for cytokines to induce PGCLCs can be bypassed by enforced expression of the transcription factor (TF) NANOG. However, the underlying mechanisms are not fully elucidated. Here, we show that Otx2 downregulation is essential to enable NANOG to induce PGCLC formation. Moreover, while previous work has shown that the direct NANOG target gene Esrrb can substitute for several functions of NANOG, enforced expression of ESRRB cannot promote PGCLC specification from EpiLCs. This appears to be due to differential downregulation of Otx2 by NANOG and ESRRB, since induction of ESRRB in Otx2+/- EpiLCs activates expression of the core PGC TFs, Blimp1, Prdm14 and Ap2{gamma} and emergence of PGCLCs. This study illuminates the interplay of TFs occurring at the earliest stages of PGC specification from a state of competence.

category: developmental biology


title: Facultative release from developmental constraints through polyphenism promotes adaptively flexible maturation

abstract: The timing of maturation, a critical fitness determinant, is influenced by developmental and energetic constraints, particularly when growth is poor in adverse conditions. Such constraints can be altered through developmental plasticity. Thus, in theory, plasticity in energetically costly sexually selected morphologies can promote life history flexibility in variable environments. We experimentally tested this hypothesis in bulb mites (Rhizoglyphus robini) that polyphenically develop as armed fighters with enlarged legs or as scramblers without modified legs. We found that (i) mites enter metamorphosis earlier if they develop as scramblers, (ii) mites accelerate the onset of metamorphosis when they sense resource limitation, and (iii) scrambler expression increases under increased competition for food, enabling males to mature early and escape juvenile mortality. We propose that life history plasticity can evolve through polyphenic release from sexually selected constraints, making the evolutionary dynamics of secondary sexual traits and life history traits, typically studied separately, interdependent.


title: A Versatile Linker for Probes Targeting Hydrolases via In Situ labeling

abstract: Hydrolases are important molecules that are involved in a wide range of biological functions and their activities are tightly regulated in healthy or diseased states. Detecting or imaging the activities of hydrolases, therefore, can reveal underlying molecular mechanisms in the context of cells to organisms, and their correlation with different physiological conditions can therefore be used in diagnosis. Due to the nature of hydrolases, substrate-based probes can be activated in their catalytic cycles, and cleavage of covalent bonds frees reporter moieties. For test-tube type bulk detection, spatial resolution is not a measure of importance, but for cell- or organism-based detection or imaging, spatial resolution is a key factor for probe sensitivity that influences signal-to-background ratio. One strategy to improve spatial resolution of the probes is to form a covalent linkage between the reporter moiety and intracellular proteins upon probe activation by the enzyme. In this work, we developed a generalizable linker chemistry that would allow in situ labeling of various imaging moieties via quinone methide species. To do so, we synthesized probes containing a monofluoromethyl or a difluoromethyl groups for {beta}-galactosidase activation, while using fluorescein as a fluorescent reporter. The labeling efficacy of these two probes was evaluated in vitro. The probe bearing a monofluormethyl group exhibited superior labeling efficiency in imaging {beta}-galactosidase activity in living cells. This study provides a versatile linker for applying quinone methide chemistry in the development of hydrolase-targeting probes involving in situ labeling.


title: Expanding Clinical Phage Microbiology:Simulating Phage Inhalation for Respiratory Tract Infections

abstract:
Phage therapy is a promising antibacterial strategy for resistant respiratory tract infections. Phage inhalation may serve this goal; however, it requires a careful assessment of their delivery by this approach. Here we present an in-vitro model to evaluate phage inhalation.

Eight phages, most of which target CF-common pathogens, were aerosolized and administered to a real-scale CT{square}derived 3D airways model with a breathing simulator. Viable phage loads reaching the output of the nebulizer and the tracheal level of the model were determined and compared to the loaded amount.

Phage inhalation resulted in a diverse range of titer reduction, primarily associated with the nebulization process. No correlation was found between phage delivery to the phage physical or genomic dimensions. These findings highlight the need for tailored simulations of phage delivery, ideally by a patient-specific model in addition to proper phage matching, to increase the potential of phage therapy success.

Take-Home MessagePhage therapy can be used against infectious diseases if personally tailored. Using a 3D airways model, we show that phage delivery by inhalation to the respiratory tract is unpredictable and also requires a precise evaluation.

category: microbiology


title: Practical observations on the use of fluorescent reporter systems in C. difficile

abstract:
ObjectivesFluorescence microscopy is a valuable tool to study a broad variety of bacterial cell components and dynamics thereof. For Clostridioides difficile, the fluorescent proteins CFPopt, mCherryOpt and phiLOV2.1, and the self-labelling tags SNAPCd and HaloTag, hereafter collectively referred as fluorescent systems, have been described to explore different cellular pathways. In this study, we sought to characterize previously used fluorescent systems in C. difficile cells.

MethodsWe performed single cell analyses using fluorescence microscopy of exponentially growing C. difficile cells harbouring different fluorescent systems, either expressing these separately in the cytosol or fused to the C-terminus of HupA, under defined conditions.

ResultsWe show that the intrinsic fluorescence of C. difficile cells increases during growth, independent from sigB or spo0A. However, when C. difficile cells are exposed to environmental oxygen autofluorescence is enhanced.

Cytosolic overexpression of the different fluorescent systems alone, using the same expression signals, showed heterogeneous expression of the fluorescent systems. High levels of mCherryOpt were toxic for C. difficile cells limiting the applicability of this fluorophore as a transcriptional reporter. When fused to HupA, C. difficile histone-like protein, the fluorescent systems behaved similarly and did not affect the HupA overproduction phenotype.

ConclusionsThe present study compares several commonly used fluorescent systems for application as transcriptional or translational reporters in microscopy and summarizes the limitations and key challenges for live-cell imaging of C. difficile. Due to independence of molecular oxygen and fluorescent signal, SNAPCd appears the most suitable candidate for live-cell imaging in C. difficile to date.

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title: Selective nuclear export of mRNAs is promoted by DRBD18 in Trypanosoma brucei

abstract: Kinetoplastids, including Trypanosoma brucei, control gene expression primarily at the posttranscriptional level. Nuclear mRNA export is an important, but understudied, step in this process. The general heterodimeric export factors, Mex67/Mtr2, function in the export of mRNAs and tRNAs in T. brucei, but RNA binding proteins (RBPs) that regulate export processes by controlling the dynamics of Mex67/Mtr2 ribonucleoprotein formation or transport have not been identified. Here, we report that DRBD18, an essential and abundant T. brucei RBP, associates with Mex67/Mtr2 in vivo, likely through its direct interaction with Mtr2. DRBD18 downregulation results in partial accumulation of poly(A)+ mRNA in the nucleus, but has no effect on localization of intron-containing or mature tRNAs. Comprehensive analysis of transcriptomes from whole cell and cytosol in DRBD18 knockdown parasites demonstrates that depletion of DRBD18 leads to impairment of nuclear export of a subset of mRNAs. CLIP experiments reveal association of DRBD18 with several of these mRNAs. Moreover, DRBD18 knockdown leads to a partial accumulation of the Mex67/Mtr2 export receptors in the nucleus. Taken together, the current study supports a model in which DRBD18 regulates the selective nuclear export of mRNAs by promoting the mobilization of export competent mRNPs to the cytosol through the nuclear pore complex.


title: A pathogenic role for histone H3 copper reductase activity in a yeast model of Friedreich's Ataxia

abstract:
Disruptions to iron-sulfur (Fe-S) clusters, essential cofactors for a broad range of proteins, cause widespread cellular defects resulting in human disease. An underappreciated source of damage to Fe-S clusters are cuprous (Cu1+) ions. Since histone H3 enzymatically produces Cu1+ to support copper-dependent functions, we asked whether this activity could become detrimental to Fe-S clusters. Here, we report that histone H3-mediated Cu1+ toxicity is a major determinant of cellular Fe-S cluster quotient. Inadequate Fe-S cluster supply, either due to diminished assembly as occurs in Friedreichs Ataxia or defective distribution, causes severe metabolic and growth defects in S. cerevisiae. Decreasing Cu1+ abundance, through attenuation of histone cupric reductase activity or depletion of total cellular copper, restored Fe-S cluster-dependent metabolism and growth. Our findings reveal a novel interplay between chromatin and mitochondria in Fe-S cluster homeostasis, and a potential pathogenic role for histone enzyme activity and Cu1+ in diseases with Fe-S cluster dysfunction.

TeaserReduction of Cu1+ production by histone H3 restores cellular deficiencies caused by insufficient supply of iron-sulfur clusters.

category: molecular biology


title: CENP-N promotes the compaction of centromeric chromatin

abstract:
The histone variant CENP-A is the epigenetic determinant for the centromere, where it is interspersed with canonical H3 to form a specialized chromatin structure that nucleates the kinetochore. The arrangement of nucleosomes at the centromere into higher order structure is unknown. Here we demonstrate that the CENP-A interacting protein CENP-N promotes the stacking of CENP-A containing mono-nucleosomes and nucleosomal arrays through a previously undefined interaction between the 6 helix of CENP-N with the DNA of a neighboring nucleosome. We describe the cryoEM structures and biophysical characterization of such CENP-N mediated nucleosome stacks and nucleosomal arrays and demonstrate that this interaction is responsible for the formation of densely packed chromatin at the centromere in the cell. Our results provide first evidence that CENP-A, together with CENP-N, promotes specific chromatin higher order structure at the centromere.

One-Sentence SummaryThe centromere-associated protein CENP-N promotes centromere-specific nucleosome stacking and higher order structures in vitro and in the cell.


title: Pharmacological inhibition of the VCP/proteasome axis rescues photoreceptor degeneration in RHOP23H rat retinal explants

abstract: 1.Rhodopsin (RHO) misfolding mutations are a common cause of the blinding disease autosomal dominant retinitis pigmentosa (adRP). The most prevalent mutation, RHOP23H, results in its misfolding and retention in the Endoplasmic Reticulum (ER). Under homeostatic conditions, misfolded proteins are selectively identified, retained at the ER, and cleared via ER-associated degradation (ERAD) and/or autophagy. Overload of these degradation processes for a prolonged period leads to imbalanced proteostasis and may eventually result in cell death. ERAD of misfolded proteins like RHOP23H includes the subsequent steps of protein recognition, targeting for ERAD, retrotranslocation, and proteasomal degradation. In the present study, we investigated and compared pharmacological modulation of ERAD at these four different major steps. We show that inhibition of the VCP/proteasome activity favors cell survival and suppresses P23H-mediated retinal degeneration in RHOP23H rat retinal explants. We suggest targeting this activity as a therapeutic approach for patients with currently untreatable adRP.


title: Localization and phosphorylation in the Snf1 network is controlled by two independent pathways

abstract: AMPK/SNF1 is the master regulator of energy homeostasis in eukaryotic cells and has a key role in glucose de-repression. If glucose becomes depleted, Snf1 is phosphorylated and activated. Activation of Snf1 is required but is not sufficient for mediating glucose de-repression indicating a second glucose-regulated step that adjusts the Snf1 pathway. To elucidate this regulation, we further explore the spatial dynamics of Snf1 and Mig1 and how they are controlled by concentrations of hexose sugars. We utilize fluorescence recovery after photobleaching (FRAP) to study the movement of Snf1 and how it responds to external glucose concentrations. We show that the Snf1 pathway reacts to the presence of glucose. Furthermore, we identify a negative feedback loop regulating Snf1 activity. Our data offer insight into the true complexity of regulation of this central signaling pathway by one signal (glucose depletion) interpreted by the cell in different ways.


title: The atypical RNA-binding protein TAF15 regulates dorsoanterior neural development through diverse mechanisms in Xenopus tropicalis.

abstract: The FET family of atypical RNA-binding proteins includes Fused in sarcoma (Fus), Ewings sarcoma (EWS), and the TATA-binding protein-associate factor 15 (TAF15). FET proteins are highly conserved, suggesting specialized requirements for each protein. Fus regulates splicing of transcripts required for mesoderm differentiation and cell adhesion in Xenopus, but roles that EWS and TAF15 play remain unknown. Here we analyze the roles of maternally deposited and zygotically transcribed TAF15, which is essential for the proper development of dorsoanterior neural tissues. By measuring changes in exon usage and transcript abundance from TAF15-depleted embryos we found TAF15 may regulate dorsoanterior neural development through fgfr4 and ventx2.1. TAF15 uses distinct mechanisms to downregulate FGFR4 expression: 1) retention of a single intron within fgfr4 when maternal and zygotic TAF15 is depleted, and 2) reduction of total fgfr4 transcript when zygotic TAF15 alone is depleted. The two mechanisms of gene regulation (post-transcriptional vs transcriptional) suggest TAF15-mediated gene regulation is target and cofactor-dependent, depending on the milieu of factors that are present at different times of development.


title: High resolution dynamic mapping of the C. elegans intestinal brush border

abstract: The intestinal brush border is made of an array of microvilli that increases the membrane surface area for nutrient processing, absorption, and host defence. Studies on mammalian cultured epithelial cells uncovered some of the molecular players and physical constrains required to establish this apical specialized membrane. However, the building and maintenance of a brush border in vivo has not been investigated in detail yet. Here, we combined super-resolution imaging, transmission electron microscopy and genome editing in the developing nematode C. elegans to build a high-resolution and dynamic localization map of known and new markers of the brush border. Notably, we show that microvilli components are dynamically enriched at the apical membrane during microvilli outgrowth and maturation but become highly stable once microvilli are built. This new mapping tool will be instrumental to understand the molecular processes of microvilli growth and maintenance in vivo as well as the effect of genetic perturbations, notably in the context of disorders affecting brush border integrity.


title: Nutrient-dependent allometric plasticity in a male-diphenic mite

abstract: Male secondary sexual traits often scale allometrically with body size. These allometries can be variable within species, and may shift depending on environmental conditions such as food quality. Such allometric plasticity has been hypothesized to initiate local adaptation and evolutionary diversification of scaling relationships, but is under-recorded, and its eco- evolutionary effects are not well understood. Here, we test for allometric plasticity in the bulb mite (Rhizoglyphus robini) in which large males tend to develop as armed adult fighters with thickened third legs, while small males become adult scramblers without thickened legs. We first examined the ontogenetic timing for size- and growth-dependent male morph determination, using experimentally amplified fluctuations in growth rate throughout juvenile development. Having established that somatic growth and body size determine male morph expression immediately before metamorphosis, we examined whether the relationship between adult male morph and size at metamorphosis shifts with food quality. We found that the threshold body size for male morph expression shifts towards lower values with deteriorating food quality, confirming food-dependent allometric plasticity. Such allometric plasticity may allow populations to track prevailing nutritional conditions, potentially facilitating rapid evolution of allometric scaling relationships.


title: Modularity and selection of nectar traits in the evolution of the selfing syndrome in Ipomoea lacunosa (Convolvulaceae)

abstract:
O_LIAlthough the evolution of the selfing syndrome often involves reductions in floral size, pollen, and nectar, few studies of selfing syndrome divergence have examined nectar. We investigate whether nectar traits have evolved independently of other floral size traits in the selfing syndrome, whether nectar traits diverged due to drift or selection, and the extent to which quantitative trait locus (QTL) analyses predict genetic correlations.
C_LIO_LIWe use F5 recombinant inbred lines (RILs) generated from a cross between Ipomoea cordatotriloba and I. lacunosa. We calculate genetic correlations to identify evolutionary modules, test whether traits have been under selection, identify QTLs, and perform correlation analyses to evaluate how well QTL properties reflect the genetic correlations.
C_LIO_LINectar and floral size traits form separate genetic clusters. Directional selection has acted to reduce nectar traits in the selfing I. lacunosa. Calculations from QTL properties are consistent with observed genetic correlations.
C_LIO_LIFloral trait divergence during mating system syndrome evolution reflects independent evolution of at least two evolutionary modules: nectar and floral size traits. This independence implies that adaptive change in these modules requires direct selection on both floral size and nectar traits. Our study also supports the expected mechanistic link between QTL properties and genetic correlations.
C_LI


title: Reconstitution of the DTX3L-PARP9 complex reveals determinants for high affinity heterodimer formation and enzymatic function

abstract: Ubiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways like DNA damage response and infection, making them attractive targets for therapeutic intervention. DTX3L, an E3 ubiquitin ligase, forms a heterodimer with PARP9. The complex has ubiquitin ligase activity and also ADP-ribosylates the C-terminus of ubiquitin on Gly76. NAD+-dependent ADP-ribosylation of ubiquitin by DTX3L-PARP9 prevents ubiquitin from conjugating to protein substrates. By using individually produced proteins, we have studied the interaction between DTX3L and PARP9. We identify that the D3 domain (230 - 510) of DTX3L mediates interaction with PARP9 with nanomolar affinity and an apparent 1:1 stoichiometry. Our results also suggest the formation of a higher molecular weight oligomer mediated by the N-terminus of DTX3L (1-200). Furthermore, we show that ADP-ribosylation of ubiquitin at Gly76 is a reversible modification that can be removed by several macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L-PARP9 mediates ADP-ribosylation and ubiquitination in an inter-regulatory manner.


title: Investigating bacterial ribosomal sequence variation in regards to future structural and antibiotic research.

abstract:
Ribosome-targeting antibiotics comprise over half of antibiotics used in medicine, but our fundamental knowledge of their binding sites is derived primarily from ribosome structures from non-pathogenic species. These include Thermus thermophilus, Deinococcus radiodurans and Haloarcula marismortui, as well as the commensal or pathogenic Escherichia coli. Advancements in electron cryomicroscopy have allowed for the determination of more ribosome structures from pathogenic bacteria, with each study highlighting species-specific differences that had not been observed in the non-pathogenic structures. These observed differences suggest that more novel ribosome structures, particularly from pathogens, are required to get a more accurate understanding of the level of diversity in the bacterial ribosome, leading to potential advancements in antibiotic research. In this study, covariance and hidden Markov models were used to annotate ribosomal RNA and protein sequences respectively from genomic sequence, allowing us to determine the underlying ribosomal sequence diversity using phylogenetic methods. This analysis provided evidence that the current non-pathogenic ribosome structures are not sufficient representatives of some pathogenic bacteria, such as Campylobacter pylori, or of whole phyla such as Bacteroidetes.

Significance StatementThe growing number of antibiotic resistance pathogenic bacteria are of critical concern to the health profession. Many of the current classes of antibiotics target the bacterial ribosome, the protein making factory for these species. However, much of our knowledge of the bacterial ribosome is based upon non-pathogenic bacteria that are highly divergent from the major pathogens of concern. We have analysed the genetic variation of the RNA and protein components of all available bacterial ribosomes. This has led us to identify the highest priority groups of bacteria that would be of the most benefit for further analysis of their ribosome structures from both a medical and evolutionary perspective.


title: Deep learning-based segmentation and quantification of podocyte foot process morphology

abstract: The kidneys constantly filter enormous amounts of fluid, with almost complete retention of albumin and other macromolecules in the plasma. Diseases of podocytes at the kidney filtration barrier reduce the intrinsic permeability of the capillary wall resulting in albuminuria. However, direct quantitative assessment of the underlying morphological changes has previously not been possible. Here we developed a deep learning-based approach for segmentation of foot processes in images acquired with optical microscopy. Our method - Automatic Morphological Analysis of Podocytes (AMAP) - accurately segments foot processes and robustly quantifies their morphology. It also robustly determined morphometric parameters, at a Pearson correlation of r > 0.71 with a previously published semi-automated approach, across a large set of mouse tissue samples. The artificial intelligence algorithm wasWe applied the analysis to a set of human kidney disease conditions allowing comprehensive quantification of various underlying morphometric parameters. These results confirmed that when podocytes are injured, they take on a more simplified architecture and the slit diaphragm length is significantly shortened, resulting in a reduction in the filtration slit area and a loss of the buttress force of podocytes which increases the permeability of the glomerular basement membrane to albumin.

category: pathology


title: Timing of meristem initiation and maintenance determines the morphology of fern gametophytes

abstract:
The alternation of generations in land plants occurs between the sporophyte phase and the gametophyte phase. The sporophytes of seed plants develop self-maintained, multicellular meristems, and these meristems determine plant architecture. The gametophytes of seed plants lack meristems and are heterotrophic. In contrast, the gametophytes of seed-free vascular plants, including ferns, are autotrophic and free-living, developing meristems to sustain their independent growth and proliferation. Compared to meristems in the sporophytes of seed plants, the cellular mechanisms underlying meristem development in fern gametophytes remain largely unknown. Here, using confocal time-lapse live imaging and computational segmentation and quantification, we determined different patterns of cell divisions associated with the initiation and proliferation of two distinct types of meristems in fern gametophytes. Our results reveal how the simple timing of a switch between two meristems has considerable consequences for the divergent gametophyte morphologies of two closely related ferns from Pteridaceae (Pteris and Ceratopteris). Our result provides evolutionary insight into the function and regulation of gametophyte meristems in seed-free vascular plants.

HighlightLive-imaging of cell growth and division in apical initials and lateral meristems reveals that the timing of a switch between the two meristem identities drives morphology variation in fern gametophytes.

category: plant biology


title: Subregions of DLPFC display graded yet distinct structural and functional connectivity

abstract: The human dorsolateral prefrontal cortex (DLPFC, approximately corresponding to Brodmann areas 9 and 46) has demonstrable roles in diverse executive functions such as working memory, cognitive flexibility, planning, inhibition, and abstract reasoning. However, it remains unclear whether this is the result of one functionally homogeneous region or whether there are functional subdivisions within the DLPFC. Here, we divided the DLPFC into seven areas along with rostral-caudal and dorsal-ventral axes anatomically and explored their respective patterns of structural and functional connectivity. In vivo probabilistic tractography and resting-state functional magnetic resonance imaging were employed to map out the patterns of connectivity from each DLPFC subregions. Structural connectivity demonstrated graded intra-regional connectivity within the DLPFC. The patterns of structural connectivity between the DLPFC subregions and other cortical areas revealed that he dorsal-rostral subregions was restricted to connect to other frontal and limbic areas, whereas the ventral-caudal region was widely connected to frontal, temporal, parietal, and limbic cortex. Functional connectivity analysis demonstrated that subregions of DLPFC were strongly interconnected to each other. The dorsal subregions were associated with the default mode network (DMN), while middle dorsal-rostral subregions were linked with the multiple demand network (MDN), respectively. Similar to the results of structural connectivity, the ventral-caudal subregion showed increased functional coupling with both DMN and MDN. Our results suggest that DLPFC may be subdivided by the diagonal axis of the dorsal-ventral axis and rostral-caudal axis, which support the patterns of connectivity the parts of the DLPFC reflects its integrative executive function.


title: Ultrafast Light Targeting for High-Throughput Precise Control of Neuronal Networks

abstract: Understanding how specific sets of neurons fire and wire together during cognitive-relevant activity is one of the most pressing questions in neuroscience. Two-photon, single-cell resolution optogenetics based on holographic light-targeting approaches enables accurate spatio-temporal control of individual or multiple neurons. Yet, currently, the ability to drive asynchronous activity in distinct cells is critically limited to a few milliseconds and the achievable number of targets to several dozens. In order to expand the capability of single-cell optogenetics, we introduce an approach capable of ultra-fast sequential light targeting (FLiT), based on switching temporally focused beams between holograms at kHz rates. We demonstrate serial-parallel photostimulation strategies capable of multi-cell sub-millisecond temporal control and many-fold expansion of the number of activated cells. This approach will be important for experiments that require rapid and precise cell stimulation with defined spatio-temporal activity patterns and optical control of large neuronal ensembles.


title: A novel and accurate full-length HTT mouse model for Huntington's disease

abstract: Here we report the generation and characterization of a novel Huntingtons disease (HD) mouse model BAC226Q by using a bacterial artificial chromosome (BAC) system, expressing full-length human HTT with [~]226 CAG-CAA repeats and containing endogenous human HTT promoter and regulatory elements. BAC226Q recapitulated a full-spectrum of age-dependent and progressive HD-like phenotypes without unwanted and erroneous phenotypes. BAC226Q mice developed normally, and gradually exhibited HD-like mood and cognitive phenotypes at 2 months. From 3-4 months, BAC226Q mice showed robust progressive motor deficits. At 11 months, BAC226Q mice showed significant reduced life span, gradual weight loss and exhibit neuropathology including significant brain atrophy specific to striatum and cortex, striatal neuronal death, widespread huntingtin inclusions and reactive pathology. Therefore, the novel BAC226Q mouse accurately recapitulating robust, age-dependent, progressive HD-like phenotypes will be a valuable tool for studying disease mechanisms, identifying biomarkers and testing gene-targeting therapeutic approaches for HD.


title: Genetic and chemical inhibition of autophagy in zebrafish induced myeloproliferation

abstract: Autophagy is an evolutionary conserved and dynamic lysosomal degradation process for cellular homeostasis and remodelling, which is essential for the development and maintenance of different hematopoietic fates. However, the roles of autophagy in definitive hematopoiesis remain elusive. Here, we exploited zebrafish (Danio rerio) to investigate the effect of knocking-out unc-51 like autophagy activating kinase 1b and 2 (ulk1b and ulk2), homologous of human ULK1 and ULK2, respectively, on definitive hematopoiesis. Upon ulk1b or ulk2 mutation, autophagosome formation was blocked in zebrafish embryos. More importantly, pan-leukocytes (lcp1), common myeloid progenitors (spi1b), neutrophils (mpx), and macrophages (mpeg1.1) significantly elevated, while the hematopoietic stem and progenitor cell (HSPC) (myb), erythroid progenitors (gata1), and embryonic hemoglobin (hbae1.1) significantly reduced in the caudal hematopoietic tissue (CHT) of ulk1b or ulk2 mutant zebrafish embryos. On the other hand, chemically modulated autophagy induction by calpeptin, a downstream autophagy inducer for ulk complex, was insufficient to ameliorate dysregulated hematopoiesis in both ulk1b and ulk2 mutants. Conversely, autophagy inhibitor 3-Methyladenine functioned parallelly with the ulk mutants to maintain defective hematopoiesis. These observations raised a link between autophagy and definitive hematopoiesis and potentiates the fact that autophagy deficiency incorporates with myeloproliferation and anemia, which warrants the significance of autophagy in regulating definitive hematopoiesis.


title: Peroxisomal ATP uptake is managed by the ABC transporters and two adenine nucleotide transporters

abstract: Peroxisomes are essential organelles involved in various metabolic processes, including fatty acid {beta}-oxidation. Their metabolic functions require a controlled exchange of metabolites and co-factors, including ATP across the peroxisomal membrane. We investigated which proteins are involved in the peroxisomal uptake of ATP in the yeast Saccharomyces cerevisiae. Using wild-type and targeted deletion strains, we measured ATP-dependent peroxisomal octanoate {beta}-oxidation, intra-peroxisomal ATP levels employing peroxisome-targeted ATP-sensing reporter proteins, and ATP uptake in proteoliposomes prepared from purified peroxisomes. We show that intra-peroxisomal ATP levels are maintained by different peroxisomal membrane proteins each with different modes of action: (1) the previously reported Ant1p protein, which catalyzes ATP/AMP exchange (2) the ABC transporter protein complex Pxa1p/Pxa2p, which mediates both acyl-CoA and ATP uptake; and; (3) the mitochondrial Aac2p protein, which catalyzes ATP/ADP exchange and was shown to have a dual localization in both mitochondria and peroxisomes. Our results provide compelling evidence for an ingenious complementary system for the uptake of ATP in peroxisomes.


title: Asynchronous rates of lineage, phenotype, and niche diversification in a continental-scale adaptive radiation

abstract: Rapidly diversifying clades are central to the study of diversification dynamics. This central importance is perhaps most apparent when rapid evolution occurs across several axes of diversification (e.g., lineage, phenotype, and niche); such clades facilitate investigations into the interplay between adaptive and non-adaptive diversification mechanisms. Yet, empirical evidence from rapidly evolving clades remains unclear about the relationships, if any, across diversification axes. This is especially apparent regarding the timing of diversification rate shifts. We address this knowledge gap through comparisons of the rate and timing of lineage, phenotypic, and niche diversification in Penstemon, a rapidly-evolving angiosperm genus. We find that diversification rate shifts in Penstemon are asynchronous; while we identify a burst and subsequent slowdown in lineage diversification rate [~]2.0-2.5 MYA, shifts in phenotypic and niche diversification rates either lagged behind temporally or did not occur at all. We posit that this asynchronicity in diversification rate shifts is the result of initial niche-neutral diversification followed by adaptive, density-dependent processes. Our findings contribute to a growing body of evidence that asynchronous shifts in diversification rates may be common and question the applicability of expectations for diversification dynamics across disparate empirical systems.


title: Mapping the Genetic Landscape of DNA Double-strand Break Repair

abstract: Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways that are critical for maintaining genomic integrity. Here we present Repair-seq, a high-throughput screening approach that measures the effects of thousands of genetic perturbations on the distribution of mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair outcomes induced by two programmable nucleases (Cas9 and Cas12a) after knockdown of 476 genes involved in DSB repair or associated processes in the presence or absence of oligonucleotides for homology-directed repair (HDR). The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. Systematic interrogation of these dependencies then uncovered unexpected relationships among DSB repair genes and isolated incompletely characterized repair mechanisms. This work provides a foundation for understanding the complex pathways of DSB repair and for optimizing genome editing across modalities.


title: I, We, and They: A Linguistic and Narrative Exploration of the Authorship Process

abstract:
IntroductionWhile authorship plays a powerful role in the academy, research indicates many authors engage in questionable practices like honorary authorship. This suggests that authorship may be a contested space where individuals must exercise agency--a dynamic and emergent process, embedded in context--to negotiate potentially conflicting norms among published criteria, disciplines, and informal practices. This study explores how authors narrate their own and others agency in making authorship decisions.

MethodWe conducted a mixed-methods analysis of 24 first authors accounts of authorship decisions on a recent multi-author paper. Authors included 14 females and 10 males in health professions education (HPE) from U.S. and Canadian institutions (10 assistant, 6 associate, and 8 full professors). Analysis took place in three phases: (1) linguistic analysis of grammatical structures shown to be associated with agency (coding for main clause subjects and verb types); (2) narrative analysis to create a "moral" and "title" for each account; and (3) integration of (1) and (2).

ResultsParticipants narrated other authors most frequently as main clause subjects (n = 191), then themselves (I; n = 151), inanimate nouns (it, the paper; n = 146), and author team (we; n = 105). Three broad types of agency were narrated: distributed (n = 15 participants), focusing on how resources and work were spread across team members; individual (n = 6), focusing on the first authors action; and collaborative (n = 3), focusing on group actions. These three types of agency contained four sub-types, e.g., supported, contested, task-based, negotiated.

DiscussionThis study highlights the complex and emergent nature of agency narrated by authors when making authorship decisions. Published criteria offer us starting point--the stated rules of the authorship game; this paper offers us a next step--the enacted and narrated approach to the game.

category: scientific communication and education


title: A thermal fuse in methionine biosynthesis arrests growth and protects Escherichia coli at elevated temperatures

abstract: Adaptive stress resistance in microbes is mostly attributed to the expression of stress response genes, such as heat shock proteins, which prevent deterioration of cellular material. Here, we report a novel response of E. coli to heat stress: induction of a growth-arrested state, caused by degradation of an enzyme in the methionine biosynthesis pathway (MetA). While MetA degradation is detrimental for proliferation, we show that the resulting growth arrest has a direct benefit for survival at high temperatures; it protects cells when temperatures rise beyond 50{degrees}C, increasing the survival chances by over 1000-fold. Using a combination of experiments and mathematical modelling, we show that degradation of MetA leads to the coexistence of growing and non-growing cells, allowing microbes to bet-hedge between continued growth if conditions remain bearable and survival if conditions worsen. We test our model experimentally and verify quantitatively how protein expression, degradation rates and environmental stresses affect the partitioning between growing and non-growing cells. Because growth arrest can be abolished with simple mutations, such as point mutations of MetA and knock-outs of proteases, we interpret the breakdown of methionine synthesis as a system that has evolved to disintegrate at high temperature and shut off growth, analogous to  thermal fuses used in engineering to shut off electricity when the device could be damaged by overheating.


title: MetA is a thermal fuse that arrests growth and protects Escherichia coli at elevated temperatures


title: Suv420 enrichment at the centromere limits Aurora B localization and function

abstract: Centromere structure and function are defined by the epigenetic modification of histones at centromeric and pericentromeric chromatin. The constitutive heterochromatin found at pericentromeric regions is highly enriched for H3K9me3 and H4K20me3. While mis-expression of the methyltransferase enzymes, Suv39 and Suv420, that regulate these marks are common in disease, the consequences of such changes are not well understood. Our data show that increased centromere localization of Suv39 and Suv420 suppress centromere transcription and compromise localization of the mitotic kinase Aurora B: decreasing microtubule dynamics and compromising chromosome alignment and segregation. We find that inhibition of Suv420 methyltransferase activity partially restores Aurora B localization to centromeres and that restoration of the Aurora B-containing CPC to the centromere is sufficient to suppress mitotic errors that result when Suv420/H4K20me3 is enriched at centromeres. Consistent with a role for Suv39 and Suv420 in negatively regulating Aurora B, high expression of these enzymes corresponds with increased sensitivity to Aurora kinase inhibition in cancer cells suggesting that increased H3K9 and H4K20 methylation may be an underappreciated source of chromosome missegregation in cancer.


title: Distinct gene expression dynamics in developing and regenerating limbs

abstract:
Regenerating animals have the ability to reproduce organs that were originally generated in the embryo and subsequently lost due to injury. Understanding whether the process of regeneration mirrors development is an open question in most regenerative species. Here we take a transcriptomics approach to examine to what extent leg regeneration shows the same temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis. We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, global patterns of gene expression during leg regeneration show a high degree of variation, related to the physiology of individual animals. A major driver of this variation is the molting cycle. After dissecting the transcriptional signals of individual physiology from regeneration, we obtain temporal signals that mark distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although both processes use largely the same genes, the temporal patterns in which these gene sets are deployed are different and cannot be systematically aligned.

HIGHLIGHTSO_LISingle-limb data on transcriptional dynamics of leg development and regeneration
C_LIO_LIDeveloping embryonic legs show stereotypic transcriptional profiles
C_LIO_LIRegenerating leg transcriptomes show a high degree on individual variation
C_LIO_LIRegenerating leg transcriptomes are influenced by adult physiology, especially molting
C_LIO_LIRegenerating leg transcriptomes reveal distinct phases of leg regeneration
C_LIO_LILeg development and regeneration use overlapping sets of genes in different temporal patterns
C_LI


title: Evolution of the nitric oxide synthase family in vertebrates and novel insights in gill development

abstract: Nitric oxide (NO) is an ancestral key signaling molecule essential for life and has enormous versatility in biological systems, including cardiovascular homeostasis, neurotransmission, and immunity. Although our knowledge of nitric oxide synthases (Nos), the enzymes that synthesize NO in vivo, is substantial, the origin of a large and diversified repertoire of nos gene orthologs in fish with respect to tetrapods remains a puzzle. The recent identification of nos3 in the ray-finned fish spotted gar, which was considered lost in the ray-finned fish lineage, changed this perspective. This prompted us to explore nos gene evolution and expression in depth, surveying vertebrate species representing key evolutionary nodes. This study provides noteworthy findings: first, nos2 experienced several lineage-specific gene duplications and losses. Second, nos3 was found to be lost independently in two different teleost lineages, Elopomorpha and Clupeocephala. Third, the expression of at least one nos paralog in the gills of developing shark, bichir, sturgeon, and gar but not in arctic lamprey, suggest that nos expression in this organ likely arose in the last common ancestor of gnathostomes. These results provide a framework for continuing research on nos genes roles, highlighting subfunctionalization and reciprocal loss of function that occurred in different lineages during vertebrate genome duplications.


title: Regulation Network of Colorectal Cancer Specific Enhancers in Progression of Colorectal Cancer

abstract: Enhancers regulate multiple genes through higher-order chromatin structure and further affect cancer progression. Epigenetic changes in cancer cells activate several cancer specific enhancers that are silenced in normal cells. These cancer specific enhancers are potential therapeutic targets of cancer. However, functions and regulation network of colorectal cancer specific enhancers are still unknown. Here in this study, we profile colorectal cancer specific enhancers and reveal the regulation network of these enhancers by analysis of HiChIP, Hi-C and RNA-seq data. We propose the regulation network of colorectal cancer specific enhancers plays important role in progression of colorectal cancer.


title: Temperature-Inducible Precision Guided Sterile Insect Technique

abstract: Releases of sterile males are the gold standard for many insect population control programs, and precise sex sorting to remove females prior to male releases is essential to the success of these operations. To advance traditional methods for scaling the generation of sterile males, we previously described a CRISPR-mediated precision-guided sterile insect technique (pgSIT), in which Cas9 and gRNA strains are genetically crossed to generate sterile males for release. While effective at generating F1 sterile males, pgSIT requires a genetic cross between the two parental strains which requires maintenance and sexing of two strains in a factory. Therefore, to further advance pgSIT by removing this crossing step, here we describe a next-generation Temperature-Inducible pgSIT (TI-pgSIT) technology and demonstrate its proof-of-concept in Drosophila melanogaster. Importantly, we were able to develop a true-breeding strain for TI-pgSIT that eliminates the requirement for sex sorting, a feature that may help further automate production at scale.

category: bioengineering


title: AN INTEGRATIVE BIOLOGY APPROACH TO QUANTIFY THE BIODISTRIBUTION OF AZIDOHOMOALANINE IN VIVO

abstract:
Identification and quantitation of newly synthesized proteins (NSPs) are critical to understanding protein dynamics in development and disease. Probing the nascent proteome can be achieved using non-canonical amino acids (ncAAs) to selectively label the NSPs utilizing endogenous translation machinery, which can then be quantitated with mass spectrometry. Since its conception, ncAA labeling has been applied to study many in vitro systems and more recently the in vivo proteomes of complex organisms such as rodents. In vivo labeling is typically achieved by introducing ncAAs into diet, which requires extended labeling times. We have previously demonstrated that labeling the murine proteome is feasible via injection of azidohomoalanine (Aha), a ncAA and methionine (Met) analog, without the need for Met depletion. With the ability to isolate NSPs without applying stress from dietary changes, Aha labeling can address biological questions wherein temporal protein dynamics are significant. However, accessing this temporal resolution requires a more complete understanding of Aha distribution kinetics in tissues. Furthermore, studies of physiological effects of ncAA administration have been limited to gross observation of animal appearance. To address these gaps, we created a deterministic, compartmental model of the biokinetic transport and incorporation of Aha in mice. Parameters were informed from literature and experimentally. Model results demonstrate the ability to predict Aha distribution and labeling under a variety of dosing paradigms and confirms the use of the model as a tool for design of future studies. To establish the suitability of the method for in vivo studies, we investigated the impact of Aha administration on normal physiology by analyzing the plasma metabolome following Aha injection. We show that Aha administration does not significantly perturb cellular functions as reflected by an unchanged plasma metabolome compared to non-injected controls.

Author SummaryAs the machinery of life, proteins play a key role in dynamic processes within an organism. As such, the response of the proteome to perturbation is increasingly becoming a critical component of biological and medical studies. Dysregulation of protein mechanisms following exposure to experimental treatment conditions can implicate physiological mechanisms of health and disease, elucidate toxin/drug response, and highlight potential targets for novel therapies. Traditionally, these questions have been probed by studying perturbations in total proteins following an experimental treatment. However, the proteome is expansive and noisy, often an early response can be indiscernible against the background of unperturbed proteins. Here, we apply a technique to selectively label newly synthesized proteins, which enables capturing early changes in protein behavior. We utilize an amino acid analog that naturally incorporates into proteins, and investigate the tissue distribution, protein labeling efficiency, and potential physiological impact of this analog in mice. Our results demonstrate that we can reproducibly predict protein labeling and that the administration of this analog does not significantly alter in vivo physiology over the course of our experimental study. We further present a computational model that can be used to guide future experiments utilizing this technique to study proteomic responses to stimuli.


title: PlasLR Enables Adaptation of Plasmid Prediction for Error-Prone Long Reads

abstract: Plasmids are extra-chromosomal genetic elements commonly found in bacterial cells that support many functional aspects including environmental adaptations. The identification of these genetic elements is vital for the further study of function and behaviour of the organisms. However it is challenging to separate these small sequences from longer chromosomes within a given species. Machine learning approaches have been successfully developed to classify assembled contigs into two classes (plasmids and chromosomes). However, such tools are not designed to directly perform classification on long and error-prone reads which have been gaining popularity in genomics studies. Assembling complete plasmids is still challenging for many long-read assemblers with a mixed input of long and error-prone reads from plasmids and chromosomes. In this paper, we present PlasLR, a tool that adapts existing plasmid detection approaches to directly classify long and error-prone reads. PlasLR makes use of both the composition and coverage information of long and error-prone reads. We evaluate PlasLR on multiple simulated and real long-read datasets with varying compositions of plasmids and chromosomes. Our experiments demonstrate that PlasLR substantially improves the accuracy of plasmid detection on top of the state-of-the-art plasmid detection tools. Moreover, we show that using PlasLR before long-read assembly helps to enhance the assembly quality in terms of plasmid recovery and near complete chromosome assembly from metagenomic datasets.

category: bioinformatics


title: Protein Domain-Based Prediction of Compound-Target Interactions and Experimental Validation on LIM Kinases

abstract:
Predictive approaches such as virtual screening have been used in drug discovery with the objective of reducing developmental time and costs. Current machine learning and network- based approaches have issues related to generalization, usability, or model interpretability, especially due to the complexity of target proteins structure/function, and bias in system training datasets. Here, we propose a new computational method "DRUIDom" to predict bio- interactions between drug candidate compounds and target proteins by utilizing the domain modularity of proteins, to overcome problems associated with current approaches. DRUIDom is composed of two methodological steps. First, ligands/compounds are statistically mapped to structural domains of their target proteins, with the aim of identifying physical or functional interactions. As such, other proteins containing the mapped domain or domain pair become new candidate targets for the corresponding compounds. Next, a million-scale dataset of small molecule compounds, including the ones mapped to domains in the previous step, are clustered based on their molecular similarities, and their domain associations are propagated to other compounds within the same clusters. Experimentally verified bioactivity data points, obtained from public databases, are meticulously filtered to construct datasets of active/interacting and inactive/non-interacting compound-target pairs ([~]2.9M data points), and used as training data for calculating parameters of compound- domain mappings, which led to 27,032 high-confidence associations between 250 domains and 8,165 compounds, and a finalized output of [~]5 million new compound-protein interactions. DRUIDom is experimentally validated by syntheses and bioactivity analyses of compounds predicted to target LIM-kinase proteins, which play critical roles in the regulation of cell motility, cell cycle progression, and differentiation through actin filament dynamics. We showed that LIMK-inhibitor-2 and its derivatives significantly block the cancer cell migration through inhibition of LIMK phosphorylation and the downstream protein cofilin. One of the derivative compounds (LIMKi-2d) was identified as a promising candidate due to its action on resistant Mahlavu liver cancer cells. The results demonstrated that DRUIDom can be exploited to identify drug candidate compounds for intended targets and to predict new target proteins based on the defined compound-domain relationships. The datasets, results, and the source code of DRUIDom are fully-available at: https://github.com/cansyl/DRUIDom.

Author SummaryDrug development comprises several interlinked steps from designing drug candidate molecules to running clinical trials, with the aim to bring a new drug to market. A critical yet costly and labor-intensive stage is drug discovery, in which drug candidate molecules that specifically interact with the intended biomolecular target (mostly proteins) are identified. Lately, data-centric computational methods have been proposed to aid experimental procedures in drug discovery. These methods have the ability to rapidly assess large molecule libraries and reduce the time and cost of the process; however, most of them suffer from problems related to producing reliable biologically relevant results, preventing them from gaining real-world usage. Here, we have developed a new method called DRUIDom to predict unknown interactions between drugs/drug candidate compounds and biological targets by utilizing the modular structure of proteins. For this, we identify the domains, i.e., the evolutionary and functional building blocks of proteins, where these potential drug compounds can bind, and utilize this information along with protein domain annotations to predict new drug targets. We have tested the biological relevance of DRUIDom on selected proteins that play critical roles in the progression of numerous types of cancer. Cell-based experimental results indicated that predicted inhibitors are effective even on drug-resistant cancer cells. Our results suggest that DRUIDom produces novel and biologically relevant results that can be directly used in the early steps of the drug discovery process.


title: Functional disruption of Transferrin expression alters reproductive physiologyin Anopheles culicifacies

abstract:
Iron metabolism is crucial to maintain optimal physiological homeostasis of every organism and any alteration of the iron concentration (i.e. deficit or excess) can have adverse consequences. Transferrins are glycoproteins that play important role in iron transportation and have been widely characterized in vertebrates, and insects, but poorly studied in blood-feeding mosquitoes. Here, we characterized a 2102 bp long transcript AcTrf1a encoding putative transferrin homolog protein from mosquito An. culicifacies. A detailed in silico analysis predicts AcTrf1a (ACUA023913-RA) encodes 624 amino acid (aa) long polypeptide that carries transferrin domain. AcTrf1a also showed a putative N-linked glycosylation site, a characteristic feature of most of the mammalian transferrins and certain non-blood feeding insects. Structure modelling prediction confers the presence of an iron binding site at the N-terminal lobe of the transferrin. Our spatial and temporal expression analysis under altered pathophysiological conditions showed that AcTrf1a abundantly express in the fat-body, ovary, and its response is significantly altered (enhanced) after blood meal uptake, and exogenous bacterial challenge. Additionally, a non-heme iron supplementation of FeCl3 at 1 mM concentration not only augmented the AcTrf1a transcript expression in fat-body, also enhanced the reproductive fecundity of gravid adult female mosquitoes. RNAi mediated knockdown of AcTrf1a causes a significant reduction in the egg laying/fecundity, confirmed important role of transferrin in oocyte maturation. Further detailed characterization may help to select this transcript as a unique target to impair the mosquito reproductive outcome.

HighlightsO_LIInsect transferrins are mostly glycoprotein of about 60-80 kDa molecular weight, involved in myriad physiological events and serve as a major iron transport protein.
C_LIO_LIHere, we identified and characterized a 2102 bp long transcript encoding putative transferrin homolog of 624 aa long peptide, carrying only one fully functional transferrin domain at N-terminal from An. culicifacies.
C_LIO_LISpatial and temporal expression analysis of AcTrf1a highlights an enriched expression in fat-body and ovary during vitellogenesis.
C_LIO_LIIron supplementation and dsRNA mediated knockdown experiments together confer that AcTrf1a may have key role in the iron homeostasis regulation during oogenesis, and egg maturation in the gravid female mosquitoes.
C_LI

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/448311v1_fig1.gif" ALT="Figure 1">
View larger version (15K):
org.highwire.dtl.DTLVardef@a87ec0org.highwire.dtl.DTLVardef@197cecorg.highwire.dtl.DTLVardef@1588092org.highwire.dtl.DTLVardef@16110b4_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOFig 1:C_FLOATNO Schematic presentation of iron transport from midgut to ovary by transferrin1 and oocyte reduction after AcTrf1a knockdown.

Mosquito acquires iron either from blood meal or iron supplementation in sugar meal. Fat-body derived transferrin proceed towards the gut surface, load iron in its N-terminal iron-binding pocket and deliver iron to ovary. This blood meal iron is required by adult female for completion of gonotrophic cycle. (a) limited iron availability in sugar meal does not support the ovary development and hence no oogenesis; (b) when sugar meal is replaced by blood meal upregulation of transferrin protein results in rapid iron transport to various organs including ovary results in healthy ovarian growth; (c) RNAi mediated knockdown of this transporter protein transferrin in fat-body followed by blood meal, may cause reduced iron transport to ovary and consequently declines in oocyte load.

C_FIG


title: Dissecting the molecular basis of human interneuron migrationin forebrain assembloids from Timothy syndrome

abstract: Defects in interneuron migration during forebrain development can disrupt the assembly of cortical circuits and have been associated with neuropsychiatric disease. The molecular and cellular bases of such deficits have been particularly difficult to study in humans due to limited access to functional forebrain tissue from patients. We previously developed a human forebrain assembloid model of Timothy Syndrome (TS), caused by a gain-of-function mutation in CACNA1C which encodes the L-type calcium channel (LTCC) Cav1.2. By functionally integrating human induced pluripotent stem cell (hiPSC)-derived organoids resembling the dorsal and ventral forebrain from patients and control individuals, we uncovered that migration is disrupted in TS cortical interneurons. Here, we dissect the molecular underpinnings of this phenotype and report that acute pharmacological modulation of Cav1.2 can rescue the saltation length but not the saltation frequency of TS migrating interneurons. Furthermore, we find that the defect in saltation length in TS interneurons is associated with aberrant actomyosin function and is rescued by pharmacological modulation of MLC phosphorylation, whereas the saltation frequency phenotype in TS interneurons is driven by enhanced GABA sensitivity and can be restored by GABA receptor antagonism. Overall, these findings uncover multi-faceted roles of LTCC function in human cortical interneuron migration in the context of disease and suggest new strategies to restore interneuron migration deficits.


title: Analyzing the differences in olfactory bulb mitral cell spiking with ortho- and retronasal stimulation

abstract:
The majority of olfaction studies focus on orthonasal stimulation where odors enter via the front nasal cavity, while retronasal olfaction, where odors enter the rear of the nasal cavity during feeding, is understudied. The processing of retronasal odors via coordinated spiking of neurons in the olfactory bulb (OB) is largely unknown. To this end, we use multi-electrode array in vivo recordings of rat OB mitral cells (MC) in response to a food odor with both modes of stimulation, and find significant differences in evoked firing rates and spike count covariances (i.e., noise correlations). To better understand these differences, we develop a single-compartment biophysical OB model that is able to reproduce key properties of important OB cell types. Prior experiments in olfactory receptor neurons (ORN) showed retro stimulation yields slower and spatially smaller ORN inputs than with ortho, yet whether this is consequential for OB activity remains unknown. Indeed with these specifications for ORN inputs, our OB model captures the trends in our OB data. We also analyze how first and second order ORN input statistics dynamically transfer to MC spiking statistics with a phenomenological linear-nonlinear filter model, and find that retro inputs result in larger temporal filters than ortho inputs. Finally, our models show that the temporal profile of ORN is crucial for capturing our data and is thus a distinguishing feature between ortho and retro stimulation, even at the OB. Using data-driven modeling, we detail how ORN inputs result in differences in OB dynamics and MC spiking statistics. These differences may ultimately shape how ortho and retro odors are coded.

Author summaryOlfaction is a key sense for many cognitive and behavioral tasks, and is particularly unique because odors can naturally enter the nasal cavity from the front or rear, i.e., ortho- and retro-nasal, respectively. Yet little is known about the differences in coordinated spiking in the olfactory bulb with ortho versus retro stimulation, let alone how these different modes of olfaction may alter coding of odors. We simultaneously record many cells in rat olfactory bulb to assess the differences in spiking statistics, and develop a biophysical olfactory bulb network model to study the reasons for these differences. Using theoretical and computational methods, we find that the olfactory bulb transfers input statistics differently for retro stimulation relative to ortho stimulation. Furthermore, our models show that the temporal profile of inputs is crucial for capturing our data and is thus a distinguishing feature between ortho and retro stimulation, even at the olfactory bulb. Understanding the spiking dynamics of the olfactory bulb with both ortho and retro stimulation is a key step for ultimately understanding how the brain codes odors with different modes of olfaction.


title: A functional network signature in the developing cerebellum: evidence from a preclinical model of autism

abstract: Autism spectrum disorders (ASD) are pervasive neurodevelopmental conditions detected during childhood when delayed language onset and social deficits are observed. Children diagnosed with ASD frequently display sensorimotor deficits associated with the cerebellum, suggesting a dysfunction of synaptic circuits. Astroglia are part of the tripartite synapses and postmortem studies reported an increased expression of the glial fibrillary acidic protein (GFAP) in the cerebellum of ASD patients. Astroglia respond to neuronal activity with calcium transients that propagate to neighboring cells, resulting in a functional network response known as a calcium wave. This form of intercellular signaling is implicated in proliferation, migration, and differentiation of neural precursors. Prenatal exposure to valproate (VPA) is a preclinical model of ASD in which premature migration and excess of apoptosis occur in the internal granular layer (IGL) of the cerebellum during the early postnatal period. In this study we tested calcium wave propagation in the IGL of mice prenatally exposed to VPA. Sensorimotor and social deficits were observed and IGL depolarization evoked a calcium wave with astrocyte recruitment. The calcium wave propagation, initial cell recruitment, and mean amplitude of the calcium transients increased significantly in VPA-exposed mice compared to the control group. Astrocyte recruitment was significantly increased in the VPA model, but the mean amplitude of the calcium transients was unchanged. Western blot and histological studies revealed an increased expression of GFAP and higher astroglial density. We conclude that the functional network of the IGL is remarkably augmented in the preclinical model of autism.


title: A species-wide genetic atlas of antimicrobial resistance in Clostridioides difficile

abstract:
Antimicrobial resistance (AMR) plays an important role in the pathogenesis and spread of Clostridioides difficile infection (CDI), the leading healthcare-related gastrointestinal infection in the world. An association between AMR and CDI outbreaks is well documented, however, data is limited to a few  epidemic strains in specific geographical regions. Here, through detailed analysis of 10,330 publicly-available C. difficile genomes from strains isolated worldwide (spanning 270 multilocus sequence types (STs) across all known evolutionary clades), this study provides the first species-wide snapshot of AMR genomic epidemiology in C. difficile. Of the 10,330 C. difficile genomes, 4,532 (43.9%) in 89 STs across clades 1 - 5 carried at least one genotypic AMR determinant, with 901 genomes (8.7%) carrying AMR determinants for three or more antimicrobial classes (multidrug-resistant, MDR). No AMR genotype was identified in any strains belonging to the cryptic clades. C. difficile from Australia/New Zealand had the lowest AMR prevalence compared to strains from Asia, Europe and North America (p<0.0001). Based on the phylogenetic clade, AMR prevalence was higher in clades 2 (84.3%), 4 (81.5%) and 5 (64.8%) compared to other clades (collectively 26.9%) (p<0.0001). MDR prevalence was highest in clade 4 (61.6%) which was over three times higher than in clade 2, the clade with the second-highest MDR prevalence (18.3%). There was a strong association between specific AMR determinants and three major epidemic C. difficile STs: ST1 (clade 2) with fluoroquinolone resistance (mainly T82I substitution in GyrA) (p<0.0001), ST11 (clade 5) with tetracycline resistance (various tet-family genes) (p<0.0001) and ST37 (clade 4) with macrolide-lincosamide-streptogramin B (MLSB) resistance (mainly ermB) (p<0.0001) and MDR (p<0.0001). A novel and previously overlooked tetM-positive transposon designated Tn6944 was identified, predominantly among clade 2 strains. This study provides a comprehensive review of AMR in the global C. difficile population which may aid in the early detection of drug-resistant C. difficile strains, and prevention of their dissemination world-wide.

Impact statementUtilising a publicly-available database of 10,330 sequence reads, this study provides the first species-wide evaluation of genotypic AMR in C. difficile. It reports the most common AMR determinants and their genomic neighbourhood, associations between important genotypes and specific strains or geographical regions, and rare AMR genotypes that may have been missed in earlier studies.

Data summaryThis study utilises publicly available raw sequence reads available at the NCBI Sequence Read Archive (SRA) as of January 2020. The details of all genomes are available in the Supplementary Data (10.6084/m9.figshare.14623533).


title: Complete metamorphosis and microbiota turnover in insects

abstract: The insects constitute the majority of animal diversity. Most insects are holometabolous: during complete metamorphosis their bodies are radically re-organized. This re-organization poses a significant challenge to the gut microbiota, as the gut is replaced during pupation, a process that does not occur in hemimetabolous insects. In holometabolous hosts, it offers the opportunity to decouple the gut microbiota between the larval and adult life stages resulting in high beta diversity whilst limiting alpha diversity. Here we studied 18 different herbivorous insect species from 5 orders of holometabolous and 3 orders of hemimetabolous insects. Comparing larval and adult specimens, we find a much higher beta-diversity and hence microbiota turnover in holometabolous insects compared to hemimetabolous insects. Alpha diversity did not differ between holo-and hemimetabolous insects nor between developmental stages within these groups. Our results support the idea that pupation offers the opportunity to change the gut microbiota and hence facilitates ecological niche shifts. This effect of niche shift facilitation could explain a selective advantage of the evolution of complete metamorphosis, which is a defining trait of the most speciose insect taxon, the holometabola.

category: evolutionary biology


title: Viral transduction and the dynamics of bacterial adaptation

abstract: Viral infections can exert a large influence on their hosts ecology by causing widespread mortality, but they also shape the evolutionary adaptation of hosts in a number of ways. A major pathway for viruses to do so is through the transfer of genetic material among individual hosts, a process known as transduction. While horizontal gene transfer is known as a major factor in prokaryotic macroevolution, its role in the microevolutionary adaptation of hosts populations is poorly known. By facilitating the transfer of beneficial alleles between host cells, transduction might facilitate and accelerate bacterial adaptation. Conversely, the risk of transferring deleterious alleles may hinder and slow it down. Here we resolve the effect of transduction on bacterial adaptation in a simple ecoevolutionary model for the combined dynamics of transduction and adaptive evolution of an ecological (resource-use) trait. The transfer of beneficial alleles by tranduction speeds up adaptation whereas the transfer of deleterious alleles causes strong stochastic fluctuations of the trait value around the adapted value. In contrast to the expected effect of recombination, which tends to oppose phenotypic diversification, viral transduction can increase host phenotypic diversity.


title: The contribution of genetic and environmental effects to Bergmanns rule and Allens rule in house mice

abstract: Distinguishing between genetic, environmental, and genotype-by-environment effects is central to understanding geographic variation in phenotypic clines. Two of the best-documented phenotypic clines are Bergmanns rule and Allens rule, which describe larger body sizes and shortened extremities in colder climates, respectively. Although numerous studies have found inter- and intraspecific evidence for both ecogeographic patterns, we still have a poor understanding of the extent to which these patterns are driven by genetics, environment, or both. Here, we measured the genetic and environmental contributions to Bergmanns rule and Allens rule across introduced populations of house mice (Mus musculus domesticus) in the Americas. First, we documented clines for body mass, tail length, and ear length in natural populations, and found that these conform to both Bergmanns rule and Allens rule. We then raised descendants of wild-caught mice in the lab and showed that these differences persisted in a common environment and are heritable, indicating that they have a genetic basis. Finally, using a full-sib design, we reared mice under warm and cold conditions. We found very little plasticity associated with body size, suggesting that Bergmanns rule has been shaped by strong directional selection in house mice. However, extremities showed considerable plasticity, as both tails and ears grew shorter in cold environments. These results indicate that adaptive phenotypic plasticity as well as genetic changes underlie major patterns of clinal variation in house mice and likely facilitated their rapid expansion into new environments across the Americas.