title: Computing the Riemannian curvature of image patch and single-cell RNA sequencing data manifolds using extrinsic differential geometry

abstract: Most high-dimensional datasets are thought to be inherently low-dimensional, that is, datapoints are constrained to lie on a low-dimensional manifold embedded in a high-dimensional ambient space. Here we study the viability of two approaches from differential geometry to estimate the Riemannian curvature of these low-dimensional manifolds. The intrinsic approach relates curvature to the Laplace-Beltrami operator using the heat-trace expansion, and is agnostic to how a manifold is embedded in a high-dimensional space. The extrinsic approach relates the ambient coordinates of a manifolds embedding to its curvature using the Second Fundamental Form and the Gauss-Codazzi equation. Keeping in mind practical constraints of real-world datasets, like small sample sizes and measurement noise, we found that estimating curvature is only feasible for even simple, low-dimensional toy manifolds, when the extrinsic approach is used. To test the applicability of the extrinsic approach to real-world data, we computed the curvature of a well-studied manifold of image patches, and recapitulated its topological classification as a Klein bottle. Lastly, we applied the approach to study single-cell transcriptomic sequencing (scRNAseq) datasets of blood, gastrulation, and brain cells, revealing for the first time the intrinsic curvature of scRNAseq manifolds.


title: Somatic mutations in CTCF zinc fingers produce cellular phenotypes explained by structure-function relationships

abstract:
BackgroundHuman cancers commonly contain mutations in transcription factors that lead to aberrant DNA binding or altered effector function at target sites. One such factor significantly mutated in cancer is the evolutionarily-conserved CCCTC-binding factor (CTCF), which has fundamental roles in maintaining chromatin architecture and transcriptional regulation. Numerous cancer genome sequencing and functional studies have revealed CTCFs role as a haploinsufficient tumour suppressor gene. However, to date, structure-function relationships of somatic CTCF mutations have not been examined.

MethodsWe collated somatic CTCF mutations from cancer genome portals and published studies to determine their nature, frequency, distribution and potential functional impact. We undertook an in-depth examination of 5 CTCF missense zinc finger (ZF) mutations occurring within key intra- and inter-ZF residues. We performed functional analyses including cell growth, colony-formation, chromatin immunoprecipitation and transcriptional reporter assays. Based on their homology, each ZF mutation was then modelled on CTCFs ZF domain crystal structure and its structural impact analysed using molecular dynamics simulations.

ResultsWe observed an enrichment of somatic missense mutations occurring in the ZF region of CTCF, compared to the unstructured N- and C-termini. Functional characterisation of CTCF ZF mutations revealed a complete (L309P, R339W, R377H) or intermediate (R339Q) abrogation as well as an enhancement (G420D) of the anti-proliferative effects of CTCF. DNA binding at select sites was disrupted and transcriptional regulatory activities abrogated. In silico mutagenesis revealed that L309P had the highest mutation energy and thus most severe impact on protein stability. Molecular docking and molecular dynamics simulations confirmed that mutations in residues specifically contacting DNA bases or backbone exhibited loss of DNA binding (R339Q, R339W, R377H). Remarkably, R339Q and G420D were stabilised by the formation of new primary DNA bonds. All mutations exhibited some loss or gain of bonds at neighbouring residues, often in adjacent zinc fingers.

ConclusionsOur data confirm the significant negative impact haploinsufficient CTCF ZF mutations have on its tumour suppressor function. A spectrum of loss-, change- and gain-of-function impacts in CTCF zinc fingers are observed in cell growth regulation and gene regulatory activities. We have established that diverse cellular phenotypes in CTCF are explained by examining structure-function relationships.

category: cancer biology


title: Genetic influences on brain activation and large-scale functional connectivity during nociceptive processing: a twin study

abstract: Nociceptive processing in the human brain is a signal that enables harm avoidance, with large interindividual variance. The relative contributions of genes and environment to the neural structures that support nociception have not been studied in twins previously. Here, we employed a classic twin-design to determine brain structures influenced by additive genetics. We found genetic influences on nociceptive processing in the midcingulate cortex, bilateral posterior insulae and thalamus. In addition to brain activations, we found genetic contributions to large-scale functional connectivity during nociceptive processing. We conclude that additive genetics influence specific aspects of nociceptive processing, which improves our understanding of human nociceptive processing.

category: neuroscience


title: Loss of PIKfyve drives the spongiform degeneration in prion diseases

abstract: Brain-matter vacuolation is a defining trait of all prion diseases, yet its cause is unknown. Here we report that prion infection and prion-mimetic antibodies deplete the phosphatidylinositol kinase PIKfyve in mouse brains, cultured cells, organotypic brain slices, and in brains of Creutzfeldt-Jakob Disease victims. We found that PIKfyve, an inositol kinase involved endolysosomal maturation, is acylated by zDHHC9 and zDHHC21, whose juxtavesicular topology is disturbed by prion infection, resulting in PIKfyve deacylation and destabilization. A protracted unfolded protein response (UPR), typical of prion diseases, also induced PIKfyve deacylation and degradation. Conversely, UPR antagonists restored PIKfyve levels in prion-infected cells. Overexpression of zDHHC9 and zDHHC21, administration of the antiprion polythiophene LIN5044, or supplementation with the PIKfyve reaction product PI(3,5)P2, suppressed prion-induced vacuolation. Thus, PIKfyve emerges as a central mediator of vacuolation and neurotoxicity in prion diseases.


title: Test-retest reliability of tone- and 40 Hz train-evoked gamma oscillations in female rats and their sensitivity to low-dose NMDA channel blockade

abstract:
RationaleSchizophrenia patients consistently show deficits in sensory-evoked broadband gamma oscillations and click-evoked entrainment at 40 Hz, called the 40 Hz auditory steady-state response (ASSR). Since such evoked oscillations depend on cortical N-methyl D-aspartic acid (NMDA)-mediated network activity, they can serve as pharmacodynamic biomarkers in the preclinical development of drug candidates engaging these circuits. However, there is little test-retest reliability data in preclinical species, a prerequisite for within-subject testing paradigms.

ObjectivesWe investigated the long-term stability of these measures in a rodent model.

MethodsFemale rats with chronic epidural implants were used to record tone- and 40 Hz click-evoked responses at multiple time points and across six sessions, spread over 3 weeks. We assessed reliability using intraclass correlation coefficients (ICC). Separately, we used mixed-effects ANOVA to examine time and session effects. Individual subject variability was determined using the coefficient of variation (CV). Lastly, to illustrate the importance of long-term measure stability for within-subject testing design, we used low to moderate doses of an NMDA antagonist MK801 (0.025-0.15 mg/kg) to disrupt the evoked responses.

ResultsWe found that 40 Hz ASSR showed good reliability (ICC=0.60-0.75) while the reliability of tone-evoked gamma ranged from fair to good (0.33-0.67). We noted time but no session effects. Subjects showed a lower variance for ASSR over tone-evoked gamma. Both measures were dose-dependently attenuated by NMDA antagonism.

ConclusionOverall, while both measures use NMDA transmission, 40 Hz ASSR showed superior psychometric properties of higher ICC and lower CV, relative to tone-evoked gamma.


title: Selective modulation of population dynamics during neuroprosthetic skill learning

abstract: Learning to control a brain-machine interface (BMI) is associated with the emergence of coordinated neural dynamics in populations of neurons whose activity serves as direct input to the BMI decoder (direct subpopulation). While previous work shows differential modification of firing rate modulation in this population relative to a population whose activity was not directly input to the BMI decoder (indirect subpopulation), little is known about how learning-rated changes in cortical population dynamics within these groups compare. To investigate this, we monitored both direct and indirect subpopulations as two macaque monkeys learned to control a BMI. We found that while the combined population increased coordinated neural dynamics, this coordination was primarily driven by changes in the direct subpopulation while the indirect subpopulation remained relatively stable. These findings indicate that motor cortex refines cortical dynamics throughout the entire network during learning, with a more pronounced effect in ensembles causally linked to behavior.


title: Ad26.COV2.S-elicited immunity protects against G614 spike variant SARS-CoV-2 infection in Syrian hamsters and does not enhance respiratory disease in challenged animals with breakthrough infection after sub-optimal vaccine dosing

abstract: Previously we have shown that a single dose of recombinant adenovirus serotype 26 (Ad26) vaccine expressing a prefusion stabilized SARS-CoV-2 spike antigen (Ad26.COV2.S) is immunogenic and provides protection in Syrian hamster and non-human primate SARS-CoV-2 infection models. Here, we investigated the immunogenicity, protective efficacy and potential for vaccine-associated enhanced respiratory disease (VAERD) mediated by Ad26.COV2.S in a moderate disease Syrian hamster challenge model, using the currently most prevalent G614 spike SARS-CoV-2 variant. Vaccine doses of 1x109 vp and 1x1010 vp elicited substantial neutralizing antibodies titers and completely protected over 80% of SARS-CoV-2 inoculated Syrian hamsters from lung infection and pneumonia but not upper respiratory tract infection. A second vaccine dose further increased neutralizing antibody titers which was associated with decreased infectious viral load in the upper respiratory tract after SARS-CoV-2 challenge. Suboptimal non-protective immune responses elicited by low-dose A26.COV2.S vaccination did not exacerbate respiratory disease in SARS-CoV-2-inoculated Syrian hamsters with breakthrough infection. In addition, dosing down the vaccine allowed to establish that binding and neutralizing antibody titers correlate with lower respiratory tract protection probability. Overall, these pre-clinical data confirm efficacy of a 1-dose vaccine regimen with Ad26.COV2.S in this G614 spike SARS-CoV-2 virus variant Syrian hamster model, show the added benefit of a second vaccine dose, and demonstrate that there are no signs of VAERD under conditions of suboptimal immunity.

category: immunology


title: Evolution in salmon life-history induced by direct and indirect effects of fishing

abstract: Understanding the drivers of evolution is a fundamental aim in biology. However, identifying the evolutionary impacts of human activities, both direct and indirect, is challenging because of lack of temporal data and limited knowledge of the genetic basis of most traits1. Atlantic salmon is a species exposed to intense anthropogenic pressures during its anadromous life cycle2. Previous research has shown that salmon age at maturity has evolved towards earlier maturation over the last 40 years, with an 18% decrease3 in the allele associated with late maturation at the large-effect vgll3 locus4; but the drivers of this change remain unknown. Here, we link genetic and phenotypic changes in a large Atlantic salmon population with salmon prey species biomass in the Barents Sea, temperature, and fishing effort in order to identify drivers of age at maturity evolution. We show that age at maturity evolution is associated with two different types of fisheries induced evolution acting in opposing directions: an indirect effect linked with commercial harvest of a salmon prey species (capelin) at sea (selection against late maturation), and a direct effect due to temporal changes in net fishing pressure in the river (surprisingly, selection against early maturation). Although the potential for direct and indirect evolutionary effects of fishing have been acknowledged, empirical evidence for induced changes at the genetic level has been lacking5. As capelin are primarily harvested to produce fish meal and oil for aquaculture6, we hereby identify an indirect path by which Atlantic salmon aquaculture may negatively affect wild populations.

category: evolutionary biology


title: APOBEC1 mediated C-to-U RNA editing: target sequence and trans-acting factor contribution to 177 RNA editing events in 119 murine transcripts in-vivo

abstract: Mammalian C-to-U RNA editing was described more than 30 years ago as a single nucleotide modification in APOB RNA in small intestine, later shown to be mediated by the RNA-specific cytidine deaminase APOBEC1. Reports of other examples of C-to-U RNA editing, coupled with the advent of genome-wide transcriptome sequencing, identified an expanded range of APOBEC1 targets. Here we analyze the cis-acting regulatory components of verified murine C-to-U RNA editing targets, including nearest neighbor as well as flanking sequence requirements and folding predictions. We summarize findings demonstrating the relative importance of trans-acting factors (A1CF, RBM47) acting in concert with APOBEC1. Using this information, we developed a multivariable linear regression model to predict APOBEC1 dependent C-to-U RNA editing efficiency, incorporating factors independently associated with editing frequencies based on 103 Sanger-confirmed editing sites, which accounted for 84% of the observed variance. Co-factor dominance was associated with editing frequency, with RNAs targeted by both RBM47 and A1CF observed to be edited at a lower frequency than RBM47 dominant targets. The model also predicted a composite score for available human C-to-U RNA targets, which again correlated with editing frequency.

category: bioinformatics


title: Cysteine is a limiting factor for glioma proliferation and survival

abstract: Nutritional intervention is becoming more prevalent as adjuvant therapy for many cancers in view of the tumor dependence on external sources for some nutrients. We report the dependence of glioma cells on exogenous cysteine/cystine, despite this amino acid being nonessential. 13C-tracing and the analysis of cystathionine synthase and cystathioninase levels revealed the metabolic landscape attributable to cysteine deprivation, and the disconnection between the methionine cycle and the transsulfuration pathway. Therefore, we explored the nutritional deprivation in a mouse model of glioma. Animals subjected to a cysteine/cystine-free diet survived longer, with concomitant reductions in glutathione and cysteine plasma levels. At the end point, however, tumors displayed the ability to synthesize glutathione, although higher levels of oxidative stress were detected. We observed a compensation from the nutritional intervention revealed as the recovery of cysteine-related metabolites in plasma. Our study highlights a time window where cysteine deprivation can be exploited for additional therapeutic strategies.


title: Genome-scale sequencing and analysis of human, wolf and bison DNA from 25,000 year-old sediment

abstract:
Archaeological sediments have been shown to preserve ancient DNA, but so far have not yielded genome-scale information of the magnitude of skeletal remains. We retrieved and analysed human and mammalian low-coverage nuclear and high-coverage mitochondrial genomes from Upper Palaeolithic sediments from Satsurblia cave, western Georgia, dated to 25,000 years ago. First, a human female genome with substantial basal Eurasian ancestry, which was an ancestry component of the majority of post-Ice Age people in the Near East, North Africa, and parts of Europe. Second, a wolf genome that is basal to extant Eurasian wolves and dogs and represents a previously unknown, likely extinct, Caucasian lineage that diverged from the ancestors of modern wolves and dogs before these diversified. Third, a bison genome that is basal to present-day populations, suggesting that population structure has been substantially reshaped since the Last Glacial Maximum. Our results provide new insights into the late Pleistocene genetic histories of these three species, and demonstrate that sediment DNA can be used not only for species identification, but also be a source of genome-wide ancestry information and genetic history.

HighlightsO_LIWe demonstrate for the first time that genome sequencing from sediments is comparable to that of skeletal remains
C_LIO_LIA single Pleistocene sediment sample from the Caucasus yielded three low-coverage mammalian ancient genomes
C_LIO_LIWe show that sediment ancient DNA can reveal important aspects of the human and faunal past
C_LIO_LIEvidence of an uncharacterized human lineage from the Caucasus before the Last Glacial Maximum
C_LIO_LI[~]0.01-fold coverage wolf and bison genomes are both basal to present-day diversity, suggesting reshaping of population structure in both species
C_LI

category: developmental biology


title: Speech-like sounds dominate the human infant landscape

abstract:
Early human infant vocalization is viewed as forming not only a critical foundation for vocal learning of language, but also a crucial realm of communication affecting emotional and social development. Although speech-like sounds are rare or absent in other ape infants, they share distress sounds (shrieks and cries) and laughter with humans, forming a potential basis for especially informative cross-species comparisons as well as potential insights regarding usage and learning of vocal sounds. A fundamental need to make such comparisons possible is empirical research to document frequency of occurrence of vocalizations of various types in natural environments.

The present work focuses on laughter in the human infant, a topic that has been viewed by many as a key factor in social development for humans and other apes. Yet we know of no research quantifying frequency of occurrence of human infant laughter in natural environments across the first year. In the past two decades it has been shown that the predominant vocalizations of the human infant are "protophones", the precursor sounds to speech. Longitudinal research has indicated unambiguously that protophones outnumber cries by a factor of at least five based on data from random-sampling of all-day recordings across the whole first year. The present work expands on the prior reports by reporting data showing that human infant laughter occurs even more rarely than cry in all-day recordings. Yet laughter is clearly a salient and important aspect of social development. We reason about the dominance of protophones in the infant vocal landscape in light of their role in illuminating human vocal learning and the origin of language.


title: Dicer promotes genome stability via the bromodomain transcriptional co-activator Brd4.

abstract:
RNA interference is essential for transcriptional silencing and genome stability, but conservation of this role in mammals has been difficult to demonstrate. Dicer1-/- mouse embryonic stem cells have microRNA-independent proliferation defects, and we conducted a CRISPR-Cas9 screen to restore viability. We identified suppressor mutations in transcriptional activators, H3K9 methyltransferases, and chromosome segregation factors, strongly resembling Dicer suppressors in fission yeast. Suppressors rescued chromosomal defects, and reversed strand-specific transcription of major satellite repeats in Dicer1-/-. The strongest suppressors were in Brd4, and in the transcriptional elongator/histone acetyltransferase Elp3. Using viable mutants and pharmaceutical inhibitors, we demonstrate that deletion of specific residues in Brd4 rescue genome instability defects of Dicer1-/- in both mammalian cells and fission yeast, implicating Dicer in coordinating transcription and replication of satellite repeats.

SummaryReplication and segregation defects in Dicer1-/- stem cells depend on centromeric transcription by Brd4, and are deeply conserved in fission yeast.

category: genetics


title: Systematically quantifying morphological features reveals constraints on organoid phenotypes

abstract: Organoids recapitulate complex 3D organ structures and represent a unique opportunity to probe the principles of self-organization. While we can alter an organoids morphology by manipulating the culture conditions, the morphology of an organoid often resembles that of its original organ, suggesting that organoid morphologies are governed by a set of tissue-specific constraints. Here, we establish a framework to identify constraints on an organoids morphological features by quantifying them from microscopy images of organoids exposed to a range of perturbations. We apply this framework to Madin-Darby Canine Kidney cysts and show that they obey a number of constraints taking the form of scaling relationships or caps on certain parameters. For example, we found that the number, but not size, of cells increases with increasing cyst size. We also find that these constraints vary with cyst age and can be altered by varying the culture conditions. We observed similar sets of constraints in intestinal organoids. This quantitative framework for identifying constraints on organoid morphologies may inform future efforts to engineer organoids.

category: systems biology


title: Aurora kinase A is essential for meiosis in mouse oocytes

abstract:
The Aurora protein kinases are well-established regulators of spindle building and chromosome segregation in mitotic and meiotic cells. In mouse oocytes, there is significant Aurora kinase A (AURKA) compensatory abilities when the other Aurora kinase homologs are deleted. Whether the other homologs, AURKB or AURKC can compensate for loss of AURKA is not known. Using a conditional mouse oocyte knockout model, we demonstrate that this compensation is not reciprocal because female oocyte-specific knockout mice are sterile and their oocytes fail to complete meiosis I. In determining the AURKA-specific functions, we demonstrate that its first meiotic requirement is to activate Polo-like kinase 1 at microtubule organizing centers (MTOCs; meiotic spindle poles). This activation induces fragmentation of the MTOCs, a step essential for building a bipolar spindle. The next step that requires AURKA is building the liquid-like spindle domain that involves TACC3. Finally, we find that AURKA is also required for anaphase I onset to trigger cohesin cleavage in an APC/C independent manner. We conclude that AURKA has multiple functions essential to completing MI that are distinct from AURKB and AURKC.

Author SummaryFemale gametes, oocytes, are uniquely prone to chromosome segregation errors in meiosis I that are associated with early miscarriages. The Aurora protein kinases are essential to control chromosome segregation in all cell types. During mitosis, Aurora kinase A (AURKA) regulates the building of the spindle, the machinery responsible for pulling chromosomes apart. Here, we use a genetic approach to demonstrate that AURKA is essential for meiosis I in mouse oocytes. AURKA is required at multiple steps in meiosis I, first to trigger fragmentation of protein structures that make up the two ends of the meiotic spindle, later to regulate building of a specialized phase-separated spindle domain, and finally to trigger efficient cleavage of cohesin, the molecular glue that holds chromosomes together until anaphase onset. These findings are the first demonstration of distinct Aurora kinase function that cannot be compensated for by the other two homologs. Therefore, this mouse model is excellent tool for pinpointing specific Aurora kinase functions and identifying AURKA target proteins critical for chromosome segregation in meiosis I.

category: cell biology


title: CYK-1/Formin activation in cortical RhoA signaling centers promotes organismal left-right symmetry breaking

abstract:
1Proper left-right symmetry breaking is essential for animal development and in many species the actin cytoskeleton plays an instrumental role in this process. Active torque generation in the actomyosin layer promotes left-right symmetry breaking in C. elegans embryos by driving chiral counter-rotating cortical flows. While both Formins and Myosins have been implied in left-right symmetry breaking, and both can rotate actin filaments in vitro, it remains unclear if active torques in the actomyosin cortex are generated by Formins, Myosins, or both. We combined the strength of C. elegans genetics with quantitative imaging and thin film, chiral active fluid theory to show that, while Non-Muscle Myosin II activity drives cortical actomyosin flows, it is permissive for chiral counter-rotation and dispensable for chiral symmetry breaking of cortical flows. Instead, we find that CYK-1/Formin activation in RhoA foci is instructive for chiral counter-rotation and promotes in-plane, active torque generation in the actomyosin cortex. Notably, we observe that artificially generated large active RhoA patches undergo rotations with consistent handedness in a CYK-1/Formin-dependent manner. Altogether, we conclude that, CYK-1/Formin-dependent active torque generation facilitates chiral symmetry breaking of actomyosin flows and drives organismal left-right symmetry breaking in the nematode worm.

2 SignificanceActive torque generation in the actin cytoskeleton has been implicated in driving left-right symmetry breaking of developing embryos, but which molecules generate the active torque and how active torque generation is organized subcellularly remains unclear. This study shows that cortical Formin, recruited to cortical regions where RhoA signaling is active, promotes active torque generation in the actomyosin layer. We find that active torque tends to locally rotate the cortex in a clockwise fashion, which drives the emergence of chiral counter-rotating flows with consistent handedness and facilitates left-right symmetry breaking of C. elegans embryos.


title: Evolvability and constraint in the primate basicranium, shoulder, and hip and the importance of multi-trait evolution

abstract: The scapula shares developmental and functional relationships with traits of the basicranium, vertebral column, humerus, and clavicle. As a limb girdle, it also shares analogous characteristics with the pelvis. Despite these relationships, studies of primate shoulder evolution often focus on traits of the scapula in isolation. Such analyses may lead to spurious conclusions, as they implicitly model the scapula as evolving independent of other anatomical regions. Traits of the shoulder girdle share genetic covariances with each other, as well as potential covariances with dimensions of other skeletal elements. To create accurate models of shoulder evolution, it is imperative to account for the constraints imposed by these sources of covariance. Here, we use evolutionary quantitative methods to test a model in which shoulder morphological evolution is influenced by its developmental and functional covariances with the basicranium in the Colobus genus. This evolutionary relationship is also assessed with morphology of the pelvis to provide context to the evolutionary covariance among traits of the basicranium and shoulder girdle. Our results indicate potential evolutionary implications arising from covariances among the basicranium, shoulder, and pelvis. We further propose that the shoulder and basicranium may be examples of developmental, functional, and genetic covariances among traits that manifest an evolutionary suite of mutually constrained morphologies. We demonstrate novel evolutionary relationships among the shoulder girdle and basicranium that affect not only models of primate shoulder evolution but have broader implications for modeling trait evolution across the skeleton.


title: Pathways and signatures of mutagenesis at targeted DNA nicks

abstract: Nicks are the most frequent form of DNA damage and a potential source of mutagenesis in human cells. By deep sequencing, we have identified factors and pathways that promote and limit mutagenic repair at targeted nicks. BRCA2 inhibits all categories of mutational events at nicks, including indels, SNVs and HDR. DNA2 and RPA promote 5 resection. Most insertions at nicks consist of a single C incorporated opposite the nick by the translesion polymerase REV1. DNA2 and REV3 inhibit these 1 bp insertions; and DNA2 also inhibits 1 bp deletions. Longer deletions are stimulated by DNA2, REV7 and POLQ. Strikingly, POLQ generates most SNVs at both nicks and double-strand breaks. These results identify mutagenic signatures of DNA2, REV1, REV3, REV7 and POLQ at nicks and highlight the potential for nicks to promote mutagenesis, especially in BRCA-deficient cells.


title: Fatty acid oxidation participates of the survival to starvation, cell cycle progression and differentiation in the insect stages of Trypanosoma cruzi

abstract:
During its complex life cycle, Trypanosoma cruzi colonizes different niches in its insect and mammalian hosts. This characteristic determined the types of parasites that adapted to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. These T. cruzi stages could be exposed to fatty acids originating from the degradation of the perimicrovillar membrane. In this study, we revisit the metabolic fate of fatty acid breakdown in T. cruzi. Herein, we show that during parasite proliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-14C]-palmitate can be taken up from the medium, leading to CO2 production via beta-oxidation. Lastly, we also show that fatty acids are degraded through beta-oxidation. Additionally, through beta-oxidation, electrons are fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylic acid cycle, which can be used to feed other anabolic pathways such as the de novo biosynthesis of fatty acids.

Author SummaryTrypanosoma cruzi is a protist parasite with a life cycle involving two types of hosts, a vertebrate one (which includes humans, causing Chagas disease) and an invertebrate one (kissing bugs, which vectorize the infection among mammals). In both hosts, the parasite faces environmental challenges such as sudden changes in the metabolic composition of the medium in which they develop, severe starvation, osmotic stress and redox imbalance, among others. Because kissing bugs feed infrequently in nature, an intriguing aspect of T. cruzi biology (it exclusively inhabits the digestive tube of these insects) is how they subsist during long periods of starvation. In this work, we show that this parasite performs a metabolic switch from glucose consumption to lipid oxidation, and it is able to consume lipids and the lipid-derived fatty acids from both internal origins as well as externally supplied compounds. When fatty acid oxidation is chemically inhibited by etomoxir, a very well-known drug that inhibits the translocation of fatty acids into the mitochondria, the proliferative insect stage of the parasites has dramatically diminished survival under severe metabolic stress and its differentiation into its infective forms is impaired. Our findings place fatty acids in the centre of the scene regarding their extraordinary resistance to nutrient-depleted environments.

category: biochemistry


title: DeepHBV: A deep learning model to predict hepatitis B virus (HBV) integration sites.

abstract:
Hepatitis B virus (HBV) is one of the main causes for viral hepatitis and liver cancer. Previous studies showed HBV can integrate into host genome and further promote malignant transformation. In this study, we developed an attention-based deep learning model DeepHBV to predict HBV integration sites by learning local genomic features automatically. We trained and tested DeepHBV using the HBV integration sites data from dsVIS database. Initially, DeepHBV showed AUROC of 0.6363 and AUPR of 0.5471 on the dataset. Adding repeat peaks and TCGA Pan Cancer peaks can significantly improve the model performance, with an AUROC of 0.8378 and 0.9430 and an AUPR of 0.7535 and 0.9310, respectively. On independent validation dataset of HBV integration sites from VISDB, DeepHBV with HBV integration sequences plus TCGA Pan Cancer (AUROC of 0.7603 and AUPR of 0.6189) performed better than HBV integration sequences plus repeat peaks (AUROC of 0.6657 and AUPR of 0.5737). Next, we found the transcriptional factor binding sites (TFBS) were significantly enriched near genomic positions that were paid attention to by convolution neural network. The binding sites of AR-halfsite, Arnt, Atf1, bHLHE40, bHLHE41, BMAL1, CLOCK, c-Myc, COUP-TFII, E2A, EBF1, Erra and Foxo3 were highlighted by DeepHBV attention mechanism in both dsVIS dataset and VISDB dataset, revealing the HBV integration preference. In summary, DeepHBV is a robust and explainable deep learning model not only for the prediction of HBV integration sites but also for further mechanism study of HBV induced cancer.

Author summaryHepatitis B virus (HBV) is one of the main causes for viral hepatitis and liver cancer. Previous studies showed HBV can integrate into host genome and further promote malignant transformation. In this study, we developed an attention-based deep learning model DeepHBV to predict HBV integration sites by learning local genomic features automatically. The performance of DeepHBV model significantly improves after adding genomic features, with an AUROC of 0.9430 and an AUPR of 0.9310. Furthermore, we enriched the transcriptional factor binding sites of proteins by convolution neural network. In summary, DeepHBV is a robust and explainable deep learning model not only for the prediction of HBV integration sites but also for the further study of HBV integration mechanism.


title: Family cluster comparisons to detect infection by Mycobacterium leprae in at-risk populations of six endemic regions in Colombia.

abstract:
IntroductionLeprosy is a chronic infectious disease, caused by Mycobacterium leprae, which is endemic in some tropical countries. It is necessary to implement strategies for its detection and elimination. We propose a strategy useful could be identifying risk factors associated with a seropositive test in leprosy.

ObjectiveWe aimed to quantify the infection rates and identify risk factors for M. leprae infection using the serological evaluations against NDO-LID in family clusters of leprosy patients, from regions with a high burden of leprosy in Colombia.

ResultsWe observed that belong to a low socioeconomic condition (OR 5.6 [95% IC 1.1-29]) and living in geographic area of residing such as Choco and Atlantico (OR 2 [95% IC 1.1-3.7]) could be risk factors related to infection by M. leprae between the members of a family cluster.

ConclusionsLeprosy is a persistent disease that affects vulnerable and large family clusters, in which the detection of antibodies against NDO-LID can be a useful tool for early detection of M. leprae in family clusters with high risk for this infection.

category: microbiology


title: FLAgellum Member 8 modulates extravascular trypanosome distribution in the mammalian host

abstract:
The African trypanosome flagellum is an essential organelle in multiple aspects of the parasites development. Here, we investigated the role of a flagellar protein termed FLAgellar Member 8 (FLAM8) that is specifically distributed along the entire flagellum in trypanosomes stages of the vertebrate host. Analyses of knockdown and knockout trypanosomes demonstrated that FLAM8 is not essential in vitro for survival, growth, motility and slender to stumpy differentiation. Functional investigations in experimental infections showed that FLAM8-deprived trypanosomes are able to establish and maintain the infection in the blood circulation, and to differentiate into insect transmissible forms. However, quantitative bioluminescence imaging revealed that FLAM8-null parasites exhibit an impaired dissemination in the extravascular compartment, that is partially restored by the addition of a single rescue copy of FLAM8. Interestingly, among all dissected organs scrutinized individually, only the skin of mice infected with FLAM8-deprived parasites showed a significant reduction in extravascular trypanosome population as compared to mice infected with parental controls. To our knowledge, FLAM8 is the first example of a flagellar protein that modulates T. brucei parasite distribution in the host tissues, contributing to the maintenance of extravascular parasite populations in mammalian anatomical niches, especially in the skin.

Take awayO_LIFLAM8 is dispensable in vitro for survival, growth, motility and differentiation of T. brucei.
C_LIO_LIFLAM8 depletion does not affect parasitemia and bloodstream form differentiation in vivo.
C_LIO_LIFLAM8 modulates the extravascular dissemination of trypanosomes in the mammalian host, especially in the skin.
C_LI


title: LuxT controls specific quorum-sensing-regulated behaviors in Vibrionaceae spp. via repression of qrr1, encoding a small regulatory RNA

abstract:
Quorum sensing (QS) is a process of chemical communication bacteria use to transition between individual and collective behaviors. QS depends on the production, release, and synchronous response to signaling molecules called autoinducers (AIs). The marine bacterium Vibrio harveyi monitors AIs using a signal transduction pathway that relies on five small regulatory RNAs (called Qrr1-5) that post-transcriptionally control target genes. Curiously, the small RNAs function redundantly making it difficult to understand the necessity for five of them. Here, we identify LuxT as a transcriptional repressor of qrr1. LuxT does not regulate qrr2-5, demonstrating that qrr genes can be independently controlled to drive unique downstream QS gene expression patterns. LuxT reinforces its control over the same genes it regulates indirectly via repression of qrr1, through a second transcriptional control mechanism. Genes dually regulated by LuxT specify public goods including an aerolysin-type pore-forming toxin. Phylogenetic analyses reveal that LuxT is conserved among Vibrionaceae and sequence comparisons predict that LuxT represses qrr1 in additional species. The present findings reveal that redundant QS regulatory RNAs can carry out both shared and unique functions to endow bacteria with plasticity in their output behaviors.

Author SummaryBacteria communicate and count their cell numbers using a process called quorum sensing (QS). In response to changes in cell density, QS bacteria alternate between acting as individuals and participating in collective behaviors. Vibrio harveyi is used as a model organism to understand QS-mediated communication. Five small RNAs lie at the heart of the V. harveyi QS system, and they regulate the target genes that underlie the QS response. The small RNAs function redundantly making it difficult to understand why V. harveyi requires five of them. Here, we discover a regulator, called LuxT, that exclusively represses the gene encoding one of the QS small RNAs. LuxT regulation of one QS small RNA enables unique control of a specific subset of QS target genes. LuxT is broadly conserved among Vibrionaceae. Our findings show how redundant regulatory components can possess both common and unique roles that provide bacteria with plasticity in their behaviors.


title: Cep55 regulation of PI3K/Akt signaling is required for neocortical development and ciliogenesis

abstract: Homozygous nonsense mutations in CEP55 are associated with several congenital malformations that lead to perinatal lethality suggesting that it plays a critical role in regulation of embryonic development. CEP55 has previously been studied as a critical regulator of cytokinesis predominantly in transformed cells and its deregulation is linked to carcinogenesis. However, its molecular functions during embryonic development in mammals have not been clearly defined. We have generated a Cep55 knockout (Cep55-/-) mouse model which demonstrated perinatal lethality associated with a wide range of neural defects. Focusing our analysis on the neocortex, we show that Cep55-/- embryos exhibited depleted neural stem/progenitor cells in the ventricular zone as a result of significantly increased cellular apoptosis. Mechanistically, we demonstrated that Cep55-loss downregulates the pGsk3{beta}/{beta}-Catenin/Myc axis in an Akt-dependent manner. The phenotype was recapitulated using human cerebral organoids and we could rescue the phenotype by inhibiting active Gsk3{beta}. Additionally, we show that Cep55-loss leads to a significant reduction of ciliated cells, highlighting its novel role in regulating ciliogenesis. Collectively, our findings demonstrate a critical role of Cep55 during brain development and provide mechanistic insights that may have important implications for genetic syndromes associated with Cep55-loss.


title: Comparative dissection of the peripheral olfactory system of the Chagas disease vectors Rhodnius prolixus and Rhodnius brethesi

abstract:
American trypanosomiasis or Chagas disease is thought to be transmitted by both domestic and sylvatic species of Triatominae. These haematophagous insects use sensory cues to find their vertebrate hosts. Among them, odorants have been shown to play a key role. Previous work revealed morphological differences in the sensory apparatus of sylvatic and domestic species of Triatomines, but to date a functional study of the olfactory system is not available. After examining the antennal sensilla with scanning electronic microscopy (SEM), we compared olfactory responses of the domestic Rhodnius prolixus and the sylvatic Rhodnius brethesi with an electrophysiological approach. In electroantennogram (EAG) recordings, we first show that the antenna of R. prolixus shows high responses to carboxylic acids, compounds found in their habitat and headspace of hosts. We then compared responses from olfactory sensory neurons (OSNs) housed in the grooved peg sensilla of both species as these are tuned to these compounds using single-sensillum recordings (SSR). In R. prolixus, the SSR responses revealed a narrower tuning breath than its sylvatic counterpart, with the latter showing responses to a broader range of chemical classes. Additionally, we observed significant differences between these two species in their response to particular volatiles, such as amyl acetate and butyryl chloride. In summary, the closely related, but ecologically differentiated R. prolixus and R. brethesi display distinct differences in their olfactory functions. Considering the ongoing rapid destruction of the natural habitat of sylvatic species and likely shifts towards environments shaped by humans, we expect that our results will contribute to the design of efficient vector control strategies in the future.

Author SummaryAmerican Tripanosomiasis, also known as Chagas disease, is a disease which no one speaks out, although there are up to eight million people infected worldwide. Its causative agent is the protozoan Tripanosoma cruzi which is transmitted by triatomine insects, alias kissing bugs. Several studies have highlighted the importance of olfaction for host-seeking behavior in these insects, which enables them to target their vertebrate hosts, and to get their vital blood meal, while infecting them at the same time. Vector control strategies have been the most efficient policy to combat the spread of Chagas disease by triatomine insects. However, recent changes in the natural habitats of these insects challenge their effectiveness, as species so far thought to be exclusive to sylvatic environments are now frequently found in peridomestic areas. In this context, to understand how sylvatic and domestic kissing bugs detect odors to locate their host and choose their habitats is highly relevant. In this study, we compare the olfactory system of the domestic kissing bug Rhodnius prolixus and its sylvatic counterpart Rhodnius brethesi at a morphological and functional level. We reveal that detection of host and habitat volatiles share many similarities, but also exhibit pronounced differences between both species.

category: ecology


title: Choosing optimal trigger points for ex situ, in toto conservation of single population threatened species

abstract: Many endangered species exist in only a single population, and almost all species that go extinct will do so from their last remaining population. Understanding how to best conserve these single population threatened species (SPTS) is therefore a distinct and important task for threatened species conservation science. As a last resort, managers of SPTS may consider taking the entire population into captivity - ex situ, in toto conservation. In the past, this choice has been taken to the great benefit of the SPTS, but it has also lead to catastrophe. Here, we develop a decision-support tool for planning when to trigger this difficult action. Our method considers the uncertain and ongoing decline of the SPTS, the possibility that drastic ex situ action will fail, and the opportunities offered by delaying the decision. Specifically, these benefits are additional time for ongoing in situ actions to succeed, and opportunities for the managers to learn about the system. To illustrate its utility, we apply the decision tool to four retrospective case-studies of declining SPTS. As well as offering support to this particular decision, our tool illustrates why trigger points for difficult conservation decisions should be formulated in advance, but must also be adaptive. A trigger-point for the ex situ, in toto conservation of a SPTS, for example, will not take the form of a simple threshold abundance.


title: RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea mays.

abstract:
In large complex plant genomes, RNA-directed DNA methylation (RdDM) ensures that epigenetic silencing is maintained at the boundary between genes and flanking transposable elements. In maize, RdDM is dependent on Modifer of Paramutation 1 (Mop1), a putative RNA dependent RNA polymerase. Here we show that although RdDM is essential for the maintenance of DNA methylation of a silenced MuDR transposon in maize, a loss of that methylation does not result in a restoration of activity. Instead, heritable maintenance of silencing is maintained by histone modifications. At one terminal inverted repeat (TIR) of this element, heritable silencing is mediated via H3K9 and H3K27 dimethylation, even in the absence of DNA methylation. At the second TIR, heritable silencing is mediated by H3K27 trimethylation, a mark normally associated with somatically inherited gene silencing. We find that a brief exposure of high temperature in a mop1 mutant rapidly reverses both of these modifications in conjunction with a loss of transcriptional silencing. These reversals are heritable, even in mop1 wild type progeny in which methylation is restored at both TIRs. These observations suggest that DNA methylation is neither necessary to maintain silencing, nor is it sufficient to initiate silencing once has been reversed. However, given that heritable reactivation only occurs in a mop1 mutant background, these observations suggest that DNA methylation is required to buffer the effects of environmental stress on transposable elements.

Author SummaryMost plant genomes are mostly transposable elements (TEs), most of which are held in check by modifications of both DNA and histones. The bulk of silenced TEs are associated with methylated DNA and histone H3 lysine 9 demethylation (H3K9me2). In contrast, epigenetically silenced genes are often associated with histone lysine 27 trimethylation (H3K27me3). Although stress can affect each of these modifications, plants are generally competent to rapidly reset them following that stress. Here we demonstrate that although DNA methylation is not required to maintain silencing of the MuDR element, it is essential for preventing heat-induced, stable and heritable changes in both H3K9me2 and H3K27me3 at this element, and for concomitant changes in transcriptional activity. These finding suggest that RdDM acts to buffer the effects of heat on silenced transposable elements, and that a loss of DNA methylation under conditions of stress can have profound and long lasting effects on epigenetic silencing in maize.


title: Distinguishing gene flow between malaria parasite populations

abstract: Measuring gene flow between malaria parasite populations in different geographic locations can provide strategic information for malaria control interventions. Multiple important questions pertaining to the design of such studies remain unanswered, limiting efforts to operationalize genomic surveillance tools for routine public health use. This report evaluates numerically the ability to distinguish different levels of gene flow between malaria populations, using different amounts of real and simulated data, where data are simulated using parameters that approximate different epidemiological conditions. Specifically, using Plasmodium falciparum whole genome sequence data and sequence data simulated for a metapopulation with different migration rates and effective population sizes, we compare two estimators of gene flow, explore the number of genetic markers and number of individuals required to reliably rank highly connected locations, and describe how these thresholds change given different effective population sizes and migration rates. Our results have implications for the design and implementation of malaria genomic surveillance efforts.

category: genomics


title: A 12 kb multi-allelic copy number variation encompassing a GC gene enhancer is associated with mastitis resistance in dairy cattle

abstract:
Clinical mastitis (CM) is an inflammatory disease occurring in the mammary glands of lactating cows. CM is under genetic control, and a prominent CM resistance QTL located on chromosome 6 was reported in various dairy cattle breeds. Nevertheless, the biological mechanism underpinning this QTL has been lacking. Herein, we mapped, fine-mapped, and discovered the putative causal variant underlying this CM resistance QTL in the Dutch dairy cattle population. We identified a ~12 kb multi-allelic copy number variant (CNV), that is in perfect linkage disequilibrium with a GWAS lead SNP, as a promising candidate variant. By implementing a genome-wide association study (GWAS) and through expression QTL mapping, we showed that the group-specific component gene (GC), a gene encoding a vitamin D binding protein, is an excellent candidate causal gene for the QTL. The multiplicated alleles are associated with increased GC expression and low CM resistance. Ample evidence from functional genomics data supports the presence of an enhancer within this CNV, which would exert cis-regulatory effect on GC. We observed that strong positive selection swept the region near the CNV, and haplotypes associated with the multiplicated allele were strongly selected for. Moreover, the multiplicated allele showed pleiotropic effects for increased milk yield and reduced fertility, hinting that a shared underlying biology for these effects may revolve around the vitamin D pathway. These findings together suggest a putative causal variant of a CM resistance QTL, where a cis-regulatory element located within a CNV can alter gene expression and affect multiple economically important traits.

Author summaryClinical mastitis (CM) is an inflammatory disease that negatively influences dairy production and compromises animal welfare. Although one major genetic locus for CM resistance was mapped on bovine chromosome 6, a mechanistic description of this association has been lacking. Herein, we report a 12-kb multiallelic copy number variant (CNV), encompassing a strong enhancer for group-specific component gene (GC), as a likely causal variant for this locus. This CNV is associated with high GC expression and low CM resistance. We speculate that upregulation of GC leads to a large amount of vitamin D binding protein, which in turn, reduces biologically available vitamin D, resulting in vitamin D deficiency and low CM resistance. Despite the negative effect on CM resistance, the CNV contributes to increased milk production, hinting at balancing selection. Our results highlight how multiplication of a regulatory element can shape economically important traits in dairy cattle, both in favourable and unfavourable directions.


title: Transposable element landscape changes are buffered by RNA silencing in aging Drosophila

abstract: Genetic mechanisms that repress transposable elements (TEs) in young animals decline during aging, as reflected by increased TE expression in aged animals. Does increased TE expression during aging lead to more genomic TE copies in older animals? To answer this question, we quantified TE Landscapes (TLs) via whole genome sequencing of young and aged Drosophila strains of wild-type and mutant backgrounds. We quantified TLs in whole flies and dissected brains and validated the feasibility of our approach in detecting new TE insertions in aging Drosophila genomes when natural defenses like RNA interference (RNAi) pathways are compromised. By also incorporating droplet digital PCR to validate genomic TE loads, we confirm TL changes can occur in a single lifespan of Drosophila when TEs are not suppressed. We also describe improved sequencing methods to quantify extra-chromosomal DNA circles (eccDNAs) in Drosophila as an additional source of TE copies that accumulate during aging. Lastly, to combat the natural progression of aging-associated TE expression, we show that knocking down PAF1, a conserved transcription elongation factor that antagonizes RNAi pathways, may bolster suppression of TEs during aging and extend lifespan. Our study suggests that RNAi mechanisms generally mitigate genomic TL expansion despite the increase in TE transcripts during aging.


title: Role of extracellular mycobacteria in blood-retinal barrier invasion in a zebrafish model of ocular TB

abstract: Intraocular inflammation following mycobacterial dissemination to the eye is common in tuberculosis (TB)-endemic countries. However, the early host-pathogen interactions during ocular dissemination are unknown. In this study, we investigate the early events during mycobacterial invasion of the blood-retinal barriers (BRB) with fluorescent tagged Mycobacterium marinum (Mm), host macrophages and retinal vasculature in a zebrafish model of ocular TB. We found that Mm invaded the vascular endothelium either in extracellular or intracellular (inside phagocytes) state, typically 3-4 days post-injection (dpi). Extracellular Mm are phagocytosed in the retinal tissue, and progress to form a compact granuloma around 6 dpi. Intracellular Mm crossing the BRB are likely to be less virulent, and either persist inside solitary macrophages (most cases), or progress to loosely arranged granuloma (rare). The early interactions between mycobacteria and host immune cells can thus determine the course of disease during mycobacterial dissemination to the eye.


title: Double drives and private alleles for localised population genetic control

abstract:
Synthetic gene drive constructs could, in principle, provide the basis for highly efficient interventions to control disease vectors and other pest species. This efficiency derives in part from leveraging natural processes of dispersal and gene flow to spread the construct and its impacts from one population to another. However, sometimes (for example, with invasive species) only specific populations are in need of control, and impacts on non-target populations would be undesirable. Many gene drive designs use nucleases that recognise and cleave specific genomic sequences, and one way to restrict their spread would be to exploit sequence differences between target and non-target populations. In this paper we propose and model a series of low threshold double drive designs for population suppression, each consisting of two constructs, one imposing a reproductive load on the population and the other inserted into a differentiated locus and controlling the drive of the first. Simple deterministic, discrete-generation computer simulations are used to assess the alternative designs. We find that the simplest double drive designs are significantly more robust to pre-existing cleavage resistance at the differentiated locus than single drive designs, and that more complex designs incorporating sex ratio distortion can be more efficient still, even allowing for successful control when the differentiated locus is neutral and there is up to 50% pre-existing resistance in the target population. Similar designs can also be used for population replacement, with similar benefits. A population genomic analysis of PAM sites in island and mainland populations of the malaria mosquito Anopheles gambiae indicates that the differentiation needed for our methods to work can exist in nature. Double drives should be considered when efficient but localised population genetic control is needed and there is some genetic differentiation between target and non-target populations.

Author summarySome disease vectors, invasive species, and other pests cannot be satisfactorily controlled with existing interventions, and new methods are required. Synthetic gene drive systems that are able to spread though populations because they are inherited at a greater-than-Mendelian rate have the potential to form the basis for new, highly efficient pest control measures. The most efficient such strategies use natural gene flow to spread a construct throughout a species range, but if control is only desired in a particular location then these approaches may not be appropriate. As some of the most promising gene drive designs use nucleases to target specific DNA sequences, it ought to be possible to exploit sequence differences between target and non-target populations to restrict the spread and impact of a gene drive. In this paper we propose using two-construct "double drive" designs that exploit pre-existing sequence differences between target and non-target populations. Our approaches maintain the efficiencies associated with only small release rates being needed and can work if the differentiated locus is selectively neutral and if the differentiation is far from complete, and therefore expand the range of options to be considered in developing genetic approaches to control pest species.

category: synthetic biology


title: CRY2 missense mutations suppress P53 and enhance cell growth

abstract: Disruption of circadian rhythms increases the risk of several types of cancer. Mammalian cryptochromes (CRY1 and CRY2) are circadian transcriptional repressors that are related to DNA repair enzymes. While CRYs lack DNA repair activity, they modulate the transcriptional response to DNA damage, and CRY2 can promote SCFFBXL3-mediated ubiquitination of c-MYC and other targets. Here, we characterize five mutations in CRY2 observed in human cancers in The Cancer Genome Atlas. We demonstrate that two orthologous mutations of mouse CRY2 (D325H and S510L) accelerate the growth of primary mouse fibroblasts expressing high levels of c-MYC. Neither mutant affects steady state levels of overexpressed c-MYC, and they have divergent impacts on circadian rhythms and on the ability of CRY2 to interact with SCFFBXL3. Unexpectedly, stable expression of either CRY2 D325H or of CRY2 S510L robustly suppresses P53 target gene expression, suggesting that this is the primary mechanism by which they influence cell growth.


title: The collapse of genetic incompatibilities in a hybridizing population

abstract: Diverging species are often genetically incompatible upon hybridization. Such incompatibilities are considered important in keeping the integrity of species from the disruption of hybrids. However, recent empirical work has shown that not all incompatibilities are gene-flow-proof, and they can collapse due to continuing hybridization. Counterintuitively, many studies found that incompatible alleles are already segregating within species, whereas they should go extinct quickly in a randomly mating population. Due to the complexity of multilocus epistasis, few general principles explain behaviors of incompatibilities under gene flow both within and between species. In the current work, we argue that the redundancy of genetic mechanisms can robustly determine the dynamics of intrinsic incompatibilities under gene flow. While higher genetic redundancy decreases the stability of incompatibilities during hybridization, it also increases the tolerance of incompatibility polymorphism within each species. We treat two general classes of incompatibilities. In the redundant class, similar to the classical Dobzhansky-Muller system, the collapse is continuous and eventually approaches quasi-neutral polymorphism between broadly-sympatric species, often as a result of isolation-by-distance. In the non-redundant class, analogous to the shifting-balance process, incompatibilities collapse abruptly with spatial traveling waves. We obtained simulated and analytical results for several incompatibility models to demonstrate the differences between the two classes. As both redundant and non-redundant genetic mechanisms of incompatibilities are common, the proposed conceptual framework may help understand the abundance of incompatibilities in natural populations.


title: The collapse of genetic incompatibilities in hybridizing populations


title: Genetic modulation of initial sensitivity to Δ9-tetrahydrocannabinol (THC) among the BXD family of mice

abstract: Cannabinoid receptor 1 activation by the major psychoactive component in cannabis, {Delta}9-tetrahydrocannabinol (THC), produces motor impairments, hypothermia, and analgesia upon acute exposure. In previous work, we demonstrated significant sex and strain differences in acute responses to THC following administration of a single dose (10 mg/kg, i.p.) in C57BL/6J (B6) and DBA/2J (D2) inbred mice. To determine the extent to which these differences are heritable, we quantified acute responses to a single dose of THC (10 mg/kg, i.p.) in males and females from 20 members of the BXD family of inbred strains derived by crossing and inbreeding B6 and D2 mice. Acute THC responses (initial sensitivity) were quantified as changes from baseline for: 1. spontaneous activity in the open field (mobility), 2. body temperature (hypothermia), and 3. tail withdrawal latency to a thermal stimulus (analgesia/antinociception). Initial sensitivity to the immobilizing, hypothermic, and antinociceptive effects of THC varied substantially across the BXD family. Heritability was highest for mobility and hypothermia traits, indicating that segregating genetic variants modulate initial sensitivity to THC. We identified genomic loci and candidate genes, including Ndufs2, Scp2, Rps6kb1 or P70S6K, Pde4d, and Pten, that may control variation in THC initial sensitivity. We also detected strong correlations between initial responses to THC and legacy phenotypes related to intake or response to other drugs of abuse (cocaine, ethanol, and morphine). Our study demonstrates the feasibility of mapping genes and variants modulating THC responses in the BXDs to systematically define biological processes and liabilities associated with drug use and abuse.


title: Distinct spermiogenic phenotypes underlie sperm elimination in the Segregation Distorter meiotic drive system

abstract: Segregation Distorter (SD) is a male meiotic drive system in Drosophila melanogaster. Males heterozygous for a selfish SD chromosome rarely transmit the homologous SD+ chromosome. It is well established that distortion results from an interaction between Sd, the primary distorting locus on the SD chromosome and its target, a satellite DNA called Rsp, on the SD+ chromosome. However, the molecular and cellular mechanisms leading to post-meiotic SD+ sperm elimination remain unclear. Here we show that SD/SD+ males of different genotypes but with similarly strong degrees of distortion have distinct spermiogenic phenotypes. In some genotypes, SD+ spermatids fail to fully incorporate protamines after the removal of histones, and degenerate during the individualization stage of spermiogenesis. In contrast, in other SD/SD+ genotypes, protamine incorporation appears less disturbed, yet spermatid nuclei are abnormally compacted, and mature sperm nuclei are eventually released in the seminal vesicle. Our analyses of different SD+ chromosomes suggest that the severity of the spermiogenic defects associates with the copy number of the Rsp satellite. We propose that when Rsp copy number is very high (> 2000), spermatid nuclear compaction defects reach a threshold that triggers a checkpoint controlling sperm chromatin quality to eliminate abnormal spermatids during individualization.


title: Chromosome-scale assembly of the Sparassis latifolia genome obtained using long-read and Hi-C sequencing

abstract:
Sparassis latifolia is a valuable edible mushroom cultivated in China. In 2018, our research group reported an incomplete and low quality genome of S. latifolia was obtained by Illumina HiSeq 2500 sequencing. These limitations in the available genome have constrained genetic and genomic studies in this mushroom resource. Herein, an updated draft genome sequence of S. latifolia was generated by Oxford Nanopore sequencing and the Hi-C technique. A total of 8.24 Gb of Oxford Nanopore long reads representing ~198.08X coverage of the S. latifolia genome were generated. Subsequently, a high-quality genome of 41.41 Mb, with scaffold and contig N50 sizes of 3.31 Mb and 1.51 Mb, respectively, was assembled. Hi-C scaffolding of the genome resulted in 12 pseudochromosomes containing 93.56% of the bases in the assembled genome. Genome annotation further revealed that 17.47% of the genome was composed of repetitive sequences. In addition, 13,103 protein-coding genes were predicted, among which 98.72% were functionally annotated. BUSCO assay results further revealed that there were 92.07% complete BUSCOs. The improved chromosome-scale assembly and genome features described here will aid further molecular elucidation of various traits, breeding of S. latifolia, and evolutionary studies with related taxa.

ImportanceSparassis latifolia Y.C. Dai et Z. Wang (Sparassidaceae, Polyporales, Agaricomycetes), collections from Asian Sparassis (Dai et al., 2006) exhibits diverse biological and pharmacologic activities. To date, total fresh fruit production in Chinese factories is over 20 tons/d. Despite the significant economic and medical value of S. latifolia, the assembly level of genome was under chromosome-scale. This study assembles a high-quality chromosome-scale reference genome of S. latifolia using Oxford Nanopore sequencing combined with Hi-C (High-through chromosome conformation capture) scaffolding. The improved reference genome will facilitate molecular breeding of S. latifolia and advance our understanding of its genetics and evolution.


title: Heparin-modified alginate microspheres enhance neovessel formation in hiPSC-derived endothelial cells and heterocellular in vitro models by controlled release of VEGF

abstract: A formidable challenge in regenerative medicine is the development of stable microvascular networks to restore adequate blood flow or to sustain graft viability and long-term function in implanted or ischemic tissues. In this work, we develop a biomimetic approach to increase the binding affinity of the extracellular matrix for the class of heparin-binding growth factors to localize and control the release of proangiogenic cues while maintaining their bioactivity. Sulfate and heparin moieties are covalently coupled to alginate, and alginate microspheres are produced and used as local delivery depots for vascular endothelial growth factor (VEGF). Release of VEGF from sulfate-alginate and heparin-alginate bulk hydrogels and microspheres was sustained over 14 days. In vitro evaluation with human induced pluripotent stem cell (hiPSC)-derived endothelial cells and aortic ring assay in a chemically-defined hydrogel demonstrates development of primitive three-dimensional vessel-like networks in the presence of VEGF released from the chemically modified alginate microspheres. Furthermore, our results suggest that the sulfate groups available on the chemically modified alginate microspheres promote some new vessel formation even in VEGF-free samples. Based on this evidence, we conclude that sulfate- and heparin-alginate hydrogels are adaptive and bioactive delivery systems for revascularization therapy and translational vascular tissue engineering.

category: bioengineering


title: AncestralClust: Clustering of Divergent Nucleotide Sequences by Ancestral Sequence Reconstruction using Phylogenetic Trees

abstract:
MotivationClustering is a fundamental task in the analysis of nucleotide sequences. Despite the exponential increase in the size of sequence databases of homologous genes, few methods exist to cluster divergent sequences. Traditional clustering methods have mostly focused on optimizing high speed clustering of highly similar sequences. We develop a phylogenetic clustering method which infers ancestral sequences for a set of initial clusters and then uses a greedy algorithm to cluster sequences.

ResultsWe describe a clustering program AncestralClust, which is developed for clustering divergent sequences. We compare this method with other state-of-the-art clustering methods using datasets of homologous sequences from different species. We show that, in divergent datasets, AncestralClust has higher accuracy and more even cluster sizes than current popular methods.

Availability and implementationAncestralClust is an Open Source program available at https://github.com/lpipes/ancestralclust.

Contactlpipes@berkeley.edu

Supplementary informationSupplementary figures and table are available online.


title: rdrugtrajectory: An R Package for the Analysis of Drug Prescriptions in Electronic Health Care Records

abstract: Primary care electronic health care records are rich with patient and clinical information. Studying electronic health care records has resulted in marked improvements to national health care processes and patient-care decision making, and is a powerful supplementary source of data for drug discovery effort. We present the R package rdrugtrajectory, designed to yield demographic and patient-level characteristics of drug prescriptions in the UK Clinical Practice Research Datalink dataset. The package operates over Clinical Practice Research Datalink Gold clinical, referral and therapy datasets and includes features such as first drug prescriptions analysis, cohort-wide prescription information, cumulative drug prescription events, the longitudinal trajectory of drug prescriptions, and a survival analysis timeline builder to identify risks related to drug prescription switching. The rdrugtrajectory package has been made freely available via the GitHub repository.


title: Semi-supervised Calibration of Risk with Noisy Event Times (SCORNET) Using Electronic Health Record Data

abstract: Leveraging large-scale electronic health record (EHR) data to estimate survival curves for clinical events can enable more powerful risk estimation and comparative effectiveness research. However, use of EHR data is hindered by a lack of direct event times observations. Occurrence times of relevant diagnostic codes or target disease mentions in clinical notes are at best a good approximation of the true disease onset time. On the other hand, extracting precise information on the exact event time requires laborious manual chart review and is sometimes altogether infeasible due to a lack of detailed documentation. Current status labels - binary indicators of phenotype status during follow up - are significantly more efficient and feasible to compile, enabling more precise survival curve estimation given limited resources. Existing survival analysis methods using current status labels focus almost entirely on supervised estimation, and naive incorporation of unlabeled data into these methods may lead to biased results. In this paper we propose Semi-supervised Calibration of Risk with Noisy Event Times (SCORNET), which yields a consistent and efficient survival curve estimator by leveraging a small size of current status labels and a large size of imperfect surrogate features. In addition to providing theoretical justification of SCORNET, we demonstrate in both simulation and real-world EHR settings that SCORNET achieves efficiency akin to the parametric Weibull regression model, while also exhibiting non-parametric flexibility and relatively low empirical bias in a variety of generative settings.


title: COMPARE, an ultra-fast and robust suite for multiparametric screening, identifies phenotypic drug responses in acute myeloid leukemia

abstract:
Multiparametric phenotypic screening of cells, for example assessing their responses to small molecules or knockdown/knockout of specific genes, is a powerful approach to understanding cellular systems and identifying potential new therapeutic strategies. However, automated tools for analyzing similarities and differences between a large number of tested conditions have not been readily available. Methods designed for clustering cells cannot identify differences between samples effectively. We introduce O_SCPLOWCOMPAC_SCPLOWRO_SCPLOWEC_SCPLOW for ultra-fast and robust analysis of multiparametric high-throughput screening. Applying a mass-aware gridding algorithm using hypercubes, O_SCPLOWCOMPAC_SCPLOWRO_SCPLOWEC_SCPLOW performs automatic and effective similarity comparison for hundreds to thousands of tests and provides information about the treatment effect. Particularly for screening data, O_SCPLOWCOMPAC_SCPLOWRO_SCPLOWEC_SCPLOW is equipped with modules to remove various sources of bias.

Benchmarking tests show that O_SCPLOWCOMPAC_SCPLOWRO_SCPLOWEC_SCPLOW can circumvent batch effects and perform a similarity analysis substantially faster than conventional analysis tools. Applying O_SCPLOWCOMPAC_SCPLOWRO_SCPLOWEC_SCPLOW to high-throughput flow cytometry screening data, we were able to distinguish subtle phenotypic drug responses in a human sample and a genetically engineered mouse model with acute myeloid leukemia (AML). O_SCPLOWCOMPAC_SCPLOWRO_SCPLOWEC_SCPLOW revealed groups of drugs with similar responses even though their mechanisms are distinct from each other. In another screening, O_SCPLOWCOMPAC_SCPLOWRO_SCPLOWEC_SCPLOW effectively circumvented batch effects and grouped samples from AML and myelodysplastic syndrome (MDS) patients using clinical flow cytometry data.


title: Anti-stress effects of the glucagon-like peptide-1 receptor agonist liraglutide in zebrafish

abstract: Stress-related disorders are extremely harmful and cause significant impacts on the individual and society. Despite the limited evidence regarding glucagon-like peptide-1 receptor (GLP-1R) and mental disorders, a few clinical and preclinical studies suggest that modulating this system could improve symptoms of stress-related disorders. This study aimed to investigate the effects of liraglutide, a GLP-1R agonist, on neurobehavioral phenotypes and brain oxidative status in adult zebrafish. Acute liraglutide promoted anxiolytic-like effects in the light/dark test, while chronic treatment blocked the impact of unpredictable chronic stress on behavioral and physiological parameters. Taken together, our study demonstrates that liraglutide is active on zebrafish brain and may counteract some of the effects induced by stress. More studies are warranted to further elucidate the potential of GLP-1R agonists for the management of brain disorders.

category: pharmacology and toxicology


title: Cofactor specificity of glucose-6-phosphate dehydrogenase isozymes in Pseudomonas putida reveals a general principle underlying glycolytic strategies in bacteria

abstract:
Glucose-6-phosphate dehydrogenase (G6PDH) is widely distributed in nature and catalyzes the first committing step in the oxidative branch of the pentose phosphate (PP) pathway, feeding either the reductive PP or the Entner-Doudoroff pathway. Besides its role in central carbon metabolism, this dehydrogenase also provides reduced cofactors, thereby affecting redox balance. Although G6PDH is typically considered to display specificity towards nicotinamide adenine dinucleotide phosphate (NADP+), some variants accept nicotinamide NAD+ similarly (or even preferentially). Furthermore, the number of G6PDH isozymes encoded in bacterial genomes varies from none to more than four orthologues. On this background, we systematically analyzed the interplay of the three G6PDH isoforms of the soil bacterium Pseudomonas putida KT2440 from a genomic, genetic and biochemical perspective. P. putida represents an ideal model to tackle this endeavor, as its genome encodes numerous gene orthologues for most dehydrogenases in central carbon metabolism. We show that the three G6PDHs of strain KT2440 have different cofactor specificities, and that the isoforms encoded by zwfA and zwfB carry most of the activity, acting as metabolic  gatekeepers for carbon sources that enter at different nodes of the biochemical network. Moreover, we demonstrate how multiplication of G6PDH isoforms is a widespread strategy in bacteria, correlating with the presence of an incomplete Embden-Meyerhof-Parnas pathway. Multiplication of G6PDH isoforms in these species goes hand-in-hand with low NADP+ affinity at least in one G6PDH isozyme. We propose that gene duplication and relaxation in cofactor specificity is an evolutionary strategy towards balancing the relative production of NADPH and NADH.

ImportanceProtein families have likely arisen during evolution by gene duplication and divergence followed by neo-functionalization. While this phenomenon is well documented for catabolic activities (typical of environmental bacteria that colonize highly polluted niches), the co-existence of multiple isozymes in central carbon catabolism remains relatively unexplored. We have adopted the metabolically-versatile soil bacterium Pseudomonas putida KT2440 as a model to interrogate the physiological and evolutionary significance of co-existing glucose-6-phosphate dehydrogenase (G6PDH) isozymes. Our results show that each of the three G6PDHs encoded in this bacterium display distinct biochemical properties, especially at the level of cofactor preference, impacting bacterial physiology in a carbon source-dependent fashion. Furthermore, the presence of multiple G6PDHs differing in NAD+- or NADP+-specificity in bacterial species strongly correlates with their predominant metabolic lifestyle. Our findings support the notion that multiplication of genes encoding cofactor-dependent dehydrogenases is a general evolutionary strategy towards achieving redox balance according to the growth conditions.


title: Host-pathogen Immune Feedbacks Can Explain Widely Divergent Outcomes from Similar Infections

abstract: A longstanding question in infection biology is why two very similar individuals, with very similar pathogen exposures, may have very different outcomes. Recent experiments have found that even isogenic Drosophila melanogaster hosts, given identical inoculations of some bacterial pathogens at suitable doses, can experience very similar initial bacteria proliferation but then diverge to either a lethal infection or a sustained chronic infection with much lower pathogen load. We hypothesized that divergent infection outcomes are a natural result of mutual negative feedbacks between pathogens and the host immune response. Here we test this hypothesis in silico by constructing process-based dynamic models for bacterial population growth, host immune induction, and the feedbacks between them, based on common mechanisms of immune system response. Mathematical analysis of a minimal conceptual model confirms our qualitative hypothesis that mutual negative feedbacks can magnify small differences among hosts into life-or-death differences in outcome. However, explaining observed features of chronic infections requires an extension of the model to include induced pathogen modifications that shield themselves from host immune responses at the cost of reduced proliferation rate. Our analysis thus generates new, testable predictions about the mechanisms underlying bimodal infection outcomes.


title: Caspase inhibition mitigates tau cleavage and neurotoxicity in iPSC-induced neurons with the V337M MAPT mutation

abstract:
Tau post-translational modifications (PTMs) are associated with progressive tau accumulation and neuronal loss in tauopathies, including forms of frontotemporal lobar degeneration (FTLD) and Alzheimers disease (AD). Proteolytic cleavage of tau by active caspases, including caspase-6, represents an underexplored tau PTM implicated in tau pathology. Caspase-cleaved tau is toxic and prone to self-aggregation in experimental models. To elucidate the presence and temporal course of caspase activation, tau cleavage, and neuronal death, we generated two neoepitope monoclonal antibodies (mAbs) against caspase-6 tau proteolytic sites and cortical neurons from induced pluripotent stem cells (iPSCs) with the frontotemporal dementia (FTD)-causing V337M MAPT mutation. FTLD V337M MAPT and AD postmortem brains showed positivity for both cleaved tau mAbs as well as active caspase-6. Relative to isogenic wild-type MAPT controls, V337M MAPT neurons showed a time-dependent increase in pathogenic tau in the form of tau oligomers, caspase-cleaved tau, and p-tau. Accumulation of toxic tau species in 3-month V337M MAPT neurons also increased vulnerability to stress, which was pharmacologically rescued by caspase inhibition. We propose a model in which time-dependent accumulation of caspase-cleaved tau in V337M MAPT neurons promotes neurotoxicity that is reversed by caspase-6 inhibition. Caspase-cleaved tau may be a biomarker of tauopathy, and caspases could be viable targets for therapeutic intervention against tau pathogenesis in FTLD and other tauopathies.

SignificanceThe mechanisms leading to tau pathology in frontotemporal dementia (FTD) and Alzheimers disease (AD) remain elusive. Experimental studies in AD demonstrate that tau cleavage by active caspase-6 contributes to tau pathology since cleaved tau may be toxic and prone to self-aggregation. Yet, the role of caspase-cleaved tau in promoting toxicity and cell death is unclear. Here, we generated two neoepitope monoclonal antibodies against caspase-6 tau and evaluated tau cleavage in postmortem human brains, iPSC-induced cortical neurons with the FTD-causing V337M MAPT mutation, and isogenic wild-type MAPT controls. Our results demonstrate a time-dependent accumulation of caspase-cleaved tau and increased neurotoxicity in the mutant iNs that is reversed by caspase-6 inhibition. Caspases could be viable therapeutic targets against tau pathology in tauopathies.


title: Effects of Ih and TASK-like shunting current on dendritic impedance in layer 5 pyramidal-tract neurons

abstract:
Pyramidal neurons in neocortex have complex input-output relationships that depend on their morphologies, ion channel distributions, and the nature of their inputs, but which cannot be replicated by simple integrate-and-fire models. The impedance properties of their dendritic arbors, such as resonance and phase shift, shape neuronal responses to synaptic inputs and provide intraneuronal functional maps reflecting their intrinsic dynamics and excitability. Experimental studies of dendritic impedance have shown that neocortical pyramidal tract neurons exhibit distance-dependent changes in resonance and impedance phase with respect to the soma. We therefore investigated how well several biophysically-detailed multi-compartment models of neocortical layer 5 pyramidal tract neurons reproduce the location-dependent impedance profiles observed experimentally. Each model tested here exhibited location-dependent impedance profiles, but most captured either the observed impedance amplitude or phase, not both. The only model that captured features from both incorporates HCN channels and a shunting current, like that produced by Twik-related acid-sensitive K+(TASK) channels. TASK-like channel activity in this model was dependent on local peak HCN channel conductance (Ih). We found that while this shunting current alone is insufficient to produce resonance or realistic phase response, it modulates all features of dendritic impedance, including resonance frequencies, resonance strength, synchronous frequencies, and total inductive phase. We also explored how the interaction of Ih and a TASK-like shunting current shape synaptic potentials and produce degeneracy in dendritic impedance profiles, wherein different combinations of Ih and shunting current can produce the same impedance profile.

New & NoteworthyWe simulated chirp current stimulation in the apical dendrites of 5 biophysically-detailed multi-compartment models of neocortical pyramidal tract neurons and found that a combination of HCN channels and TASK-like channels produced the best fit to experimental measurements of dendritic impedance. We then explored how HCN and TASK-like channels can shape the dendritic impedance as well as the voltage response to synaptic currents.


title: Human gut bacteria produce TH17-modulating bile acid metabolites

abstract: The microbiota plays a pivotal role in gut immune homeostasis. Bacteria influence the development and function of host immune cells, including T helper cells expressing interleukin-17a (TH17 cells). We previously reported that the bile acid metabolite 3-oxolithocholic acid (3-oxoLCA) inhibits TH17 cell differentiation1. While it was suggested that gut-residing bacteria produce 3-oxoLCA, the identity of such bacteria was unknown. Furthermore, it was not clear whether 3-oxoLCA and other immunomodulatory bile acids are associated with gut inflammatory pathologies in humans. Using a high-throughput screen, we identified human gut bacteria and corresponding enzymes that convert the secondary bile acid lithocholic acid into 3-oxoLCA as well as the abundant gut metabolite isolithocholic acid (isoLCA). Like 3-oxoLCA, isoLCA suppressed TH17 differentiation by inhibiting ROR{gamma}t (retinoic acid receptor-related orphan nuclear receptor {gamma}t), a key TH17 cell-promoting transcription factor. Levels of both 3-oxoLCA and isoLCA and the 3-hydroxysteroid dehydrogenase (3-HSDH) genes required for their biosynthesis were significantly reduced in patients with inflammatory bowel diseases (IBD). Moreover, levels of these bile acids were inversely correlated with expression of TH17 cell-associated genes. Overall, our data suggest that bacterially produced TH17 cell-inhibitory bile acids may reduce the risk of autoimmune and inflammatory disorders such as IBD.


title: Local Shifts in Inflammatory and Resolving Lipid Mediators in Response to Tendon Overuse

abstract: Tendon inflammation has been implicated in both adaptive connective tissue remodeling and overuse-induced tendinopathy. Lipid mediators control the initiation and resolution of inflammation, but their roles within tendon are largely unknown. Here we profiled local shifts in intratendinous lipid mediators via liquid chromatography-tandem mass spectrometry in response to synergist ablation-induced plantaris tendon overuse. Sixty-four individual lipid mediators were detected in homogenates of habitually loaded plantaris tendons from healthy ambulatory rats. This included many bioactive metabolites of the cyclooxygenase (COX), lipoxygenase (LOX), and epoxygenase (CYP) pathways. Synergist ablation induced a robust inflammatory response at day 3 post-surgery characterized by epitenon infiltration of polymorphonuclear leukocytes (PMNs) and macrophages (M{Phi}), heightened expression of inflammation-related genes, and increased intratendinous concentrations of the pro-inflammatory eicosanoids thromboxane B2 (TXB2) and prostaglandin E2 (PGE2). By day 7, M{Phi} became the predominant myeloid cell type in tendon and there were further delayed increases in other COX metabolites including PGD2, PGF2 and PGI2. Specialized pro-resolving mediators (SPMs) including protectin D1 (PD1) and resolvin D6 (RvD6), as well as related pathway markers of D-resolvins (17-HDoHE), E-resolvins (18-HEPE) and lipoxins (15-HETE) were also increased locally in response to tendon overuse, as were many anti-inflammatory fatty acid epoxides of the CYP pathway (e.g. EpETrEs). Nevertheless, intratendinous prostaglandins remained markedly increased even following 28 days of tendon overuse together with a lingering M{Phi} presence. These data reveal a delayed and prolonged local inflammatory response to tendon overuse characterized by an overwhelming predominance of pro-inflammatory eicosanoids and a relative lack of pro-resolving lipid mediators.


title: The effect of root exudates on the transcriptome of rhizosphere Pseudomonas spp.

abstract: Plants live in association with microorganisms that positively influence plant development, vigor, and fitness in response to pathogens and abiotic stressors. The bulk of the plant microbiome is concentrated belowground at the plant root-soil interface. Plant roots secrete carbon-rich rhizodeposits containing primary and secondary low molecular-weight metabolites, lysates, and mucilages. These exudates provide nutrients for soil microorganisms and modulate their affinity to host plants, but molecular details of this process are largely unresolved. We addressed this gap by focusing on the molecular dialogue between eight well-characterized beneficial strains of the Pseudomonas fluorescens group and Brachypodium distachyon, a model for economically important food, feed, forage, and biomass crops of the grass family. We collected and analyzed root exudates of B. distachyon and demonstrated the presence of multiple carbohydrates, amino acids, organic acids and phenolic compounds. The subsequent screening of bacteria by Biolog Phenotype MicroArrays revealed that many of these metabolites provide carbon and energy for the Pseudomonas strains. RNA-seq profiling of bacterial cultures amended with root exudates revealed changes in the expression of genes encoding numerous catabolic and anabolic enzymes, transporters, transcriptional regulators, stress response, and conserved hypothetical proteins. Almost half of the differentially expressed genes mapped to the variable part of the strains pangenome, reflecting the importance of the variable gene content in the adaptation of P. fluorescens to the rhizosphere lifestyle. Our results collectively reveal the diversity of cellular pathways and physiological responses underlying the establishment of mutualistic interactions between these beneficial rhizobacteria and their plant hosts.


title: Targeting the artemisinin resistant malaria by repositioning of the anti-Hepatitis C Virus drug Alisporivir

abstract: The rapid emergence of P. falciparum-resistant strains raises an urgent need to find new antimalarial drug candidates. This study reports the rational repositioning of the anti-Hepatitis C Virus drug, Alisporivir, a non-immunosuppressive analog of cyclosporin A (CsA) against multiple, drug-resistant strains of P. falciparum. Alisporivir being non-hemolytic has been proven to be a better drug than CsA. Indeed, our study also demonstrated the same. Alisporivir inhibited chloroquine-sensitive parasite growth with an IC50 of 196.6nM. Alisporivir also inhibited the growth of chloroquine-resistant parasites with an IC50 of 422.1nM. Alisporivir exhibited, anti-malarial activity in in vivo. Further, we exploited the Cyclophilins targeting potential of Alisporivir against artemisinin-resistant malaria parasite owing to the fact that PfCyP-19B is one of the genes that is overexpressed in artemisinin-resistant parasite revealed by a population transcriptomic study. Our semiquantitative real-time transcript and immunofluorescence analysis confirmed the overexpression of PfCyP-19B in Artemisinin-resistant P. falciparum (PfKelch13R539T). Artemisinin resistance is attributed to slow clearance of ring stage parasites. Ring survival assay (RSA) is designed to access the potency of compounds on these dormant slow clearing parasites leading to drug resistance. Thus, the potency of Alisporivir against PfKelch13R539T was evaluated by RSA. A 2.5-fold decrease in parasite survival was detected with Alisporivir. Further, combination of Alisporivir with DHA found to potentiate the efficacy of DHA by 4.55-fold. These results support the hypothesis that targeting of resistance mechanism is a potential approach to deal with resistant parasite. Overall, this study demonstrates the rational reposition of Alisporivir against resistant malaria resistance.


title: Biotechnological exploitation of Saccharomyces jurei and its hybrids in craft beer fermentation uncovers new aroma combinations.

abstract: Hybridization is an important evolutionary mechanism to bring about novel phenotypes and may produce new hybrids with advantageous combinations of traits of industrial importance. Within the Saccharomyces group, Saccharomyces jurei is a newly discovered species and its biotechnological potential has not yet been fully explored. This yeast was found to be able to grow well in synthetic wort and at low temperatures, qualities necessary in good candidates for fermentated bevarages. Here, we analysed its fermentation and aroma profile and created novel non-GMO hybrids between S. jurei and S. cerevisiae ale yeasts to develop new starter strains with interesting flavours for the craft beer industry. Pilot beer fermentations with specific hybrids showed a good fermentation performance, similar to the ale parent strain, while presenting a better sugar attenuation and a more complex flavour profile. This study exploits the genetic diversity of yeasts and show how inter-specific hybridisation and clone selection can be effectively used in brewing to create new products and to eliminate or increase specific traits.


title: Controlled spatial organization of bacterial clusters reveals cell filamentation is vital for Xylella fastidiosa biofilm formation

abstract: The morphological plasticity of bacteria to form filamentous cells commonly represents an adaptive strategy induced by stresses. In contrast, for diverse pathogens filamentous cells have been observed during biofilm formation, with function yet to be elucidated. To identify prior hypothesized quorum sensing as trigger of such cell morphogenesis, spatially controlled cell adhesion is pivotal. Here, we demonstrate highly-selective cell adhesion of the biofilm-forming phytopathogen Xylella fastidiosa to gold-patterned SiO2 substrates with well-defined geometries and dimensions. The consequent control of both cell density and distances between cell clusters using these patterns provided evidence of quorum sensing governing filamentous cell formation. While cell morphogenesis is induced by cell cluster density, filamentous cell growth is oriented towards neighboring cell clusters and distance-dependent; large interconnected cell clusters create the early biofilm structural framework. Together, our findings and investigative platform could facilitate therapeutic developments targeting biofilm formation mechanisms of X. fastidiosa and other pathogens.


title: Gut microbiome features are associated with sepsis onset and outcomes

abstract:
BackgroundEpidemiologic studies have linked antibiotic exposure to subsequent sepsis, suggesting that microbiome disruption may be in the causal pathway and an independent risk factor. This study tests whether variation in the gut microbiota associates with risk of sepsis onset and its outcomes.

MethodsUsing a validated surveillance definition, patients with an archived rectal swab from intensive care and hematology units were screened for sepsis. After confirmation by chart review, cases were matched to controls in a 1:2 ratio based on age, gender, and collection date. Relative taxon abundance was measured by sequence analysis of 16S rRNA gene amplicons; total bacterial abundance was measured by qPCR of the 23S rRNA gene. Conditional logistic regression identified clinical and microbiota variables associated with sepsis.

ResultsThere were 103 sepsis cases matched to 206 controls. In a final model adjusting for exposure to broad-spectrum antibiotics and indwelling vascular catheters, high relative abundance (RA) of Enterococcus (Odds Ratio (OR) 1.36 per 10% increase, P=.016) and high total bacterial abundance (OR 1.50 per 10-fold increase in 23S copies/L, P =.001) were independently associated with sepsis. Decreased RA of butyrate-producing bacteria also independently associated with sepsis (OR 1.20 for 10% decrease in RA, P =.041), and mortality in unadjusted analysis (OR=1.47 for 10% decrease in RA, P=.034).

ConclusionsThis study indicates that the microbiota is altered at sepsis onset. The decreased RA of butyrate-producing bacteria in sepsis also associates with mortality, suggesting a therapeutic role for prebiotics and probiotics in the prevention and treatment of sepsis.

ImportanceEarly detection of patients at risk for sepsis could enable interventions to prevent or rapidly treat this life-threatening condition. Prior antibiotic treatment is associated with sepsis, suggesting that disruption of the bacterial population in the gut (the intestinal microbiome) could be an important step leading to disease. To investigate this theory, we matched hospitalized patients with and without sepsis and characterized the patients microbiomes close to or at onset of sepsis. We found that several microbiome alterations, including having more total bacteria in the gut was associated with onset, regardless of prior antibiotic treatment. This signature of microbiome disruption brings us closer to identifying the biological causes of sepsis and could be used to develop new diagnostic tests to identify patients at risk of sepsis.