title: Cigarette smoke preferentially induces full length ACE2 exposure in primary human airway cells but does not alter susceptibility to SARS-CoV-2 infection

abstract: Cigarette smoking has multiple serious negative health consequences. However, the epidemiological relationship between cigarette smoking and SARS-CoV-2 infection is controversial; and the interaction between cigarette smoking, airway expression of the ACE2 receptor and the susceptibility of airway cells to infection is unclear. We exposed differentiated air-liquid interface cultures derived from primary human airway stem cells to cigarette smoke extract (CSE) and infected them with SARS-CoV-2. We found that CSE increased expression of full-length ACE2 (flACE2) but did not alter the expression of a Type I-interferon sensitive truncated ACE2 that lacks the capacity to bind SARS-CoV-2 or a panel of interferon-sensitive genes. Importantly, exposure to CSE did not increase viral infectivity despite the increase in flACE2. Our data are consistent with epidemiological data suggesting current smokers are not at excess risk of SARS-CoV-2 infection. This does not detract from public health messaging emphasising the excess risk of severe COVID-19 associated with smoking-related cardiopulmonary disease.


title: Mct11 deficiency alters hepatic glucose metabolism and energy homeostasis

abstract: Genetic variation at the SLC16A11 locus contributes to the disproportionate impact of type 2 diabetes (T2D) on Latino populations. We recently demonstrated that T2D risk variants reduce SLC16A11 liver expression and function of MCT11, the monocarboxylate transporter encoded by the SLC16A11 gene. Here, we show that SLC16A11 expression within the liver is primarily localized to the low oxygen pericentral region, and that T2D risk variants disrupt oxygen-regulated SLC16A11 expression in human hepatocytes. Under physiologic oxygen conditions, MCT11 deficiency alters hepatocyte glucose metabolism, resulting in elevated intracellular lactate and a metabolic shift toward triacylglycerol accumulation. We also demonstrate an impact of Mct11 deficiency on glucose and lipid metabolism in Slc16a11 knockout mice, which display physiological changes that are observed in individuals with T2D. Our findings provide mechanistic insight into how SLC16A11 disruption impacts hepatic energy metabolism and T2D risk, and highlight MCT11-mediated regulation of lactate levels as a potential therapeutic target.

category: genetics


title: A redox cycle with complex II promotes sulfide quinone oxidoreductase dependent H2S oxidation

abstract:
The dueling roles of H2S as an endogenously synthesized respiratory substrate and as a toxin, raise questions as to how it is cleared when the electron transport chain is inhibited. Sulfide quinone oxidoreductase (SQOR) is a mitochondrial inner membrane flavoprotein that catalyzes the first step in the H2S oxidation pathway and uses coenzyme Q (CoQ) as an electron acceptor. However, complex IV poisoning by H2S inhibits complex III-dependent recycling of CoQH2, which is needed to sustain H2S oxidation. We have discovered that under these conditions, reversal of complex II activity using fumarate as an electron acceptor, establishes a new redox cycle with SQOR. The purine nucleotide cycle and the malate aspartate shuttle are sources of fumarate in H2S treated cells, which accumulate succinate. Complex II knockdown decreases the efficiency of H2S clearance and increases recovery time to the basal respiration rate in H2S treated cells. In contrast, attenuation of complex I, which is a major competitor for the mitochondrial CoQ pool, has the opposite effects. Targeted knockout of complex II in murine intestinal epithelial cells that are routinely exposed to microbiota derived H2S, decreases serum, urine, and fecal thiosulfate, a product of H2S oxidation. Our study identifies a metabolic reprogramming response to H2S that furnishes fumarate as an alternate electron acceptor and supports H2S oxidation independent of complex IV activity. Complex II-linked redox cycling of SQOR has important implications for gut H2S metabolism as colonocytes are routinely exposed to high concentrations of this gas derived from the microbiota.

One Sentence SummaryReversal of complex II sustains and prioritizes H2S oxidation when respiration is poisoned.

category: biochemistry


title: Interplay between mitochondria and reactive oxygen and nitrogen species in metabolic adaptation to hypoxia in facioscapulohumeral muscular dystrophy: potential therapeutic targets.

abstract:
Facioscapulohumeral muscular dystrophy (FSHD) is characterised by descending skeletal muscle weakness and wasting. FSHD is caused by mis-expression of the transcription factor DUX4, which is linked to oxidative stress, a condition especially detrimental to skeletal muscle with its high metabolic activity and energy demands. Oxidative damage characterises FSHD and recent work suggests metabolic dysfunction and perturbed hypoxia signalling as novel pathomechanisms. However, redox biology of FSHD remains poorly understood, and integrating the complex dynamics of DUX4-induced metabolic changes is lacking.

Here we pinpoint the kinetic involvement of altered mitochondrial RONS metabolism and impaired mitochondrial function in aetiology of oxidative stress in FSHD. Transcriptomic analysis in FSHD muscle biopsies reveals strong enrichment for pathways involved in mitochondrial complex I assembly, nitrogen metabolism, oxidative stress response and hypoxia signalling. We found elevated ROS levels correlate with increases in steady-state mitochondrial membrane potential in FSHD myogenic cells. DUX4 triggers mitochondrial membrane polarisation prior to oxidative stress generation and apoptosis through mitochondrial ROS, and affects NO{middle dot} bioavailability via mitochondrial peroxidation. We identify complex I as the primary target for DUX4-induced mitochondrial dysfunction, with strong correlation between complex I-linked respiration and cellular oxygenation/hypoxia signalling activity in environmental hypoxia. Thus, FSHD myogenesis is uniquely susceptible to hypoxia-induced oxidative stress as a consequence of metabolic mis-adaptation. Importantly, mitochondria-targeted antioxidants rescue FSHD pathology more effectively than conventional antioxidants, highlighting the central involvement of disturbed mitochondrial RONS metabolism. This work provides a pathomechanistic model by which DUX4-induced changes in oxidative metabolism impair muscle function in FSHD, amplified when metabolic adaptation to varying O2 tension is required.

HighlightsO_LITranscriptomics data from FSHD muscle indicates enrichment for disturbed mitochondrial pathways
C_LIO_LIDisturbed RONS metabolism correlates with mitochondrial membrane polarisation and myotube hypotrophy
C_LIO_LIDUX4-induced changes in mitochondrial function precede oxidative stress through mitoROS and affect hypoxia signalling via complex I
C_LIO_LIFSHD is sensitive to environmental hypoxia, which increases ROS levels in FSHD myotubes
C_LIO_LIHypotrophy in hypoxic FSHD myotubes is efficiently rescued with mitochondria-targeted antioxidants
C_LI

Graphical abstract

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category: pathology


title: Characterization of the Endogenous DAF-12 Ligand and Its Use as an Anthelmintic Agent in Strongyloides stercoralis

abstract: A prevalent feature of Strongyloides stercoralis is a life-long and potentially lethal infection that is due to the nematode parasites ability to autoinfect and, thereby, self-replicate within its host. Here, we investigated the role of the parasites nuclear receptor, Ss-DAF-12, in governing infection. We identified {Delta}7-DA as the endogenous Ss-DAF-12 ligand and elucidated the hormones biosynthetic pathway. Genetic loss of function of the ligands rate-limiting enzyme demonstrated that {Delta}7-DA synthesis is necessary for parasite reproduction, whereas its absence is required for development of infectious larvae. Availability of the ligand permits Ss-DAF-12 to function as an on/off switch governing autoinfection, making it vulnerable to therapeutic intervention. In a preclinical model of hyperinfection, pharmacologic activation of DAF-12 suppressed autoinfection and markedly reduced lethality. Moreover, when {Delta}7-DA was administered with ivermectin, the current but limited drug of choice for treating strongyloidiasis, the combinatorial effects of the two drugs resulted in a near cure of the disease.

category: pharmacology and toxicology


title: High-Dimensional Gene Expression and Morphology Profiles of Cells across 28,000 Genetic and Chemical Perturbations

abstract: Populations of cells can be perturbed by various chemical and genetic treatments and the impact on the cells gene expression (transcription, i.e. mRNA levels) and morphology (in an image-based assay) can be measured in high dimensions. The patterns observed in this profile data can be used for more than a dozen applications in drug discovery and basic biology research, but both types of profiles are rarely available for large-scale experiments. We provide a collection of four datasets with both gene expression and morphological profile data useful for developing and testing multi-modal methodologies. Roughly a thousand features are measured for each of the two data types, across more than 28,000 thousand chemical and genetic perturbations. We define biological problems that can be investigated using the shared and complementary information in these two data modalities, provide baseline analysis and evaluation metrics for multi-omic applications, and make the data resource publicly available (http://broad.io/rosetta).

category: systems biology


title: Targeted isolation of panels of diverse human broadly neutralizing antibodies against SARS-like viruses

abstract: The emergence of current SARS-CoV-2 variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy 1-7. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed 8, 9. Notably, several recent studies have revealed that vaccination of recovered COVID-19 donors results in enhanced nAb responses compared to SARS-CoV-2 infection or vaccination alone 10-13. Here, we utilized a targeted donor selection strategy to isolate a large panel of broadly neutralizing antibodies (bnAbs) to sarbecoviruses from two such donors. Many of the bnAbs are remarkably effective in neutralization against sarbecoviruses that use ACE2 for viral entry and a substantial fraction also show notable binding to non-ACE2-using sarbecoviruses. The bnAbs are equally effective against most SARS-CoV-2 VOCs and many neutralize the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor binding domain (RBD) as opposed to strain-specific nAbs to the receptor binding site that are commonly elicited in SARS-CoV-2 infection and vaccination 14-18. Consistent with targeting of conserved sites, select RBD bnAbs exhibited in vivo protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model. The generation of a large panel of potent bnAbs provides new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and, importantly, provides a molecular basis for effective design of pan-sarbecovirus vaccines.

category: immunology


title: Targeted isolation of panels of diverse human protective broadly neutralizing antibodies against SARS-like viruses


title: Listeria monocytogenes TcyKLMN cystine/cysteine transporter facilitates glutathione synthesis and virulence gene expression

abstract:
Listeria monocytogenes (Lm) is a saprophyte and a human intracellular pathogen. Upon invasion into mammalian cells, it senses multiple metabolic and environmental signals that collectively trigger its transition to the pathogenic state. One of these signals is the tripeptide glutathione, which acts as an allosteric activator of Lms master virulence regulator, PrfA. While glutathione synthesis by Lm was shown to be critical for PrfA activation and virulence gene expression, it remains unclear how this tripeptide is synthesized under changing environments, especially in light of the observation that Lm is auxotrophic to one of its precursors, cysteine. Here, we show that the ABC transporter TcyKLMN is a cystine/cysteine importer that supplies cysteine for glutathione synthesis, hence mediating the induction of the virulence genes. Further, we demonstrate that this transporter is negatively regulated by three metabolic regulators: CodY, CymR and CysK, which sense and respond to changing concentrations of branched chain amino acids (BCAA) and cysteine. The data indicate that under low concentrations of BCAA, TcyKLMN is up-regulated, driving the production of glutathione by supplying cysteine, thereby facilitating PrfA activation. These findings provide molecular insight into the coupling of Lm metabolism and virulence, connecting BCAA sensing to cysteine uptake and glutathione biosynthesis, as a mechanism that controls virulence gene expression. This study exemplifies how bacterial pathogens sense their intracellular environment and exploit essential metabolites as effectors of virulence.

ImportanceBacterial pathogens sense the repertoire of metabolites in the mammalian niche and use this information to shift into a pathogenic state to accomplish successful infection. Glutathione is a virulence-activating signal that is synthesized by L. monocytogenes during infection of mammalian cells. In this study, we show that cysteine uptake via TcyKLMN drives glutathione synthesis and virulence gene expression. The data emphasize the intimate cross-regulation between metabolism and virulence in bacterial pathogens.

category: microbiology


title: Circadian patterns of energy metabolism are only partially reconstituted in gnotobiotic mice

abstract: The capacity of the intestinal microbiota to degrade otherwise indigestible diet components is known to greatly improve the recovery of energy from food. This has led to the hypothesis that increased digestive efficiency may underlie the contribution of the microbiome to obesity. OligoMM12-colonized gnotobiotic mice have a consistently higher fat-mass than germ-free or fully colonized counterparts. We therefore investigated their food intake, digestion efficiency, energy expenditure and respiratory quotient using a novel isolator-housed metabolic cage system which allows long-term measurements without contamination risk. This demonstrated that microbiota- released calories are perfectly balanced by decreased food intake in fully colonized versus gnotobiotic OligoMM12 and germ-free mice fed a standard chow diet, i.e., microbiota-released calories can in fact be well-integrated into appetite control. We also observed no significant difference in energy expenditure per gram lean mass between the different microbiota groups, suggesting that cumulative very small differences in energy balance, or altered energy storage must underlie fat accumulation in OligoMM12 mice. Consistent with altered energy storage, major differences were observed in the type of respiratory substrates used in metabolism over the circadian cycle: in germ-free mice the respiratory exchange ratio was consistently lower than that of fully colonized mice at all times of day, indicative of more reliance on fat and less on glucose metabolism. Intriguingly the RER of OligoMM12-colonized gnotobiotic mice phenocopied fully colonized mice during the dark (active/eating) phase but phenocopied germ-free mice during the light (fasting/resting) phase. Further, OligoMM12-colonized mice showed a germ-free-like drop in liver glycogen storage during the light cycle and both liver and plasma metabolomes of OligoMM12 mice clustered closely with germ-free mice. This implies the existence of microbiota functions that are required to maintain normal host metabolism during the resting/fasting phase of circadian cycle, and which are absent in the OligoMM12 consortium.


title: Metabolic reconstitution by a gnotobiotic microbiota varies over the circadian cycle


title: CRISPR-Cas12a induced DNA double-strand breaks are repaired by locus-dependent and error-prone pathways in a fungal pathogen

abstract: CRISPR-Cas mediated genome engineering has revolutionized functional genomics. However, basic questions remain regarding the mechanisms of DNA repair following Cas-mediated DNA cleavage. We developed CRISPR-Cas12a ribonucleoprotein genome editing in the fungal plant pathogen, Magnaporthe oryzae, and found frequent donor DNA integration despite the absence of long sequence homology. Interestingly, genotyping from hundreds of transformants showed that frequent non-canonical DNA repair outcomes predominated the recovered genome edited strains. Detailed analysis using sanger and nanopore long-read sequencing revealed five classes of DNA repair mutations, including single donor DNA insertions, concatemer donor DNA insertions, large DNA deletions, deletions plus donor DNA insertions, and infrequently we observed INDELs. Our results show that different error-prone DNA repair pathways resolved the Cas12a-mediated double-strand breaks (DSBs) based on the DNA sequence of edited strains. Furthermore, we found that the frequency of the different DNA repair outcomes varied across the genome, with some tested loci resulting in more frequent large-scale mutations. These results suggest that DNA repair pathways provide preferential repair across the genome that could create biased genome variation, which has significant implications for genome engineering and the genome evolution in natural populations.


title: An ultrasensitive microfluidic approach reveals correlations between the physico-chemical and biological activity of experimental peptide antibiotics

abstract: Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, peptide antibiotics hold particular promise. These agents hit multiple targets in bacteria starting with their most exposed regions - their membranes. However, suitable assays to quantify the efficacy of peptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure-activity relationships of two experimental series of peptide antibiotics. We reveal strong correlations between each peptides physicochemical activity at the membrane level and biological activity at the cellular level by assaying the membranolytic activities of the antibiotics on hundreds of individual giant lipid vesicles, and quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials.


title: Methods for the targeted sequencing and analysis of integrons and their gene cassettes from complex microbial communities

abstract: Integrons are bacterial genetic elements that can integrate mobile gene cassettes. They are mostly known for spreading antibiotic resistance cassettes among human pathogens. However, beyond clinical settings, gene cassettes encode an extraordinarily diverse range of functions important for bacterial adaptation. The recovery and sequencing of cassettes has promising applications, including: surveillance of clinically important genes, particularly antibiotic resistance determinants; investigating the functional diversity of integron-carrying bacteria; and novel enzyme discovery. Although gene cassettes can be directly recovered using PCR, there are no standardised methods for their amplification and, importantly, for validating sequences as genuine integron gene cassettes. Here, we present reproducible methods for the PCR amplification, sequence processing, and validation of gene cassette amplicons from complex communities. We describe two different PCR assays that either amplify cassettes together with integron integrases, or gene cassettes together within cassette arrays. We compare the use of Nanopore and Illumina sequencing, and present bioinformatic pipelines that filter sequences to ensure that they represent amplicons from genuine integrons. Using a diverse set of environmental DNAs, we show that our approach can consistently recover thousands of unique cassettes per sample and up to hundreds of different integron integrases. Recovered cassettes confer a wide range of functions, including antibiotic resistance, with as many as 300 resistance cassettes found in a single sample. In particular, we show that class 1 integrons appear to be collecting and concentrating antibiotic resistance genes out of the broader diversity of cassette functions. The methods described here can be applied to any environmental or clinical microbiome sample.


title: Pathogenic, but not non-pathogenic, Rickettsia manipulate inflammasome-dependent IL-1 responses to facilitate their replication and host dissemination.

abstract:
Rickettsia species (spp.) are strict obligate intracellular bacteria, with some being pathogenic in their mammalian host, including humans. One critical feature of these stealthy group of pathogens is their ability to manipulate hostile cytosolic environments to their benefits. Although our understanding of Rickettsia cell biology and pathogenesis are evolving, the mechanisms by which pathogenic Rickettsia spp. evade host innate immune detection remains elusive. Here, we showed that disease severity in wild-type (WT) C57BL/6J mice infected with R. typhi (etiologic agent of murine typhus) and R. rickettsii (etiologic agent of Rocky Mountain Spotted Fever), but not with non-pathogenic R. montanensis, correlated with levels of bacterial burden as detected in the spleens, as well as the serum concentrations of pro-inflammatory cytokine IL-1 and to a lesser extent IL- 1{beta}. Antibody-mediated neutralization of IL-1 confirmed a key role in controlling mortality rates and bacterial burdens of rickettsiae-infected WT mice. As macrophages are a primary source of both IL-1 and IL-1{beta} cytokines, we determined the mechanism of the anti-rickettsial activities using bone-marrow-derived macrophages. We found that pathogenic R. typhi and R. rickettsii, but not non-pathogenic R. montanensis, eluded pro- IL-1 induction and benefited pre-dominantly from the reduced IL-1 secretion, via a Caspase-11-Gsdmd-dependent pathway, to facilitate intracytosolic replication. Adoptative transfer experiments identified that IL-1 secretion by macrophages was critical for controlling rickettsiosis in WT mice. In sum, we identified a previously unappreciated pathway by which pathogenic, unlike non-pathogenic, rickettsiae preferentially target the Caspase-11-Gsdmd-IL-1 signaling axis in macrophages thus supporting their replication within the host.

IMPORTANCECurrently, no vaccines are available to prevent rickettsioses, while vector-borne rickettsial infections in humans are on the rise globally. In fact, the insufficient understanding of how pathogenic Rickettsia species circumvent host immune defense mechanisms has significantly hindered the development of more effective therapeutics. Here, we identified a previously unappreciated role for the Caspase-11-Gsdmd-IL-1 signaling axis, to limiting the replication of pathogenic R. rickettsia and R. typhi species in murine macrophages and wild-type (WT) C57BL/6J mice. Adoptative transfer studies further identified IL-1-secreting macrophages as critical mediators in controlling rickettsial infection in WT mice. Collectively, these findings provide insight into the potential mechanism of how pathogenic, but not non-pathogenic Rickettsia spp., benefit from a reduction in the Caspase-11-Gsdmd-mediated release of IL-1 to support host colonization.


title: Pathogenic, but not non-pathogenic, Rickettsia evade inflammasome-dependent IL-1 responses to establish an intracytosolic replication niche.


title: Artemisia annua hot-water extracts show potent activity in vitro against Covid-19 variants including delta

abstract:
Ethnopharmacological relevanceFor millennia in Southeast Asia, Artemisia annua L. was used to treat "fever". This medicinal plant is effective against numerous infectious microbial and viral diseases and is used by many global communities as a source of artemisinin derivatives that are first-line drugs to treat malaria.

Aim of the StudyThe SARS-CoV-2 (Covid-19) global pandemic has killed millions and evolved numerous variants, with delta being the most transmissible to date and causing break-through infections of vaccinated individuals. We further queried the efficacy of A. annua cultivars against new variants.

Materials and MethodsUsing Vero E6 cells, we measured anti-SARS-CoV-2 activity of dried-leaf hot-water A. annua extracts of four cultivars, A3, BUR, MED, and SAM, to determine their efficacy against five fully infectious variants of the virus: alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), and kappa (B.1.617.1).

ResultsIn addition to being effective against the original wild type WA1, A. annua cultivars A3, BUR, MED and SAM were also potent against all five variants. IC50 and IC90 values based on measured artemisinin content ranged from 0.3-8.4 M and 1.4-25.0 M, respectively. The IC50 and IC90 values based on dried leaf weight (DW) used to make the tea infusions ranged from 11.0-67.7 g DW and 59.5-160.6 g DW, respectively. Cell toxicity was insignificant at a leaf dry weight of [≤]50 g in the extract of any cultivar.

ConclusionsResults suggest that oral consumption of A. annua hot-water extracts (tea infusions), could provide a cost-effective therapy to help stave off the rapid global spread of these variants, buying time for broader implementation of vaccines.


title: Dysfunction of novel energy sensor NFE2L1 leads to uncontrollable AMPK signal and glucose metabolism reprogramming

abstract: NFE2L1 (also called Nrf1) acts a core regulator of redox signaling and metabolism homeostasis, and thus its dysfunction results in multiple systemic metabolic diseases. However, the molecular mechanism(s) by which NFE2L1 regulates glycose and lipid metabolism is still elusive. Here, we found that the loss of NFE2L1 in human HepG2 cells led to a lethal phenotype upon glucose deprivation. The uptake of glucose was also affected by NFE2L1 deficiency. Further experiments unveiled that although the glycosylation of NFE2L1 was monitored through the glycolysis pathway, it enabled to sense the energy state and directly interacted with AMPK. These indicate that NFE2L1 can serve as a dual sensor and regulator of glucose homeostasis. In-depth sights into transcriptome, metabolome and seahorse data further unraveled that glucose metabolism was reprogrammed by disruption of NFE2L1, so as to aggravate the Warburg effect in NFE2L1-silenced hepatoma cells, along with the mitochondrial damage observed under the electron microscope. Collectively, these demonstrate that disfunction of NFE2L1 triggers the uncontrollable signaling by AMPK towards glucose metabolism reprogramming in the liver cancer development.

category: molecular biology


title: Dysfunction of an energy sensor NFE2L1 triggers uncontrollable AMPK signal and glucose metabolism reprogramming


title: Integrating multiple single-cell phenotypes links stress acclimation to prior life history in yeast

abstract: Stress defense and cell growth are inversely related in bulk culture analyses; however, these studies do not capture cellular heterogeneity, thus obscuring true phenotypic relationships. Here, we devised a microfluidics system to characterize multiple phenotypes in single yeast cells responding dynamically to stress. We simultaneously followed cell and colony growth, cell size and volume, and cell-cycle phase plus nuclear trans-localization of two transcription factors: stress-responsive activator Msn2 and repressor Dot6 that are co-regulated during stress. Coordinated activation reflects a systemic stress response, whereas decoupled behavior indicates factor-specific responses. We scored these features before, during, and after salt stress. Modeling of multi-cell phenotypes revealed surprising new information, including unexpected discordance between Msn2 and Dot6 behavior that revealed subpopulations of cells with distinct growth properties. Although past work connected Msn2 activation to growth rate, we instead found stronger correlations with Dot6 behavior. Post-stress growth rate could be partly predicted by integrating multiple cellular phenotypes, with higher accuracy than considering any single feature alone. Our results underscore that life-history experiences partially predict how cells will respond to stress.


title: Host- and age-dependent transcriptional changes in Mycobacterium tuberculosis cell envelope biosynthesis genes after exposure to human alveolar lining fluid

abstract: Tuberculosis (TB) infection, caused by the airborne pathogen Mycobacterium tuberculosis (M.tb), resulted in almost 1.4 million deaths in 2019 and the number of deaths is predicted to increase by 20% over the next 5 years due to the COVID-19 pandemic. Upon reaching the alveolar space, M.tb comes in close contact with the lung mucosa before and after its encounter with host alveolar compartment cells. Our previous studies show that homeostatic innate soluble components of the alveolar lining fluid (ALF) can quickly alter the cell envelope surface of M.tb upon contact, defining subsequent M.tb-host cell interactions and infection outcomes in vitro and in vivo. We also demonstrated that ALF from 60+ year old elders (E-ALF) vs. healthy 18- to 45-year-old adults (A-ALF) is dysfunctional with loss of homeostatic capacity and impaired innate soluble responses linked to high local oxidative stress. In this study, a targeted transcriptional assay demonstrates that M.tb exposure to human ALF alters the expression of its cell envelope genes. Specifically, our results indicate that A-ALF-exposed M.tb upregulates cell envelope genes associated with lipid, carbohydrate, and amino acid metabolism, as well as genes associated with redox homeostasis and transcriptional regulators. Conversely, M.tb exposure to E-ALF shows lesser transcriptional response, with most of the M.tb genes unchanged or downregulated. Overall, this study indicates that M.tb responds and adapts to the lung alveolar environment upon contact, and that the host ALF status determined by factors such as age might play an important role in determining infection outcome.


title: Intercellular crosstalk regulating ARRB2/RARRES1 is involved in transition from fibrosis to cancer

abstract:
Progressive fibrogenesis in chronic liver injury is often associated with cancer development. Beta-arrestin-2 (ARRB2) is a regulator of the profibrotic Angiotensin II type 1 receptor (AGTR1). The role of ARRB2 in liver fibrosis and in the transition from fibrosis to cancer is not fully understood and was investigated in this study.

This study demonstrates that upregulation of the retinoic acid receptor responder 1 (RARRES1) in HSC mediated by ARRB2 leads to fibrosis. This process is driven by exosomal ARRB2 transfer to HSC, major fibrosis contributors, from injured hepatocytes, which highly express ARRB2. By contrast, downregulation of RARRES1 in hepatocytes induces malignant transformation and hepatocellular carcinoma (HCC) development. Consequently, Arrb2-deficient mice show higher number and size of liver tumors than wild-type mice in a hepatocellular carcinoma model with fibrosis. The identified relationship between ARRB2 and RARRES1 was observed in at least two species, including human cells and tissues in fibrosis and HCC and has a predictive value for survival in cancer patients. This study describes the discovery of a novel molecular pathway mediating the transition from fibrosis to cancer offering potential diagnostics and therapeutics.


title: A CRISPR-Cas assisted shotgun mutagenesis method for evolutionary genome engineering

abstract: Genome mutagenesis drives the evolution of organisms. Here, we developed a CRISPR-Cas assisted random mutation (CARM) technology for whole genome mutagenesis. The method leverages an entirely random gRNA library and SpCas9-NG to randomly damage genomes in a controllable shotgun-like manner that then triggers diverse and abundant mutations via low-fidelity repair. As a proof-of-principle, CARM was applied to evolve the capacity of Saccharomyces cerevisiae BY4741 to produce {beta}-carotene. After seven rounds of iterative evolution over two months, a {beta}-carotene hyper-producing strain, C7-143, was isolated with a 10.5-fold increase in {beta}-carotene production and 857 diverse genomic mutants that comprised indels, duplications, inversions, and chromosomal rearrangements. Transcriptomic analysis revealed that the expression of 2,541 genes of strain C7-143 were significantly altered, suggesting that the metabolic landscape of the strain was deeply reconstructed. In addition, CARM was applied to evolve the industrially relevant Saccharomyces cerevisiae CEN.PK2-1C, the S-adenosyl-L-methionine production of which was increased to 2.28 times after just one round. Thus, CARM is a user-friendly and practical strategy for genetic remodeling and reverse engineering to investigate complicated organismal metabolism.

category: evolutionary biology


title: Deep sampling of ancestral genetic diversity reveals Saccharomyces cerevisiae pre-domestication life histories

abstract: The ecology and genetic diversity of model yeast Saccharomyces cerevisiae prior to human domestication remain poorly understood. Taiwan is regarded as part of this yeasts geographic birthplace where the most divergent natural lineage was discovered. Here, we deep sampled the broad-leaf forests across this continental island to probe the ancestral species diversity. We found that S. cerevisiae is distributed ubiquitously at low abundance in the forests. Whole-genome sequencing of 121 isolates revealed nine distinct lineages, the highest known in any region. Three lineages are endemic to Taiwan and six are widespread in Asia. Molecular dating placed the divergence of the Taiwanese and Asian lineages during the Pleistocene, when a transient continental shelf land bridge connected Taiwan to other major landmasses. Extensive historical and recent admixture events were detected between natural lineages. In particular, the genetic component from a lineage associated with fruits that spanned the widest geographical range was present in most admixed isolates. Collectively, Taiwanese isolates harbor genetic diversity comparable to that of the whole Asia continent, and different lineages have coexisted at a fine spatial scale even on the same tree. Patterns of variations within each lineage revealed that S. cerevisiae is highly clonal and predominantly reproduces asexually in nature. We detected prevalent purifying selection genome-wide, with lineage-specific signals of positive or directional selection independent between lineages. This study establishes that S. cerevisiae has rich natural diversity sheltered from human influences, making it a powerful model system in microbial ecology.

category: genomics


title: Semisynthesis of functional transmembrane proteins in GUVs

abstract: Cellular transmembrane (TM) proteins are essential sentries of the cell facilitating cell-cell communication, internal signaling, and solute transport. Reconstituting functional TM proteins into model membranes remains a challenge due to the difficulty of expressing hydrophobic TM domains and the required use of detergents. Herein, we use a intein-mediated ligation strategy to semisynthesize bitopic TM proteins in synthetic membranes. We have adapted the trans splicing capabilities of split inteins for a native peptide ligation between a synthetic TM peptide embedded in the membrane of giant unilamellar vesicles (GUVs) and an expressed soluble protein. We demonstrate that the extracellular domain of programmed cell death protein 1 (PD-1), a mammalian transmembrane immune checkpoint receptor, retains its function for binding its ligand PD-L1 at a reconstituted membrane interface after ligation to a synthetic TM peptide in GUV membranes. We envision that the construction of full-length TM proteins using orthogonal split intein-mediated semisynthetic protein ligations will expand applications of membrane protein reconstitution in pharmacology, biochemistry, biophysics, and artificial cell development.


title: A systematic analysis of diet-induced nephroprotection reveals overlapping and conserved changes in cysteine catabolism

abstract: Caloric Restriction (CR) extends lifespan and augments cellular stress-resistance from yeast to primates, making CR an attractive strategy for organ protection in the clinic. Translation of CR to patients is complex, due to problems regarding adherence, feasibility and safety concerns in frail patients. Novel tailored dietary regimens, which modulate the dietary composition of macro- and micronutrients rather than reducing calorie intake promise similar protective effects and increased translatability. However, a direct head-to-head comparison to identify the most potent approach for organ protection as well as overlapping metabolic consequences has not been performed. We systematically analyzed six dietary preconditioning protocols - fasting mimicking diet (FMD), ketogenic diet (KD), dietary restriction of branched chained amino acids (BCAA), two dietary regimens restricting sulfur-containing amino acids (SR80/100) and CR - in a rodent model of renal ischemia-reperfusion injury (IRI) to quantify diet-induced resilience in kidneys. Of the administered diets, FMD, SR80/100 and CR efficiently protect from kidney damage after IRI. Interestingly, these approaches show overlapping changes in oxidative and hydrogen sulfide (H2S)-dependent cysteine catabolism as a potential common mechanism of organ protection. Importantly, these metabolic changes can be recapitulated in patients preconditioned by a diet limiting sulfur-containing amino acids indicating conserved diet-induced mechanisms of stress-resistance that may ultimately lead to clinical application.


title: Interleukin-6 Signaling Mediates Cartilage Degradation and Pain in Post-Traumatic Osteoarthritis

abstract:
Osteoarthritis (OA) and post-traumatic OA (PTOA) are prevalent joint disorders and leading causes of chronic pain. The disease pathology of OA/PTOA is caused by imbalanced catabolic and anabolic responses and pro-inflammatory changes; however, their connection to pain is not well studied. Since IL-6 is involved in cartilage degradation and conditions of inflammatory pain, we set out to identify whether IL-6 and IL-6 signaling mechanisms contribute to both PTOA-associated cartilage degradation and pain. We performed a modified destabilization of the medial meniscus (DMM) surgery, a model of PTOA, on conventional IL-6 KO and control mice and assessed both cartilage degradation and pain-associated phenotypes. Genetic removal of Il6 in males attenuates PTOA-associated cartilage catabolism, decreases innervation of soft tissues associated with the knee joint, and reduces nociceptive pain signaling, without improving subchondral bone sclerosis or chondrocyte apoptosis. We further demonstrate that specific downstream mediators of IL-6 signaling, the Janus kinases (JAKs), are critical in regulating both cartilage catabolism and pain signaling. We identified STAT3 as a key regulator of cartilage catabolism downstream of JAK; however, inhibition of STAT3 decreases cartilage anabolism while enhancing pain signals. ERK was found to be important for neurite outgrowth and pain signaling; however, inhibition of ERK was less effective in reducing cartilage catabolism. Therefore, our data demonstrate that IL-6 mediates both PTOA-associated cartilage degradation and pain, and provides critical details regarding the downstream mediators of IL-6 signaling as therapeutic targets for disease-modifying osteoarthritis drugs.

Single Sentence Summary

IL-6 mediates PTOA-associated cartilage degradation and pain via specific downstream signaling mechanisms in a gender specific manner.


title: Classification of antimicrobial mechanism of action using bacterial time-lapse imaging

abstract: Antimicrobial resistance is a major threat to human health. Basic knowledge of antimicrobial mechanism of action (MoA) is imperative for patient care and for identification of novel antimicrobials. However, the process of antimicrobial MoA identification is relatively laborious. Here, we developed a simple, quantitative time-lapse fluorescence imaging method, Dynamic Bacterial Morphology Imaging (DBMI), to facilitate this process. It uses a membrane dye and a nucleoid dye to track the morphological changes of single Bacillus subtilis cells in response to antimicrobials for up to 60 min. DBMI of bacterial cells facilitated assignment of the MoAs of 14 distinct, known antimicrobial compounds to the five main classes. Using this method, we found that the poorly studied antimicrobial, harzianic acid, a secondary metabolite that we purified from the fungal culture of Oidiodendron flavum, targets the cell envelope. We conclude that DBMI is a simple method, which facilitates rapid classification of the MoA of antimicrobials in functionally distinct classes.


title: Time-lapse imaging of bacterial morphology facilitates classification of antimicrobial agents


title: Disruption of fish gut microbiota composition and holobiont metabolome by cyanobacterial blooms

abstract:
BackgroundCyanobacterial blooms are one of the most common stress encountered by metazoans living in freshwater lentic systems such as lakes and ponds. Blooms reportedly impair fish health, notably through oxygen depletion and production of bioactive compounds including cyanotoxins. However, in the times of the "microbiome revolution", it is surprising that so little is still known regarding the influence of blooms on fish microbiota. In this study, an experimental approach is used to demonstrate that blooms affect fish microbiome composition and functions, as well as the metabolome of holobionts. To this end, the model teleost Oryzias latipes is exposed to simulated Microcystis aeruginosa blooms of various intensities in a microcosm setting, and the response of bacterial gut communities is evaluated in terms of composition, metagenome-encoded functions and metabolome profiling.

ResultsThe gut bacterial community of O. latipes exhibits marked responses to the presence of M. aeruginosa blooms in a dose-dependent manner. Notably, abundant gut-associated Firmicutes almost disappear, while potential opportunists increase. The holobionts gut metabolome displays major changes, while functions encoded in the metagenome of bacterial partners are more marginally affected. Bacterial communities tend to return to original composition after the end of the bloom suggesting post-bloom resilience, and remain sensitive in case of a second bloom, reflecting a highly reactive gut community.

ConclusionIn the context of increasingly frequent and intense blooms worldwide, results point to the relevance of accounting for short- and long-term microbiome-related effects in fish ecology, with potential outcomes relevant to conservation biology as well as aquaculture.


title: SARS-CoV-2 expresses a microRNA-like small RNA able to selectively repress host genes

abstract:
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease (COVID-19), continues to be a pressing health concern. In this study, we investigated the impact of SARS-CoV-2 infection on host microRNA (miRNA) populations in three human lung-derived cell lines, as well as in nasopharyngeal swabs from SARS-CoV-2 infected individuals. We did not detect any major and consistent differences in host miRNA levels after SARS-CoV-2 infection. However, we unexpectedly discovered a viral miRNA-like small RNA, named vmiR-5p (for viral miRNA), derived from the SARS-CoV-2 ORF7a transcript. Its abundance ranges from low to moderate as compared to host miRNAs. vmiR-5p functionally associates with Argonaute proteins -- core components of the RNA interference pathway -- leading to downregulation of host transcripts. One such host messenger RNA encodes Basic Leucine Zipper ATF-Like Transcription Factor 2 (BATF2), which is linked to interferon signaling. We demonstrate that vmiR-5p production relies on cellular machinery, yet is independent of Drosha protein, and is enhanced by the presence of a strong and evolutionarily conserved hairpin formed within the ORF7a sequence.

Significance statementWe discovered that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses a small viral non-coding RNA, named vmiR-5p (for viral miRNA), derived from the ORF7a transcript. vmiR-5p associates with the cellular RNA interference machinery to regulate host transcripts likely via target silencing. The production of vmiR-5p relies on cellular machinery and the formation of a strong hairpin within ORF7a sequences. This newly-described vmiR-5p may contribute to SARS-CoV-2 pathogenesis and could become a target for therapeutic intervention.


title: The spliced leader RNA silencing (SLS) pathway in Trypanosoma brucei is induced by perturbations of endoplasmic reticulum, Golgi, or mitochondrial proteins factors and functional analysis of SLS inducing kinase, PK3

abstract: In the parasite Trypanosoma brucei, the causative agent of human African sleeping sickness, all mRNAs are trans-spliced to generate a common 5 exon derived from the spliced leader RNA (SL RNA). Perturbations of protein translocation across the endoplasmic reticulum (ER) induce the spliced leader RNA silencing (SLS) pathway. SLS activation is mediated by a serine-threonine kinase, PK3, which translocates from the cytosolic face of the ER to the nucleus, where it phosphorylates the TATA binding protein TRF4, leading to the shut-off of SL RNA transcription, followed by induction of programmed cell death. Here, we demonstrate that SLS is also induced by depletion of the essential ER resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi-localized quiescin sulfhydryl oxidase (QSOX1). Most strikingly, silencing of Rhomboid-like 1(TIMRHOM1) involved in mitochondrial protein import, also induces SLS. The PK3 kinase, which integrates SLS signals, is modified by phosphorylation on multiple sites. To determine which of the phosphorylation events activate PK3, several individual mutations or their combination were generated. These mutations failed to completely eliminate the phosphorylation or translocation of the kinase to the nucleus. The structure of PK3 kinase and its ATP binding domain were therefore modeled. A conserved phenylalanine at position 771 was proposed to interact with ATP, and the PK3F771L mutation completely eliminated phosphorylation under SLS, suggesting that the activation involves most if not all the phosphorylation sites. The study suggests that the SLS occurs broadly in response to failures in protein sorting, folding, or modification across multiple compartments.


title: Efficient transgenesis and homology-directed gene targeting in monolayers of primary human small intestinal and colonic epithelial stem cells

abstract:
Background & Aims2D monolayers of primary intestinal and colonic epithelial cells represent next-generation in vitro models of the gut. Efficient transgenesis and gene-editing in human intestinal stem cells (hISCs) would significantly improve utility of these models by enabling generation of reporter and loss/gain-of-function hISCs, but no published methods exist for transfecting 2D hISC monolayers. Electroporation has proven effective in other difficult-to-transfect cells; thus we applied this method to hISCs.

MethodsTwenty-four electroporation parameters were tested, and the optimal condition for efficiency and viability was validated on hISCs from six anatomical regions along the small intestine and colon. PiggyBac transposase and Cas9 ribonucleoprotein (RNP) complexes were used for stable genomic integration of reporter genes. High-throughput methods for clone isolation, expansion, and screening were developed. An hISC OLFM4-emGFP reporter was generated and validated by qPCR, organoid assays, and hISC compartmentalization on a planar crypt-microarray (PCM) device.

ResultsMaximum electroporation efficiency was 79.9% with a mean survival of 65%. Transfection of 105 hISCs produced [~]142 (0.14%) stable transposase-mediated clones. Transfection of OLFM4-targetting RNPs yielded [~]35% editing and 99/220 (45%) of antibiotic-resistant colonies analyzed expressed emGFP. OLFM4-emGFP hISCs applied to PCMs remained emGFP+ and proliferative in high-Wnt3a/R-spondin3/Noggin zones yet differentiated to emGFP-/KRT20+ cells outside engineered crypt zones. OLFM4-emGFP levels correlated with endogenous OLFM4. Olfm4-emGFPhigh cells were LGR5high/KRT20low, and demonstrated high organoid-forming potential.

ConclusionsElectroporation of hISCs is highly efficient for stable transgenesis and transgenic lines can be generated in 3-4 weeks. Workflows mirror conventional culture methods, facilitating rapid integration into established tissue-culture operations. OLFM4high is a robust hISC marker with functional properties in culture.


title: Homocysteine-induced endoplasmic reticulum stress activates FGF21 via CREBH, resulting in browning and atrophy of white adipose tissue in Bhmt knockout mice.

abstract: Betaine-homocysteine methyltransferase (BHMT) catalyzes the transfer of methyl-groups from betaine to homocysteine (Hcy) producing methionine and dimethylgycine. In this work, we characterize Bhmt wildtype (WT) and knockout (KO) mice that were fully backcrossed to a C57Bl6/J background. Consistent with our previous findings, Bhmt KO mice had decreased body weight, fat mass and adipose tissue weight compared to WT. Histological analyses and gene expression profiling indicate that adipose browning was activated in KO mice and contributed to the adipose atrophy observed. BHMT is not expressed in adipose tissue but is abundant in liver, thus, a signal must be originating from the liver that modulates adipose tissue. We found that, in Bhmt KO mice, homocysteine-induced endoplasmic reticulum (ER) stress, with activation of hepatic transcription factor cyclin AMP response element binding protein (CREBH), mediated an increase in hepatic and plasma concentrations of fibroblast growth factor 21 (FGF21), which is known to induce adipose browning. CREBH binds to the promoter regions of FGF21 to activate its expression. Taken together, our data indicate that deletion of a single gene in one-carbon metabolism modifies adipose biology and energy metabolism. It would be interesting to determine whether people with functional polymorphisms in BHMT exhibit a similar adipose atrophy phenotype.

category: cell biology


title: Polycomb group protein CBX7 represses cardiomyocyte proliferation via modulation of the TARDBP/Rbm38 axis

abstract: Cardiomyocyte (CM) proliferation notably decreases during the perinatal period. At present, regulatory mechanisms for this loss of proliferative capacity is poorly understood. CBX7, a polycomb group (PcG) protein, regulates the cell cycle but its role in CM proliferation is unknown. Here, we report that CBX7 inhibits proliferation of perinatal CMs by controlling TARDBP/Rbm38 pathway. Gene expression profiling demonstrated that CBX7 expression in the heart was low during the prenatal period, abruptly increased during the perinatal period, and sustained constantly throughout the adulthood. CBX7, when overexpressed via adenoviral transduction in neonatal CMs, reduced proliferation and promoted multinucleation of the CMs. Mutant mice carrying targeted inhibition of CBX7 in CMs exhibited cardiomegaly with increased proliferation of CMs at postnatal stages. Mechanistically, CBX7 interacted with TAR DNA-binding protein 43 (TARDBP) and positively regulated its downstream target, RNA Binding Motif Protein 38 (RBM38). Rbm38 was upregulated in the postnatal hearts and overexpression of RBM38 reduced proliferation of neonatal CMs. Together, this study provides a novel insight into the role of CBX7 in regulation of CM proliferation during the perinatal period.

category: developmental biology


title: Developing an ethanol utilisation pathway based NADH regeneration system in Escherichia coli

abstract: Many industrially relevant biotransformation in whole-cells are dependent on cofactors such as NADH or NADPH. Cofactor regeneration is an established approach for providing a cheap source of cofactors in support of the main biotransformation reaction in biocatalysis. In essence, cofactor regeneration uses a sacrificial substrate to help regenerate a cofactor consumed by the main biotransformation reaction. Enzymatic in nature, alternative cofactor regeneration systems with high efficiency and which utilises low cost sacrificial substrate are of interest. Glucose dehydrogenase system has been dominant in NADH regeneration. But, in its current incarnation, glucose dehydrogenase system is relatively inefficient in regenerating NADH with theoretical yield of one NADH per glucose molecule. This work sought to explore the utility of a two-gene ethanol utilisation pathway in NADH regeneration. Comprising the first step that takes ethanol to acetaldehyde, and a second step that converts acetaldehyde to acetyl-CoA, NADH from both steps could be mined for supporting biotransformation reaction in cofactor regeneration mode. Theoretically, ethanol utilisation pathway (EUP) affords a higher NADH yield of two NADH per ethanol molecule, and is therefore more efficient than glucose dehydrogenase (GDH) system. In this project, the EUP pathway was coupled to a cpsADH (an alcohol dehydrogenase from Candida parapsilosis) mediated ketone to alcohol anaerobic biotransformation with concentration of alcohol product as marker for efficiency of cofactor regeneration. Experiment tests showed that EUP was more efficient than GDH. Further, EUP could support biotransformation of both butanone and acetophenone in single and two-phase biotransformation, respectively. Additional work conducted to improve biotransformation efficiency revealed that ethanol provision positively correlated with biotransformation efficiency. Growing cell biotransformation was also found to improve biotransformation efficiency compared to resting cell due largely to the driving force generated by cell growth. Tests of a growth medium effect also found that cells cultivated in M9 ethanol medium delivered higher biotransformation efficiency compared to those cultivated in LB medium. This could arise due to the lower expression of NADH dependent enzymes during growth in M9 ethanol medium compared to LB medium that allowed more NADH to be diverted to support ketone biotransformation. However, a persistent problem with the experimental system is the relatively poor consumption of ethanol that points to need for further engineering of the system. Collectively, pathway-based NADH regeneration is possible with ethanol utilisation, with biotransformation efficiency dependent on mode of biotransformation (resting cell versus growing cell) and growth medium used.

category: bioengineering


title: Off-target expression of Cre-dependent adeno-associated viruses in wild type C57BL/6J mice

abstract:
Adeno-associated viruses (AAVs) are a commonly used tool in neuroscience to efficiently label, trace, and/or manipulate neuronal populations. Highly specific targeting can be achieved through recombinase-dependent AAVs in combination with transgenic rodent lines that express Cre-recombinase in specific cell types. Visualization of viral expression is typically achieved through fluorescent reporter proteins (e.g., GFP or mCherry) packaged within the AAV genome. Although non-amplified fluorescence is usually sufficient to observe viral expression, immunohistochemical amplification of the fluorescent reporter is routinely used to improve viral visualization. In the present study, Cre-dependent AAVs were injected into the hippocampus and cortex of wild-type C57BL/6J mice. While we observed weak but consistent non-amplified off-target DIO expression in C57BL/6J mice, antibody amplification of the GFP or mCherry reporter revealed extensive Cre-independent viral expression. Off-target expression of DIO constructs in wild-type C57BL/6J mice occurred independent of vendor, AAV serotype or promoter. We also evaluated whether Cre-independent expression had functional effects via Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). The DREADD agonist C21 had no effect on contextual fear conditioning or cFos expression in DIO-hM3Dq-mCherry+ cells of C57BL/6J mice. Taken together, our results indicate that DIO constructs have considerable off-target expression in wild type subjects. Our findings are particularly important for the design of experiments featuring sensitive systems and/or quantitative measurements that could be negatively impacted by off-target expression.

Significance StatementAdeno-associated viruses (AAV) are widely used in neuroscience because of their safety and ease of use. Combined with specific promoters, Cre/loxP, and stereotaxic injections, highly specific targeting of cells and circuits within the brain can be achieved. In the present study we injected Cre-dependent AAVs into wild-type C57BL/6J mice and found considerable Cre-independent viral expression of AAVs encoding mCherry, GFP, or hM3Dq following immunohistochemical amplification of the fluorescent reporter protein. Importantly, we observed no functional effects of the Cre-independent expression in the hippocampus, as C21 had no detectable effect on DIO-hM3Dq-mCherry infected neurons in C57BL/6J mice. Given the widespread use of DIO rAAVs by the neuroscience community, our data supports careful consideration when using DIO constructs in control animals.

category: neuroscience


title: Land Use Cover changes in the western escarpment of Rift Valley in the Gamo Zone, Southern Ethiopia

abstract: LULC changes are caused by natural and human alterations of the landscape that could largely affect forest biodiversity and the environment. The aim of the study was to analyzed LULC change dynamics in the western escarpment of the rift valley of the Gamo Zone, Southern Ethiopia. Digital satellite images downloaded from USGS were analyzed using ERDAS Imagine (14) and Arc GIS 10.2 software and supervised image classification was used to generate LULC classification, accuracy assessment and Normalized Difference Vegetation Index (NDVI). Drivers of LULC change were identified and analyzed. Four land classes were identified such as forest, farmland, settlement and water-wetland. Settlement and farmlands have increased by 7.83% and 5.88%, respectively. On the other hand, both forest and water bodies and wetland decreased by aerial coverage of 11.03% and 2.68%, respectively. The overall accuracy of the study area was 92.86%, 94.22% and 94.3% with a kappa value of 0.902, 0.92 and 0.922, respectively. NDVI values ranged between -0.42 to 0.73. Agricultural expansion (31.4%), expansion of settlement (25.7%) and Fuelwood collection and Charcoal production (22.9%) were the main driving forces that jeopardize forest biodiversity of the study area. Integrated land use and policy to protect biodiversity loss, forest degradation and climate changes are deemed necessary.

category: ecology


title: Maternal SMCHD1 regulates Hox gene expression andpatterning in the mouse embryo

abstract: Parents transmit genetic and epigenetic information to their offspring. Maternal effect genes regulate the offspring epigenome to ensure normal development. Here we report that the epigenetic regulator SMCHD1 has a maternal effect on Hox gene expression and skeletal patterning. Maternal SMCHD1, present in the oocyte and preimplantation embryo, prevents precocious activation of Hox genes postimplantation. Without maternal SMCHD1, highly penetrant posterior homeotic transformations occur in the embryo. Hox genes are decorated with Polycomb marks H2AK119ub and H3K27me3 from the oocyte throughout early embryonic development; however, loss of maternal SMCHD1 does not alter these marks. Therefore, we propose maternal SMCHD1 acts downstream of Polycomb marks to establish a chromatin state necessary for persistent epigenetic silencing and appropriate Hox gene expression later in the developing embryo. This is a striking role for maternal SMCHD1 in long-lived epigenetic effects impacting offspring phenotype.


title: Whole-genome sequence analysis unveils different origins of European and Asiatic mouflon and domestication-related genes in sheep

abstract: The domestication and subsequent development of sheep are crucial events in the history of human civilization and the agricultural revolution. However, the impact of interspecific introgression on the genomic regions under domestication and subsequent selection remains unclear. Here, we analyze the whole genomes of domestic sheep and all their wild relative species. We found introgression from wild sheep such as the snow sheep and its American relatives (bighorn and thinhorn sheep) into urial, Asiatic and European mouflons. We observed independent events of adaptive introgression from wild sheep into the Asiatic and European mouflons, as well as shared introgressed regions from both snow sheep and argali into Asiatic mouflon before or during the domestication process. We revealed European mouflons arose through hybridization events between a now extinct sheep in Europe and feral domesticated sheep around 6,000 - 5,000 years BP. We also unveiled later introgressions from wild sheep to their sympatric domestic sheep after domestication. Several of the introgression events contain loci with candidate domestication genes (e.g., PAPPA2, NR6A1, SH3GL3, RFX3 and CAMK4), associated with morphological, immune, reproduction or production traits (wool/meat/milk). We also detected introgression events that introduced genes related to nervous response (NEURL1), neurogenesis (PRUNE2), hearing ability (USH2A) and placental viability (PAG11 and PAG3) to domestic sheep and their ancestral wild species from other wild species.


title: Spinal reconsolidation engages non-ionotropic NMDA receptor signaling to reverse pain hypersensitivity

abstract: Reconsolidation enables the activity-dependent modification of memory traces and has been used to reverse addiction, fear memory, and pain hypersensitivity in animal models. We demonstrate that non-ionotropic NMDA receptor signalling in the spinal dorsal horn is sufficient to reverse pain hypersensitivity and necessary for pain modulation by spinal reconsolidation. These findings reveal a key process by which reconsolidation disrupts memory traces that may be exploited in the treatment of pain and other disorders.


title: eQTLs are key players in the integration of genomic and transcriptomic data for phenotype prediction

abstract:
Multi-omics represent a promising link between phenotypes and genome variation. Few studies yet address their integration to understand genetic architecture and improve predictability. Our study used 241 poplar genotypes, phenotyped in two common gardens, with their xylem and cambium RNA sequenced at one site, yielding large phenotypic, genomic and transcriptomic datasets. For each trait, prediction models were built with genotypic or transcriptomic data and compared to concatenation integrating both omics. The advantage of integration varied across traits and, to understand such differences, we made an eQTL analysis to characterize the interplay between the genome and the transcriptome and classify the predicting features into CIS or TRANS relationships. A strong and significant negative correlation was found between the change in predictability and the change in predictor importance for eQTLs (both TRANS and CIS effects) and CIS regulated transcripts, and mostly for traits showing beneficial integration and evaluated in the site of transcriptomic sampling. Consequently, beneficial integration happens when redundancy of predictors is decreased, leaving the stage to other less prominent but complementary predictors. An additional GO enrichment analysis appeared to corroborate such statistical output. To our knowledge, this is a novel finding delineating a promising way to explore data integration.

One-sentence summarySuccessful multi-omics integration when predicting phenotypes makes redundant the predictors that are linked to ubiquitous connections between the omics, according to biological and statistical approaches


title: Interpretable deep learning for chromatin-informed inference of transcriptional programs driven by somatic alterations across cancers

abstract: Cancer is a disease of gene dysregulation, where cells acquire somatic and epigenetic alterations that drive aberrant cellular signaling. These alterations adversely impact transcriptional programs and cause profound changes in gene expression. Interpreting somatic alterations within context-specific transcriptional programs will facilitate personalized therapeutic decisions but is a monumental task. Toward this goal, we develop a partially interpretable neural network model called Chromatin-informed Inference of Transcriptional Regulators Using Self-attention mechanism (CITRUS). CITRUS models the impact of somatic alterations on transcription factors and downstream transcriptional programs. Our approach employs a self-attention mechanism to model the contextual impact of somatic alterations. Furthermore, CITRUS uses a layer of hidden nodes to explicitly represent the state of transcription factors (TFs) to learn the relationships between TFs and their target genes based on TF binding motifs in the open chromatin regions of tumor samples. We apply CITRUS to genomic, transcriptomic, and epigenomic data from 17 cancer types profiled by The Cancer Genome Atlas. CITRUS predicts patient-specific TF activities and reveals transcriptional program variations between and within tumor types. We show that CITRUS yields biological insights into delineating TFs associated with somatic alterations in individual tumors. Thus, CITRUS is a promising tool for precision oncology.

category: bioinformatics


title: Experimental characterization and automatic identification of stridulatory sounds inside wood

abstract: The propagation of animal vocalizations in water and in air is a well-studied phenomenon, but sound produced by bark and wood boring insects, which feed and reproduce inside trees, is poorly understood. Often being confined to the dark and chemically-saturated habitat of wood, many bark- and woodborers have developed stridulatory mechanisms to communicate acoustically. Despite their ecological and economic importance and the unusual medium used for acoustic communication, very little is known about sound production in these insects, or their acoustic interactions inside trees. Here, we use bark beetles (Scolytinae) as a model system to study the effects of wooden tissue on the propagation of insect stridulations and propose algorithms for their automatic identification. We characterize distance-dependence of the spectral parameters of stridulatory sounds, propose data-based models for the power decay of the stridulations in both outer and inner bark, provide optimal spectral ranges for stridulation detectability, and develop automatic methods for their detection and identification. We also discuss the acoustic discernibility of species cohabitating the same log. The species tested can be acoustically identified with 99% of accuracy at distances up to 20 cm and detected to the greatest extent in the 2-6 kHz frequency band. Phloem was a better medium for sound transmission than bark.

category: biophysics


title: Simultaneous Cardiac and Neurological Monitoring to Assess Chemical Exposures and Drug Toxicity in Xenopus Laevis

abstract: Simultaneous monitoring of electrocardiogram (ECG) and electroencephalogram (EEG) under chemical exposure requires innovative engineering techniques that can capture minute physiological changes in studied animal models. However, this is often administered with a bulky system that may cause signal distortions and discomfort for animals. We develop an integrated bioelectronic sensing system to provide simultaneous ECG and EEG assessment in real-time under chemical exposure for Xenopus laevis. The microelectrode array (MEA) membrane with integrated ECG and EEG sensing offers an opportunity to achieve multichannel noninvasive electrophysiological monitoring with favorable dimensions and spatial resolution. To validate the performance of our system, we assessed the ECG and EEG of Xenopus under exposure of Pentylenetetrazol (PTZ), an epilepsy-inducing drug. Effects of PTZ were detected with clear ECG and EEG alterations, including frequent ictal and interictal EEG events, 30 dB average EEG amplitude elevations, abnormal ECG morphology, and heart rate changes. Overall, our Xenopus-based real-time electrophysiology monitoring system holds high potential for many applications in drug screening and remote environmental toxicity monitoring.

category: physiology


title: LIFE-Seq: A universal Large Integrated DNA Fragment Enrichment Sequencing strategy for transgene integration in genetically modified organisms

abstract: Molecular characterisation of genetically modified organisms (GMOs) yields basic information on exogenous DNA integration, including integration sites, entire inserted sequences and structures, flanking sequences and copy number, providing key data for biosafety assessment. However, there are few effective methods for deciphering transgene integration, especially for large DNA fragment integration with complex rearrangement, inversion, and tandem repeats. Herein, we developed a universal Large Integrated DNA Fragments Enrichment strategy combined with PacBio Sequencing (LIFE-Seq) for deciphering transgene integration in GMOs. Universal tilling DNA probes targeting transgenic elements and exogenous genes facilitate specific enrichment of large inserted DNA fragments associated with transgenes from plant genomes, followed by PacBio sequencing. LIFE-Seq were evaluated using six GM events and four crop species. Target DNA fragments averaging [~]6275 bp were enriched and sequenced, generating [~]26,352 high fidelity reads for each sample. Transgene integration structures were determined with high repeatability and sensitivity. Compared with whole-genome sequencing, LIFE-Seq achieved better data integrity and accuracy, greater universality, and lower cost, especially for transgenic crops with complex inserted DNA structures. LIFE-Seq could be applied in molecular characterisation of transgenic crops and animals, and complex DNA structure analysis in genetics research.

category: plant biology


title: Concurrent Multimodal Data Acquisition During Brain Scanning is within Reach

abstract:
BackgroundPrevious brain-scanning research exploring the neural mechanisms underpinning visuomotor planning and control has mostly been done without simultaneous motion-tracking and eye-tracking. Employing concurrent methodologies would enhance understanding of the brain mechanisms underlying visuomotor integration of cognitive, visual, ocular, and motor aspects of reaching and grasping behaviours. Therefore, this work presents the methods and validation for a high-speed, multimodal and synchronized system to holistically examine neural processes that are involved in visually-guided movement.

MethodsThe multimodal methods included high speed 3D motion tracking (Qualisys), 2D eye-tracking (SR Research), and magnetoencephalography (MEG; Elekta) that were synchronized to millisecond precision. Previous MRIs were taken to provide improved spatial localization. The methods section describes the system layout and acquisition parameters to achieve multimodal synchronization. Pilot results presented here are preliminary data from a larger study including 29 participants. Using a pincer grip, five people (3 male, 2 female, ages 30-32) reached for and grasped a translucent dowel 50 times, after it was pseudorandomly illuminated. The object illumination was the Go cue. Seven discrete time points (events) throughout the task were chosen for investigation of simultaneous brain, hand and eye activity associated with specific visual (Go cue), oculomotor (1st saccade after Go), motor (Reaction Time; RT, Maximum Velocity: MV, Maximum Grip Width; MGW) or cognitive (Ready, End) mechanisms. Time-frequency analyses were performed on the MEG data sourced from the left precentral gyrus to explore task-related changes time-locked to these chosen events.

Pilot resultsBasic kinematic parameters including RT, MV, MGW, Movement Time, and Total Time were similar to previous, seminal research by Castiello, Paulignan and Jeannerod, (1991), using a similar task. Although no gaze instructions were given, eye-tracking results indicated volunteers mostly gazed at or near the target object when Ready (72%), and then hardly looked away throughout the rest of the task at the important events sampled here (92% - 98%). At the End event, when lifting the dowel, on average, participants gazed at or near the target object 100% of the time. Although saccades > 100 ms after Go, but prior to RT were made on average in about one fourth (M = 13, SD = 6) of trials, a mixed model (REML) indicated their latency in timing after the Go was significantly (F = 13.376, p = .001) associated with RT scores on those trials (AIC = 724, R m2 = 0.407, Rc2= 0.420). Neural activity relative to baseline in the beta band was desynchronized for the visually guided reach periods, beginning prior to Go, and remaining sustained until beyond End, after the grasp and lift were executed.

ConclusionThis study presents the layout, acquisition parameters and validation for a multimodal, synchronized system designed to record data from the hand, eye and brain simultaneously, with millisecond precision during an ecologically-valid prehension task with physical, 3D objects. The pilot results align with previous research made with single or bimodal data recordings. This multimodal method enables full-brain modelling that can holistically map the precise location and timing of neural activity involved in the visual, oculomotor, motor and cognitive aspects of reach-to-grasp planning and control.


title: Beyond traditional visual sleep scoring: massive feature extraction and unsupervised clustering of sleep time series

abstract: Sleep is classically measured with electrophysiological recordings, which are then scored based on guidelines tailored for the visual inspection of these recordings. As such, these rules reflect a limited range of features easily captured by the human eye and do not always reflect the physiological changes associated with sleep. Here we present a novel analysis framework that characterizes sleep using over 7700 time-series features from the hctsa software. We used clustering to categorize sleep epochs based on the similarity of their features, without relying on established scoring conventions. The resulting structure overlapped substantially with that defined by visual scoring and we report novel features that are highly discriminative of sleep stages. However, we also observed discrepancies as hctsa features unraveled distinctive properties within traditional sleep stages. Our framework lays the groundwork for a data-driven exploration of sleep and the identification of new signatures of sleep disorders and conscious sleep states.


title: CaPTure: Calcium Peak Toolbox for analysis of in vitro calcium imaging data

abstract:
BackgroundCalcium imaging is a powerful technique for recording cellular activity across large populations of neurons. However, analysis methods capable of single-cell resolution in cultured neurons, especially for cultures derived from human induced pluripotent stem cells (hiPSCs), are lacking. Existing methods lack scalability to accommodate high-throughput comparisons between multiple lines, across developmental timepoints, or across pharmacological manipulations.

ResultsWe developed a scalable, automated Ca2+ imaging analysis pipeline called CaPTure (https://github.com/LieberInstitute/CaPTure). This method detects neurons, classifies and quantifies spontaneous activity, quantifies synchrony metrics, and generates cell- and network-specific metrics that facilitate phenotypic discovery. The method is compatible with parallel processing on computing clusters without requiring significant user input or parameter modification.

ConclusionCaPTure allows for rapid assessment of neuronal activity in cultured cells at cellular resolution, rendering it amenable to high-throughput screening and phenotypic discovery. The platform can be applied to both human- and rodent-derived neurons and is compatible with many imaging systems.


title: EEG-Based Spectral Analysis ShowingBrainwave Changes Related to ModulatingProgressive Fatigue During a ProlongedIntermittent Motor Task

abstract: Repeatedly performing a submaximal motor task for a prolonged period of time leads to muscle fatigue comprising a central and peripheral component, which demands a gradually increasing effort. However, the brain contribution to the enhancement of effort to cope with progressing fatigue lacks a complete understanding. The intermittent motor tasks (IMTs) closely resemble many activities of daily living (ADL), thus remaining physiologically relevant to study fatigue. The scope of this study is therefore to investigate the EEG-based brain activation patterns in healthy subjects performing IMT until self-perceived exhaustion. Fourteen participants (median age 51.5 years; age range 26-72 years; 5 males) repeated elbow flexion contractions at 40% maximum voluntary contraction by following visual cues displayed on an oscilloscope screen until subjective exhaustion. Each contraction lasted for approximately 5 s with a 2-s rest between trials. The force, EEG, and surface EMG (from elbow joint muscles) data were simultaneously collected. After preprocessing, we selected a subset of trials at the beginning, middle, and end of the study session representing brain activities germane to mild, moderate, and severe fatigue conditions, respectively, to compare and contrast the changes in the EEG time-frequency (TF) characteristics across the conditions. The outcome of channel- and source-level TF analyses reveals that the theta, alpha, and beta power spectral densities vary in proportion to fatigue levels in cortical motor areas. We observed a statistically significant change in the band-specific spectral power in relation to the graded fatigue from both the steady- and post-contraction EEG data. The findings would enhance our understanding on the etiology and physiology of voluntary motor-action-related fatigue and provide pointers to counteract the perception of muscle weakness and lack of motor endurance associated with ADL. The study outcome would help rationalize why certain patients experience exacerbated fatigue while carrying out mundane tasks, evaluate how clinical conditions such as neurological disorders and cancer treatment alter neural mechanisms underlying fatigue in future studies, and develop therapeutic strategies for restoring the patients ability to participate in ADL by mitigating the central and muscle fatigue.


title: Genome-wide transcriptional profiling uncovers a similar oligodendrocyte-related transcriptional response to acute and chronic alcohol drinking in the amygdala

abstract:
Alcohol intake progressively increases after prolonged consumption of alcohol, but relatively few new therapeutics targeting development of alcohol use disorder (AUD) have been validated. Here, we conducted a genome-wide RNA-sequencing (RNA-seq) analysis in mice exposed to different modes (acute vs chronic) of ethanol drinking. We focused on transcriptional profiles in the amygdala including the central and basolateral subnuclei, a brain area previously implicated in alcohol drinking and seeking, demonstrating distinct gene expression patterns and canonical pathways induced by both acute and chronic intake. Surprisingly, both drinking modes triggered similar transcriptional changes, including up-regulation of ribosome-related/translational pathways and myelination pathways, and down-regulation of chromatin binding and histone modification. Notably, multiple genes that were significantly regulated in mouse amygdala with alcohol drinking, including Atp2b1, Slc4a7, Nfkb1, Nts, and Hdac2, among others had previously been associated with human AUD via GWAS or other genomic studies. In addition, analyses of hub genes and upstream regulatory pathways predicted that voluntary ethanol consumption affects epigenetic changes via histone deacetylation pathways, oligodendrocyte and myelin function, and oligodendrocyte-related transcriptional factor, Sox17.

Overall, our results suggest that the transcriptional landscape in the central and basolateral subnuclei of the amygdala is sensitive to voluntary alcohol drinking. They provide a unique resource of gene expression data for future translational studies examining transcriptional mechanisms underlying the development of AUD due to alcohol consumption.


title: The vacuolar iron transporter mediates iron detoxification in Toxoplasma gondii

abstract: Iron is essential to living cells, acting as a cofactor in a number of essential enzymes in metabolism; however, iron requires proper storage or it can be dangerous to the cell. In both yeast and plants, iron is stored in a vacuole through the action of a vacuolar iron transporter (VIT). This transporter is conserved in the apicomplexan family of obligate intracellular parasites, including in Toxoplasma gondii, a pathogen of medical and veterinary importance. Here, we assess the role of VIT in T. gondii. We show that deletion of VIT causes a slight growth defect in vitro, however leads to hypersensitivity in the presence of excess iron, confirming its essential role in iron detoxification in the parasite. In the absence of VIT, parasites contain less iron and are at a growth disadvantage when moving into an iron-depleted environment. We show parasite VIT expression is regulated by environmental iron levels at both the transcript and protein level, and by altering the distribution of VIT within the cell. In the absence of VIT, we find that the T. gondii responds by altering expression of genes with a role in iron metabolism and by increasing the activity of the antioxidant protein catalase. We also show that iron detoxification has an important role both in parasite survival within macrophages and in pathogenesis in a mouse model. Together, by demonstrating a critical role for VIT during iron detoxification in T. gondii, we reveal the importance of iron storage in the parasite and provide the first insight into the machinery involved.


title: Riparian forest restoration as sources of biodiversity and ecosystem functions in anthropogenic landscapes

abstract:
O_LIRestoration of tropical riparian forests is challenging, since these ecosystems are the most diverse, dynamic, and complex physical and biological terrestrial habitats. This study tested whether biodiversity can predict ecosystem functions in a human-impacted tropical riparian forest.
C_LIO_LIWe explored the effects of several biodiversity components (taxonomic or functional groups) on different ecosystem functions associated with restored riparian forests
C_LIO_LIOverall, 49% of the biodiversity components showed positive effects on ecosystem functions, each component to a different degree. In general, our results showed that both taxonomic and functional biodiversity had strong effects on ecosystem functions indicating that floral and faunal biodiversity enhanced the multifunctionality of these restored riparian tropical forests.
C_LIO_LIThese findings indicate that in restored riparian forests, recovery of biodiversity is followed by improvement in important ecosystem functions that are the basis for successful restoration. Future research and policy for restoration programs must focus on restoring elementary faunal and floral components of biodiversity in order to promote ecosystem multifunctionality.
C_LI


title: Population genomics of Bacillus anthracis from an anthrax hyperendemic area reveals transmission processes across spatial scales and unexpected within-host diversity

abstract:
Genomic sequencing has revolutionized our understanding of bacterial disease epidemiology, but remains underutilized for zoonotic pathogens in remote endemic settings. Anthrax, caused by the spore-forming bacterium Bacillus anthracis, remains a threat to human and animal health and rural livelihoods in low- and middle-income countries. While the global genomic diversity of B. anthracis has been well-characterized, there is limited information on how its populations are genetically structured at the scale at which transmission occurs, critical for understanding the pathogens evolution and transmission dynamics. Using a uniquely rich dataset, we quantified genome-wide single nucleotide polymorphisms (SNPs) among 73 B. anthracis isolates derived from 33 livestock carcasses sampled over one year throughout the Ngorongoro Conservation Area, Tanzania, an area hyperendemic for anthrax. Genome-wide SNPs distinguished 22 unique B. anthracis genotypes within the study area. However, phylogeographic structure was lacking, as identical SNP profiles were found throughout the study area, likely the result of the long and variable periods of spore dormancy and long-distance livestock movements. Significantly, divergent genotypes were obtained from spatio-temporally linked cases and even individual carcasses. The high number of SNPs distinguishing isolates from the same host is unlikely to have arisen during infection, as supported by our simulation models. This points to an unexpectedly wide transmission bottleneck for B. anthracis, with an inoculum comprising multiple variants being the norm. Our work highlights that inferring transmission patterns of B. anthracis from genomic data will require analytical approaches that account for extended and variable environmental persistence as well as co-infection.

ImportancePathogens transmitted between animals and people affect the health and livelihoods of farmers, particularly in developing countries dependent on livestock. Understanding over what distances these pathogens are transmitted and how they evolve is important to inform control strategies towards reducing disease impacts. Information on the circulation of Bacillus anthracis, which causes the often-lethal disease anthrax, is lacking for settings where the disease is commonplace. Consequently, we examined its genetic variability in an area in Tanzania where anthrax is widespread. We found no clear link between how closely cases were sampled and their genetic similarity. We suspect this lack of congruence is primarily driven by large-scale livestock movements, which control efforts should take into consideration. Another significant finding was the co-occurrence of multiple B. anthracis types within individual hosts, suggesting animals are commonly infected with a mixture of variants. This needs to be accounted for when investigating possible connections between cases.


title: The response of Oligo-Miocene bivalve fauna of the Kutch Basin (western India) to regional changes in tectonics and climate

abstract:
Tectonic changes has influenced the evolution of the marine community by changing the land and seaway configuration through time. Two such tectonic events during Oligo-Miocene times -- the closure of the Tethyan seaway due to development of the Gomphotherium-Landbridge leading to separation of the Arabian Sea from proto-Mediterranean Sea ([~]19 Ma) and significant uplift of the Tibetan plateu marking the initiation of the monsoon ([~]16 Ma) -- represent a classic case of tectonic shift influencing the regional environment of the Indian subcontinent. We investigated the taxonomic and body size related response of the shallow marine fauna to this regional change using bivalves from 11 time-constrained shellbeds of the Kutch Basin (western India) from three formations -- Maniyara Fort (Chattian), Khari Nadi (Aquitanian) and Chhasra (Burdigalian-Langian) representing a time span of [~]9 Ma (24.4 - 15 Ma).

Our collection of over 2000 individuals represents a total of 15 families and 61 morphospecies. The fossils are predominantly calcitic in nature and families of aragonitic composition are often preserved as molds indicating a potential negative effect of diagenesis. The taphonomic nature, however, does not vary substantially across shellbeds and hence, less likely produced a temporal pattern. The five most abundant species, Ostrea latimarginata, Ostrea angulata, Talochlamys articulata, Anomia primaeva and Placuna lamellata occur in all the formations. The species composition of the Maniyara Fort formation is substantially different from those of the younger formations implying the possible effect of biogeographic separation. Moreover, the absence of proto-Mediterranean taxa in Oligocene shellbeds support a limited faunal exchange as early as [~]24.4Ma (Chattian) ago. We observed a monotonic increase in the overall rarefied species richness and a decrease in evenness from the Maniyara Fort to the Chhasra Formation. However, shellbed analyses show a dominantly conservative behavior of diversity and body size without a strong directional trend through time. Although it is difficult to rule out the negative influence of taphonomy on the diversity of the studied fauna, Oligo-Miocene marine bivalve fauna of the Kutch Basin demonstrate little or no influence of the Tethyan closure and Himalayan upliftment.

category: paleontology


title: Population modelling insights of extinct environments: the case of the Kem Kem palaeocommunity

abstract: The Kem Kem beds are well-known palaeontological deposits. Among the species that lived there, there are some large theropods, such as Deltadromeus agilis, Carcharodontosaurus saharicus, and Spinosaurus aegyptiacus. It is possible that these large predators were facultative scavengers, and they could compete for carrion. In the present paper, I simulate a small community module of this environment, consisting of Carrion, Fishes, Spinosaurus, and a functional group composed of large terrestrial Theropods. I assume that these top predators feed on carrion, but they also have exclusive food sources. I show that these exclusive food sources could have assured the possibility of coexistence, and in their absence, one top predator could be locally extinct.


title: Identification of a stress-sensitive anorexigenic neurocircuit from medial prefrontal cortex to lateral hypothalamus in humans and mice

abstract: Animal models that examine neural circuits controlling food intake often lack translational relevance. To address this limitation, we identified neural network dynamics related to homeostatic state and BMI in humans. This approach predicted a novel pathway projecting from the medial prefrontal cortex (mPFC) to the lateral hypothalamus (LH) in humans. We then dissected the mechanistic underpinnings of this human-relevant mPFC-LH circuit in mice. Chemogenetic or optogenetic activation of the mPFC-LH pathway in mice suppressed food intake and motivated sucrose-seeking. Fibre photometry demonstrated this pathway was active in response to acute stress or prior to novel environment or object exposure, suggesting a role in the predictive assessment of potential threat. Food consumption suppressed mPFC-LH neuronal activity, independent of metabolic state or palatability. Finally, inhibition of this circuit increased feeding and motivated behaviour under mild stress and chronic ablation caused weight gain. These studies identify the mPFC-LH as a novel stress-sensitive anorexigenic neural pathway involved in the cortical control of food intake and motivated reward-seeking.


title: The chromatin-remodeling enzyme Smarca5 regulates erythrocyte aggregation via Keap1-Nrf2 signaling

abstract: Although thrombosis has been extensively studied using various animal models, however, our understanding of the underlying mechanism remains elusive. Here, using zebrafish model, we demonstrated that smarca5-deficient red blood cells (RBCs) formed blood clots in the caudal vein plexus that mimics venous thrombosis. We further used the anti-thrombosis drugs to treat smarca5zko1049a embryos and found that a thrombin inhibitor, argatroban, partially prevented blood clot formation in smarca5zko1049a. To explore the regulatory mechanism of smarca5 in RBC homeostasis, we profiled the chromatin accessibility landscape and transcriptome features in RBCs from smarca5zko1049a and their siblings and found that both the chromatin accessibility at the keap1a promoter and expression of keap1a were decreased. Keap1 is a suppressor protein of Nrf2, which is a major regulator of oxidative responses. We further identified that the expression of hmox1a, a downstream target of Keap1-Nrf2 signaling pathway, was markedly increased upon smarca5 deletion. Importantly, overexpression of keap1a or knockdown of hmox1a partially rescued the blood clot formation, suggesting that the disrupted Keap1-Nrf2 signaling is responsible for the venous thrombosis-like phenotypes in smarca5 mutants. Together, our study using zebrafish smarca5 mutants not only characterizes a novel role for smarca5 in blood clot formation, but also provides a new venous thrombosis animal model to support drug screening and pre-clinical therapeutic assessments to treat thrombosis.


title: Identification and characterisation of Botrylloides species from Aotearoa New Zealand coasts

abstract: Botryllid ascidians possess diverse biological abilities like whole-body regeneration (WBR), hibernation/aestivation, blastogenesis, metamorphosis, and natural chimerism. However, the absence of distinctive morphological features often makes identification problematic. Botrylloides diegensis is a botryllid ascidian that has been misidentified in previous studies and is recorded in GenBank as Botrylloides leachii due to the high morphological similarity between the sister species. More available sequences and strategies around identification would help resolve some of the confusion currently surrounding its ambiguous nature. We collected several Botrylloides samples from 7 locations around New Zealand and barcoded the species based on Cytochrome Oxidase I, Histone 3, 18S, and 28S ribosomal RNA markers. Network and Bayesian trees confirmed three Botrylloides species: B. diegensis, B. jacksonianum, and B. aff. anceps. Additionally, recognition assays were applied to analyse the histocompatibility between distinct morphs qualitatively.

category: genetics


title: Genome sequencing and analysis of two early-flowering cherry (Cerasus x kanzakura) varieties, 'Kawazu-zakura' and 'Atami-zakura'

abstract: To gain genetic insights into the early-flowering phenotype of ornamental cherry, also known as sakura, we determined the genome sequences of two early-flowering cherry (Cerasus x kanzakura) varieties,  Kawazu-zakura and  Atami-zakura. Since the two varieties are interspecific hybrids, likely derived from crosses between Cerasus campanulata (early-flowering species) and Cerasus speciosa, we employed the haplotype-resolved sequence assembly strategy. Genome sequence reads obtained from each variety by single molecule real-time sequencing (SMRT) were split into two subsets, based on the genome sequence information of the two probable ancestors, and assembled to obtain haplotype-phased genome sequences. The resultant genome assembly of  Kawazu-zakura spanned 519.8 Mb with 1,544 contigs and an N50 value of 1,220.5 kb, while that of  Atami-zakura totaled 509.6 Mb with 2,180 contigs and an N50 value of 709.1 kb. A total of 72,702 and 72,528 potential protein-coding genes were predicted in the genome assemblies of  Kawazu-zakura and  Atami-zakura, respectively. Gene clustering analysis identified 2,634 clusters uniquely presented in the C. campanulata haplotype sequences, which might contribute to its early-flowering phenotype. Genome sequences determined in this study provide fundamental information for elucidating the molecular and genetic mechanisms underlying the early-flowering phenotype of ornamental cherry tree varieties and their relatives.


title: Supervised Promoter Recognition: A Benchmark Framework

abstract:
AO_SCPLOWBSTRACTC_SCPLOWDeep learning has become a prevalent method in identifying genomic regulatory sequences such as promoters. In a number of recent papers, the performance of deep learning models have continually been reported as an improvement over alternatives for sequence-based promoter recognition. However, the performance improvements in these models do not account for the different datasets that models are being evaluated on. The lack of a consensus dataset and procedure for benchmarking purposes has made the comparison of each models true performance difficult to assess.

We present a framework called Supervised Promoter Recognition Framework ( SUPR REF) capable of streamlining the complete process of training, validating, testing, and comparing promoter recognition models in a systematic manner. SUPR REF includes the creation of biologically relevant benchmark datasets to be used in the evaluation process of deep learning promoter recognition models. We showcase this framework by comparing the models performance on alternative datasets, and properly evaluate previously published models on new benchmark datasets. Our results show that the reliability of deep learning ab initio promoter recognition models on eukaryotic genomic sequences is still not at a sufficient level, as precision is severely lacking. Furthermore, given the observational nature of these data, cross-validation results from small datasets need to be interpreted with caution.

AvailabilitySource code and documentation of the framework is available online at https://github.com/ivanpmartell/suprref