title: Dystrophin modulates focal adhesion tension and YAP-mediated mechanotransduction

abstract: Dystrophin is an essential muscle protein that contributes to cell membrane stability by linking the actin cytoskeleton to the extracellular matrix. The absence or impaired function of dystrophin causes muscular dystrophy. Focal adhesions are mechanosensitive adhesion complexes that also connect the cytoskeleton to the extracellular matrix. However, the interplay between dystrophin and focal adhesion force transmission has not been investigated. Using a bioluminescent tension sensor, we measured focal adhesion tension in transgenic C2C12 myoblasts expressing wild type (WT) dystrophin, a non-pathogenic SNP (I232M), or two missense mutations associated with Duchenne (L54R), or Becker muscular dystrophy (L172H). We found that myoblasts expressing WT or nonpathogenic I232M dystrophin showed increased focal adhesion tension compared to non-transgenic myoblasts, while myoblasts expressing L54R or L172H dystrophin presented with decreased focal adhesion tension. Moreover, myoblasts expressing L54R or L172H dystrophin showed decreased YAP activation and exhibited slower and less directional migration compared to cells expressing WT or I232M dystrophin. Our results suggest that disease-causing missense mutations in dystrophin may disrupt a cellular tension sensing pathway in dystrophic skeletal muscle.


title: Leaf area index estimation of even-aged oak (Quercus petraea) forests using in situ stand dendrometric parameters

abstract:
The leaf area index (LAI) is a key characteristic of forest stand aboveground net productivity (ANP), and many methods have been developed to estimate the LAI. However, every method has flaws, e.g., methods may be destructive, require means or time and/or show intrinsic bias and estimation errors.

A relationship using basal area (G) and stand age to estimate LAI was proposed by Sonohat et al. (2004). We used literature data in addition to data form measurements campaign made in the northern half of France to build a data set with large ranges of pedoclimatic conditions, stand age and measured LAI. We validated the Sonohat et al. (2004) relationship and attempted to improve or modify it using other stand/dendrometric characteristics that could be predictors of the LAI.

The result is a series of three models using the G, age and/or quadratic mean diameter (Dg), and the models were able to estimate the LAI of an oak only even-aged forest stand with good confidence (root mean square error, RMSE < 0.75) While G is the main predictor here, age and Dg could be used conjointly or exclusively given the available data, with variable precision in the estimations.

Although these models could not, by construction, relate to the interannual variability of the LAI, they may provide the theoretical LAI of an untouched forest (no meteorological, biotic or anthropogenic perturbation) in recent years. additionally, the use of this model may be more interesting than an LAI measurement campaign, depending on the means to be invested in such a campaign.

category: ecology


title: The Arabidopsis HOP2 Gene Has a Role in Preventing illegitimate Exchanges between Nonhomologous Chromosomes

abstract:
Meiotic homologous chromosomes pair up and undergo crossing over. In many eukaryotes both intimate pairing and crossing over require the induction of double stranded breaks (DSBs) and subsequent repair via Homologous Recombination (HR). In these organisms, two key proteins are the recombinases RAD51 and DMC1. Recombinase-modulators HOP2 and MND1 have been identified as proteins that assist RAD51 and DMC1 and are needed to promote stabilized pairing. We have probed the nature of the genetic lesions seen in hop2 mutants and looked at the role of HOP2 in the fidelity of genetic exchanges. Using {gamma}H2Ax as a marker for unrepaired DSBs we found that hop2-1 and mnd1 mutants have different appearance/disappearance for DSBs than wild type, but all DSBs are repaired by mid-late pachytene. Therefore, the bridges and fragments seen from metaphase I onward are due to mis-repaired DSBs, not unrepaired ones. Studying Arabidopsis haploid meiocytes we found that wild type haploids produced the expected five univalents, but hop2-1 haploids suffered many illegitimate exchanges that were stable enough to produce bridged chromosomes during segregation. Our results suggest that HOP2 has a significant active role in preventing nonhomologous associations. We also found evidence that HOP2 plays a role in preventing illegitimate exchanges during repair of radiation-induced DSBs in rapidly dividing petal cells. Thus, HOP2 plays both a positive role in promoting homologous chromosome synapsis and a separable role in preventing nonhomologous chromosome exchanges. Possible mechanisms for this second important role are discussed.

SIGNIFICANCE STATEMENTThe fidelity of Homologous Recombination (HR) during meiosis is essential to the production of viable gametes and for maintaining genome integrity in vegetative cells. HOP2 is an important protein for accurate meiotic HR in plants. We find high levels of illegitimate repairs between nonhomologous chromosomes during meiosis and in irradiated petal cells in HOP2 mutants. We consider mechanisms of how this second role might be accomplished.

category: genetics


title: Using adopted individuals to partition maternal genetic effects into prenatal and postnatal effects on offspring phenotypes

abstract: Maternal genetic effects can be defined as the effect of a mothers genotype on the phenotype of her offspring, independent of the offsprings genotype. Maternal genetic effects can act via the intrauterine environment during pregnancy and/or via the postnatal environment. In this manuscript, we present a simple extension to the basic adoption design that uses structural equation modelling (SEM) to partition maternal genetic effects into prenatal and postnatal effects. We assume that in biological families, offspring phenotypes are influenced prenatally by their mothers genotype and postnatally by both parents genotypes, whereas adopted individuals phenotypes are influenced prenatally by their biological mothers genotype and postnatally by their adoptive parents genotypes. Our SEM framework allows us to model the (potentially) unobserved genotypes of biological and adoptive parents as latent variables, permitting us in principle to leverage the thousands of adopted singleton individuals in the UK Biobank. We examine the power, utility and type I error rate of our model using simulations and asymptotic power calculations. We apply our model to polygenic scores of educational attainment and birth weight associated variants, in up to 5178 adopted singletons, 983 trios, 3650 mother-offspring pairs, 1665 father-offspring pairs and 350330 singletons from the UK Biobank. Our results show the expected pattern of maternal genetic effects on offspring birth weight, but unexpectedly large prenatal maternal genetic effects on offspring educational attainment. Sensitivity and simulation analyses suggest this result may be at least partially due to adopted individuals in the UK Biobank being raised by their biological relatives. We show that accurate modelling of these sorts of cryptic relationships is sufficient to bring type I error rate under control and produce unbiased estimates of prenatal and postnatal maternal genetic effects. We conclude that there would be considerable value in following up adopted individuals in the UK Biobank to determine whether they were raised by their biological relatives, and if so, to precisely ascertain the nature of these relationships. These adopted individuals could then be incorporated into informative statistical genetics models like the one described in our manuscript to further elucidate the genetic architecture of complex traits and diseases.


title: Broad domains of histone marks in the highly compact Paramecium macronuclear genome

abstract: The unicellular ciliate Paramecium contains a large vegetative macronucleus with several unusual characteristics including an extremely high coding density and high polyploidy. As macronculear chromatin is devoid of heterochromatin our study characterizes the functional epigenomic organisation necessary for gene regulation and proper PolII activity. Histone marks (H3K4me3, H3K9ac, H3K27me3) revealed no narrow peaks but broad domains along gene bodies, whereas intergenic regions were devoid of nucleosomes. Our data implicates H3K4me3 levels inside ORFs to be the main factor to associate with gene expression and H3K27me3 appears to occur as a bistable domain with H3K4me3 in plastic genes. Surprisingly, silent and lowly expressed genes show low nucleosome occupancy suggesting that gene inactivation does not involve increased nucleosome occupancy and chromatin condensation. Due to a high occupancy of Pol II along highly expressed ORFs, transcriptional elongation appears to be quite different to other species. This is supported by missing heptameric repeats in the C-terminal domain of Pol II and a divergent elongation system. Our data implies that unoccupied DNA is the default state, whereas gene activation requires nucleosome recruitment together with broad domains of H3K4me3. This could represent a buffer for paused Pol II along ORFs in absence of elongation factors of higher eukaryotes.

category: genomics


title: GEPSi: A Python Library to Simulate GWAS Phenotype Data

abstract:
MotivationMany computational methods aim to identify genetic variants associated with diseases and complex traits. Due to the absence of ground truth data, simulated genotype and phenotype data is needed to benchmark these methods. However, phenotypes are frequently simulated as an additive function of randomly selected variants, neglecting biological complexity such as non-random occurrence of causal SNPs, epistatic effects, heritability and dominance. Including such features would improve benchmarking studies and accelerate the development of methods for genetic analysis.

ResultsHere, we describe GEPSi (GWAS Epistatic Phenotype Simulator), a user-friendly python package to simulate phenotype data based on user-supplied genotype data for a population. GEPSi incorporates diverse biological parameters such as heritability, dominance, population stratification and epistatic interactions between SNPs. We demonstrate the use of this package to compare machine learning methods for GWAS analysis.

Availability and ImplementationGEPSi is freely available under an Apache 2.0 license, and can be downloaded from https://github.com/clara-parabricks/GEPSi.

Supplementary informationSupplementary data are available online.


title: A Bayesian Analysis of Steady-State Enzyme Data leads to Estimates of Rate Constants and Uncertainties in a Multi-Step Reaction

abstract: The microscopic rate constants that govern an enzymatic reaction are only directly measured under certain experimental set-ups, such as stopped flow, quenched flow, or temperature-jump assays; the majority of enzymology proceeds from steady state conditions which leads to a set of more easily-observable parameters such as kcat, KM, and observed Kinetic Isotope Effects (Dkcat). This paper further develops a model from Toney (2013) to estimate microscopic rate constants from steady-state data for a set of reversible, four-step reactions. This paper uses the Bayesian modeling software Stan, and demonstrates the benefits of Bayesian data analysis in the estimation of these rate constants. In contrast to the optimization methods employed often in the estimation of kinetic constants, a Bayesian treatment is more equipped to estimate the uncertainties of each parameter; sampling from the posterior distribution using Hamiltonian Monte Carlo immediately gives parameter estimates as mean or median of the posterior, and also confidence intervals that express the uncertainty of each parameter.

category: biophysics


title: Global emergence and dissemination of Neisseria gonorrhoeae ST-9363 isolates with reduced susceptibility to azithromycin

abstract: Neisseria gonorrhoeae multi-locus sequence type (ST) 9363 genogroup isolates have been associated with reduced azithromycin susceptibility (AZMrs) and show evidence of clonal expansion in the U.S. Here we analyze a global collection of ST-9363 genogroup genomes to shed light on the emergence and dissemination of this strain. The global population structure of ST-9363 genogroup falls into three lineages: Basal, European, and North American; with 32 clades within all lineages. Although, ST-9363 genogroup is inferred to have originated from Asia in the mid-19th century; we estimate the three modern lineages emerged from Europe in the late 1970s to early 1980s. The European lineage appears to have emerged and expanded from around 1986 to 1998, spreading into North America and Oceania in the mid-2000s with multiple introductions, along with multiple secondary reintroductions into Europe. Our results suggest two separate acquisition events of mosaic mtrR and mtrR promoter alleles: first during 2009-2011 and again during the 2012-2013 time, facilitating the clonal expansion of this genogroup with AZMrs in the U.S. By tracking phylodynamic evolutionary trajectories of clades that share distinct demography as well as population-based genomic statistics, we demonstrate how recombination and selective pressures in the mtrCDE efflux operon granted a fitness advantage to establish ST-9363 as a successful gonococcal lineage in the U.S. and elsewhere. Although it is difficult to pinpoint the exact timing and emergence of this young genogroup, it remains critically important to continue monitoring it, as it could acquire additional resistance markers.

category: microbiology


title: PixR, a novel activator of conjugative transfer of IncX4 resistance plasmids, mitigates the fitness cost of mcr-1 carriage in Escherichia coli

abstract:
The emergence of the plasmid-borne colistin resistance gene mcr-1 threats public health. IncX4-type plasmids are one of the most epidemiologically successful vehicles for spreading mcr-1 worldwide. Since MCR-1 is known for imposing a fitness cost to its host bacterium, the successful spread of mcr-1-bearing plasmids might be linked to high conjugation frequency, which would enhance the maintenance of the plasmid in the host without antibiotic selection. However, the mechanism of IncX4 plasmids conjugation remains unclear. In this study, we used high-density transposon mutagenesis to identify factors required for IncX4 plasmid transfer and 18 genes were identified, including five with annotations unrelated to conjugation. The Cappable-seq and RNA-seq analysis confirmed that a novel transcriptional regulator gene, pixR, directly regulates the transfer of IncX4 plasmids by binding the promoter of 13 essential transfer genes to increase their transcription. Plasmid invasion and co-culture competition assays revealed that pixR is essential for the spread and persistence of mcr-1-bearing IncX4 plasmids in bacterial populations, and effective conjugation is crucial for alleviating the fitness cost exerted by mcr-1 carriage. The existence of the IncX4-specific pixR gene increases plasmid transmissibility while promoting the invasion and persistence of mcr-1-bearing plasmids in bacterial populations, which helps explain their global prevalence.

IMPORTANCEThe spread of clinical important antibiotic resistance genes is frequently related to some epidemic plasmids. However, the underlying molecular mechanisms contributing to the successful spread of these epidemic plasmids remains unclear. The significant of our research indicated that efficient conjugation could promote the invasion and persistence of plasmids within a bacterial population, resulting in the successful dissemination of epidemic plasmids in nature. Our data also highlight the importance of developing plasmid conjugation inhibitors to solve the antibiotic resistance crisis.


title: Direct reprogramming of human fibroblasts into insulin-producing cells by transcription factors

abstract: Direct lineage reprogramming of one somatic cell into another bypassing an intermediate pluripotent state has emerged as an alternative to embryonic or induced pluripotent stem cell differentiation to generate clinically relevant cell types. One cell type of clinical interest is the pancreatic {beta} cell that secretes insulin and whose loss and/or dysfunction leads to diabetes. Generation of functional {beta}-like cells from developmentally related somatic cell types (pancreas, liver, gut) has been achieved via enforced expression of defined sets of transcription factors. However, clinical applicability of these findings is challenging because the starting cell types are not easily obtainable. Skin fibroblasts are accessible and easily manipulated cells that could be a better option, but available studies indicate that their competence to give rise to {beta} cells through similar direct reprogramming approaches is limited. Here, using human skin fibroblasts and a protocol that ensures high and consistent expression of adenovirus-encoded reprogramming factors, we show that the transcription factor cocktail consisting of Pdx1, Ngn3, MafA, Pax4 and Nkx2-2 activates key {beta} cell genes and down-regulates the fibroblast transcriptional program. The converted cells produce insulin and exhibit intracellular calcium responses to glucose and/or membrane depolarization. Furthermore, they secrete insulin in response to glucose in vitro and after transplantation in vivo. These findings demonstrate that transcription factor-mediated direct reprogramming of human fibroblasts is a feasible strategy to generate insulin-producing cells.

category: developmental biology


title: Effect of EMS induced mutation in rice cultivar Nagina 22 on salinity tolerance

abstract:
Salinized hydroponic culture experiment with three salinity levels (EC control, 6 and 12 dS/m) was performed to screen salt tolerant mutants of aerobic rice cultivar Nagina 22 and to study the nature of salt tolerance from a total of 432 EMS induced M4 mutants. Plants were harvested 30 days after sowing. Growth parameters viz. root weight, shoot weight, root length, shoot length, Na and K concentrations in shoot and roots were measured. Combined Factor scores of growth parameters was computed by Principle Component Analysis using Minitab software.

At EC 12 dS/m 10 mutants out of 432 were able to survive. At moderate salinity, some mutant lines produced higher shoot weight compared to their respective control showing inverse trend and the effectiveness of EMS induced mutation in inducing salinity tolerance to these mutants. At high salinity only10 mutants survived (remained green) up to the time of 30 days harvest. These mutants performed well in terms of overall growth recording 2.1-2.5 times higher factor score and 8-14 times higher shoot weight compared to the N 22 check. One mutant N22-L-1010 almost completely excluded Na at xylem parenchyma level. Two other mutants N22-L-1013 and N22-L-806 maintained Na exclusion compared to the N22 check. N22 check and mutant N22-L-1009 maintained similar degree of Na exclusion though the N22 check died because it cannot maintain adequate K in shoot.

We conclude that EMS has induced salinity tolerance in some mutants. The study can be advanced further to characterize the putative mutants through molecular genetics approaches.


title: The mycobacterial guaB1 gene encodes a guanosine 5'-monophosphate reductase with a cystathione-β-synthase domain

abstract: Purine metabolism plays a pivotal role in bacterial life cycle, however, regulation of the de novo and purine salvage pathways have not been extensively detailed in mycobacteria. By gene knockout, biochemical and structural analyses, we identified Mycobacterium smegmatis (Msm) and Mycobacterium tuberculosis (Mtb) guaB1 gene product as a novel type of guanosine 5-monophosphate reductase (GMPR), which recycles guanosine monophosphate to inosine monophosphate within the purine salvage pathway and contains cystathione {beta}-synthase (CBS) domains with atypical orientation in the octamer. CBS domains share a much larger interacting area with a conserved catalytic domain in comparison with the only known CBS containing protozoan GMPR and closely related inosine monophosphate dehydrogenase structures. Our results revealed essential effect of pH on allosteric regulation of Msm GMPR activity and oligomerization with adenine and guanosine nucleotides binding to CBS domains.Bioinformatic analysis indicated the presence of GMPRs containing CBS domains across the entire Actinobacteria phylum.

category: biochemistry


title: Structural insights into recognition of chemokine receptors by Staphylococcus aureus leukotoxins

abstract: Staphylococcus aureus (SA) leukocidin LukED belongs to a family of bicomponent pore forming toxins that play important roles in SA immune evasion and nutrient acquisition. LukED targets specific G protein-coupled chemokine receptors to lyse human erythrocytes and leukocytes. The first recognition step of receptors is critical for specific cell targeting and lysis. The structural and molecular bases for this mechanism are not well understood but could constitute essential information to guide antibiotic development. Here, we characterized the interaction of LukE with chemokine receptors ACKR1, CCR2 and CCR5 using a combination of structural, pharmacological and computational approaches. First, crystal structures of LukE in complex with a small molecule mimicking sulfotyrosine side chain (p-cresyl sulfate) and with peptides containing sulfotyrosines issued from receptor sequences revealed the location of receptor sulfotyrosine binding sites in the toxins. Then, by combining the available experimental information with protein docking, classical and accelerated weight histogram (AWH) molecular dynamics we propose models of the ACKR1-LukE and CCR5-LukE complexes. This work provides novel insights into chemokine receptor recognition by leukotoxins and suggests that the conserved sulfotyrosine binding pocket could be a target of choice for future drug development.


title: Sift-PULs: A public repository for specific functional polysaccharide utilization loci

abstract:
BackgroundPolysaccharide utilization loci (PULs) were bacterial gene clusters encoding genes responsible for polysaccharide utilization process. PUL studies are blooming in recent years but the biochemical characterization speed is relative slow. There is a growing demand for PUL database with function annotations.

ResultsUsing signature genes corresponding for specific polysaccharide, 10422 PULs specific for 6 polysaccharides (agar, alginate, pectin, carrageenan, chitin and {beta}-manan) from various bacterial phyla were predicted. Then online website of specific functional polysaccharide utilization loci (Sift-PULs) was constructed. Sift-PULs provides a repository where users could browse, search and download interested PULs without registration.

ConclusionsThe key advantage of Sift-PULs is to assign a function annotation of each PUL, which is not available in existing PUL databases. PULs functional annotation lays a foundation for studying novel enzymes, new pathways, PUL evolution or bioengineering. The website is available on https://sift-puls.org

category: bioinformatics


title: Microtubule rescue at midzone edges promotes overlap stability and prevents spindle collapse during anaphase B

abstract: During anaphase B, molecular motors slide interpolar microtubules to elongate the mitotic spindle, contributing to the separation of chromosomes. However, sliding of antiparallel microtubules reduces their overlap, which may lead to spindle breakage, unless microtubules grow to compensate sliding. How sliding and growth are coordinated is still poorly understood. In this study, we have used the fission yeast S. pombe to measure microtubule dynamics during anaphase B. We report that the coordination of microtubule growth and sliding relies on promoting rescues at the midzone edges. This makes microtubules stable from pole to midzone, while their distal parts including the plus ends alternate between assembly and disassembly. Consequently, the midzone keeps a constant length throughout anaphase, enabling sustained sliding without the need for a precise regulation of microtubule growth speed. Additionally, we found that in S. pombe, which undergoes closed mitosis, microtubule growth speed decreases when the nuclear membrane wraps around the spindle midzone.

category: cell biology


title: Characterization and clustering of kinase isoform expression in metastatic melanoma

abstract:
Mutations to the human kinome are known to play causal roles in cancer. The kinome regulates numerous cell processes including growth, proliferation, differentiation, and apoptosis. In addition to aberrant expression, aberrant alternative splicing of cancer-driver genes is receiving increased attention as it could create loss or gain of functional domains, altering a kinases downstream effects.

The present study quantifies changes in gene expression and isoform ratios in the kinome of metastatic melanoma cells relative to primary tumors. We contrast 538 total kinases and 3042 known kinase isoforms between 103 primary tumor and 367 metastatic samples from The Cancer Genome Atlas (TCGA). We find strong evidence of differential expression (DE) at the gene level in 123 genes (23%). Additionally, of the 468 genes with alternative isoforms, 60 (13%) had differential isoform ratios (DIR). Notably, DE and DIR have little correlation; for instance, although DE highlights enrichment in receptor tyrosine kinases (RTKs), DIR identifies altered splicing in non-receptor tyrosine kinases (nRTKs). Using exon junction mapping, we identify five examples of splicing events favored in metastatic samples.

We cluster isoform expression data and identify subgroups that correlate with genomic subtypes and anatomic tumor locations. Notably, distinct DE and DIR patterns separate samples with BRAF hotspot mutations and (N/K/H)RAS hotspot mutations, the latter of which lacks effective kinase inhibitor treatments. DE in RAS mutants concentrates in CMGC kinases (a group including cell cycle and splicing regulators) rather than RTKs as in BRAF mutants. Furthermore, isoforms in the RAS kinase subgroup show enrichment for cancer-related processes such as angiogenesis and cell migration. Our results reveal a new approach to therapeutic target identification and demonstrate how different mutational subtypes may respond differently to treatments highlighting possible new driver events in cancer.


title: Heterochromatin diversity modulates genome compartmentalization and loop extrusion barriers

abstract:
Two dominant processes organizing chromosomes are loop extrusion and the compartmental segregation of active and inactive chromatin. The molecular players involved in loop extrusion during interphase, cohesin and CTCF, have been extensively studied and experimentally validated. However, neither the molecular determinants nor the functional roles of compartmentalization are well understood. Here, we distinguish three inactive chromatin states using contact frequency profiling, comprising two types of heterochromatin and a previously uncharacterized inactive state exhibiting a neutral interaction preference. We find that heterochromatin marked by long continuous stretches of H3K9me3, HP1 and HP1{beta} correlates with a conserved signature of strong compartmentalization and is abundant in HCT116 colon cancer cells. We demonstrate that disruption of DNA methyltransferase activity dramatically remodels genome compartmentalization as a consequence of the loss of H3K9me3 and HP1 binding. Interestingly, H3K9me3-HP1/{beta} is replaced by the neutral inactive state and retains late replication timing. Furthermore, we show that H3K9me3-HP1/{beta} heterochromatin is permissive to loop extrusion by cohesin but refractory to CTCF, explaining a paucity of visible loop extrusion-associated patterns in Hi-C. Accordingly, CTCF loop extrusion barriers are reactivated upon loss of H3K9me3-HP1/{beta}, not as a result of canonical demethylation of the CTCF binding motif but due to an intrinsic resistance of H3K9me3-HP1/{beta} heterochromatin to CTCF binding. Together, our work reveals a dynamic structural and organizational diversity of the inactive portion of the genome and establishes new connections between the regulation of chromatin state and chromosome organization, including an interplay between DNA methylation, compartmentalization and loop extrusion.

HighlightsO_LIThree inactive chromatin states are distinguishable by long-range contact frequencies in HCT116, respectively associated with H3K9me3, H3K27me3 and a H3K9me2 state with neutral contact preferences.
C_LIO_LIH3K9me3-HP1/{beta} heterochromatin has a high degree of homotypic affinity and is permissive to loop extrusion but depleted in extrusion barriers.
C_LIO_LIDisrupting DNA methylation causes widespread loss of H3K9me3-HP1/{beta} and dramatic remodeling of genome compartmentalization.
C_LIO_LIH3K9me3-HP1/{beta} is replaced by the neutral inactive state, which gains CTCF loop extrusion barriers and associated contact frequency patterns.
C_LIO_LIDNA methylation suppresses CTCF binding via two distinct mechanisms.
C_LI


title: Heavy isotope labeling and mass spectrometry reveal unexpected remodeling of bacterial cell wall expansion in response to drugs

abstract: Antibiotics of the {beta}-lactam (penicillin) family inactivate target enzymes called D,D-transpeptidases or penicillin-binding proteins (PBPs) that catalyze the last cross-linking step of peptidoglycan synthesis. The resulting net-like macromolecule is the essential component of bacterial cell walls that sustains the osmotic pressure of the cytoplasm. In Escherichia coli, bypass of PBPs by the YcbB L,D-transpeptidase leads to resistance to these drugs. We developed a new method based on heavy isotope labeling and mass spectrometry to elucidate PBP- and YcbB-mediated peptidoglycan polymerization. PBPs and YcbB similarly participated in single-strand insertion of glycan chains into the expanding bacterial side wall. This absence of any transpeptidase-specific signature suggests that the peptidoglycan expansion mode is determined by other components of polymerization complexes. YcbB did mediate {beta}-lactam resistance by insertion of multiple strands that were exclusively cross-linked to existing tripeptide-containing acceptors. We propose that this unprecedented mode of polymerization depends upon accumulation of linear glycan chains due to PBP inactivation, formation of tripeptides due to cleavage of existing cross-links by a {beta}-lactam-insensitive endopeptidase, and concerted cross-linking by YcbB.


title: Evolution of the folding landscape of effector caspases

abstract: Caspases are a family of cysteinyl proteases that control programmed cell death and maintain homeostasis in multicellular organisms. The caspase family is an excellent model to study protein evolution because all caspases are produced as zymogens (procaspases) that must be activated to gain full activity; the protein structures are conserved through hundreds of millions of years of evolution; and some allosteric features arose with the early ancestor while others are more recent evolutionary events. The apoptotic caspases evolved from a common ancestor into two distinct subfamilies: monomers (initiator caspases) or dimers (effector caspases). Differences in activation mechanisms of the two subfamilies, and their oligomeric forms, play a central role in the regulation of apoptosis. Here, we examine changes in the folding landscape by characterizing human effector caspases and their common ancestor. The results show that the effector caspases unfold by a minimum three-state equilibrium model at pH 7.5, where the native dimer is in equilibrium with a partially folded monomeric (procaspase-7, common ancestor) or dimeric (procaspase-6) intermediate. In comparison, the unfolding pathway of procaspase-3 contains both oligomeric forms of the intermediate. Overall, the data show that the folding landscape was first established with the common ancestor and was then retained for >650 million years. Partially folded monomeric or dimeric intermediates in the ancestral ensemble provide mechanisms for evolutionary changes that affect stability of extant caspases. The conserved folding landscape allows for the fine-tuning of enzyme stability in a species-dependent manner while retaining the overall caspase-hemoglobinase fold.


title: Developing a Novel Positronium Biomarker for Cardiac Myxoma Imaging

abstract:
Here, positronium imaging is presented to determine cardiac myxoma (CM) extracted from patients undergoing urgent cardiac surgery due to unexpected atrial masses. Positronium is an atom build from an electron and a positron, produced copiously in intra-molecular voids during the PET imaging. CM, the most common cardiac tumor in adults, accounts for 50-75% of benign cardiac tumors. We aimed to assess if positronium serves as a biomarker for diagnosing CM. Perioperative examinations and histopathology staining in six patients confirmed the primary diagnosis of CM. We observed significant differences in the mean positronium lifetime between tumor and normal tissues, with an average value of 1.92(02) ns and 2.72(05) ns for CM and the adipose tissue, respectively. Our findings, combined with positronium lifetime imaging, reveals the novel emerging positronium biomarker for cardiovascular imaging.

One-Sentence SummaryPositronium may serve as an imaging biomarker for cancer diagnostics.


title: iRhom2 regulates ERBB signalling to promote KRAS-driven oncogenesis

abstract: Dysregulation of the ERBB/EGFR signalling pathway causes multiple types of cancer (1, 2). Accordingly, ADAM17, the primary shedding enzyme that releases and activates ERBB ligands, is tightly regulated. It has recently become clear that iRhoms, inactive members of the rhomboid-like superfamily, are regulatory cofactors for ADAM17 (3, 4). Here we show that oncogenic KRAS mutants target the cytoplasmic domain of iRhom2 to induce ADAM17-dependent shedding and the release of ERBB ligands. Activation of ERK1/2 by oncogenic KRAS induces the phosphorylation of iRhom2, recruitment of the phospho-binding 14-3-3 proteins, and consequent ADAM17-dependent shedding of ERBB ligands. In addition, cancer-associated mutations in iRhom2 act as sensitisers in this pathway by further increasing KRAS-induced shedding of ERBB ligands. This mechanism is conserved in lung cancer cells, where iRhom activity is required for tumour xenograft growth. In this context, the activity of oncogenic KRAS is modulated by the iRhom2-dependent release of ERBB ligands, thus placing iRhom2 as a central component of a positive feedback loop in lung cancer cells. Overall, the cytoplasmic domain of iRhom2 is a critical component of KRAS-induced oncogenesis of lung cancer cells. Both ADAM17 and iRhom2 have also been implicated in a wide range of other cancers (5-10), so the mechanism we have revealed may also have wider oncogenic significance.

category: cancer biology


title: Quantitative analysis of tumour spheroid structure

abstract: Tumour spheroids are common in vitro experimental models of avascular tumour growth. Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour microenvironment where spatial differences in nutrient availability strongly influence growth. We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids. We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region. Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density. Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size. Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines.


title: Histone H3K36me2-specific methyltransferase ASH1L is required for the MLL-AF9-induced leukemogenesis

abstract: ASH1L and MLL1 are two histone methyltransferases that facilitate transcriptional activation during normal development. However, the roles of ASH1L and its enzymatic activity in the development of MLL-rearranged leukemias are not fully elucidated in the Ash1L gene knockout animal models. In this study, we used an Ash1L conditional knockout mouse model to show that loss of ASH1L in hematopoietic progenitor cells impaired the initiation of MLL-AF9-induced leukemic transformation in vitro. Furthermore, genetic deletion of ASH1L in the MLL-AF9-transformed cells impaired the maintenance of leukemic cells in vitro and largely blocked the leukemia progression in vivo. Importantly, the loss of ASH1L function in the Ash1L-deleted cells could be rescued by wild-type but not the catalytic-dead mutant ASH1L, suggesting the enzymatic activity of ASH1L was required for its function in promoting MLL-AF9-induced leukemic transformation. At the molecular level, ASH1L enhanced the MLL-AF9 target gene expression by directly binding to the gene promoters and modifying the local histone H3K36me2 levels. Thus, our study revealed the critical functions of ASH1L in promoting the MLL-AF9-induced leukemogenesis, which provides a molecular basis for targeting ASH1L and its enzymatic activity to treat MLL-arranged leukemias.


title: Unraveling the Connections between Calreticulin and Myeloproliferative Neoplasms via Calcium Signalling

abstract: Mutations in the form of insertions and deletions (INDEL) in the calreticulin gene lead to essential thrombocythemia which is characterized by the formation of thrombosis. However, the connection between calreticulin INDEL and essential thrombocythemia remains largely elusive. Through combined molecular dynamics simulation and in-vitro studies on the wild type and mutated isoforms of calreticulin, the mechanism underlying the calreticulin INDEL induced essential thrombocythemia was investigated at the molecular level. Our results demonstrate that mutations in exon-9 could lead to significant conformational variations of calreticulin structure and thereby reducing its interaction with calcium ions due to decreased electrostatic contributions. The consequence of mutations on calreticulins structural integrity was revealed by identifying the key residues and their roles in calcium binding. Furthermore, mutations implemented by CRISPR-Cas9 in exon-9 showed diminished calcium signaling in HEK-293T cells, which agree well with our in-silico findings. The current study might help in understanding of the interactions between calreticulin exon-9 INDEL and calcium ions mediated by the structural variations of calreticulin. The results provide useful information for designing novel therapeutic approaches targeting essential thrombocythemia.


title: Proteomic landscape of multi-layered breast cancer internal tumor heterogeneity

abstract: Despite extensive research, internal tumor heterogeneity presents enormous challenges to achieve complete therapeutic responses. Changes in protein expression are central determinants of cancer phenotypes that reflect potential therapeutic targets. However, previous proteomic studies did not address internal heterogeneity, therefore, masked the necessary spatial resolution to achieve a comprehensive understanding of cancer complexity. Here we present the first large-scale multi-focal breast cancer proteomic study of 330 tumor regions which associated cancer cell function, pathological parameters, and spatial localization of each tumor region. We found marked internal proteomic heterogeneity even within tumors presenting homogeneous receptor expression. Additionally, analysis of the internal heterogeneity, based on coexisting receptor expression or histological patterns in single tumors, showed significant functional differences between homogeneous and heterogeneous tumors related to cancer metabolism, immunogenicity, and proliferation. We anticipate that this study will serve as a starting point towards the development of improved cancer therapy and diagnostics.


title: Fat specific adipose triglyceride lipase is necessary for iron-mediated lipolysis and lipid mobilization in response to negative energy balance

abstract: Maintenance of energy balance is essential for the overall health of an organism. In mammals, both negative and positive energy balance are associated with disease states. To maintain their energy balance within a defined homeostatic setpoint, mammals have evolved complex regulatory mechanisms that control energy intake and expenditure. Traditionally, studies have focused on understanding the role of macronutrient physiology in energy balance. In the present study, we examined the role of the essential micronutrient iron in regulating energy balance. Using a dietary model, we found that a short course of excess dietary iron caused a negative energy balance resulting in a severe whole body wasting phenotype. This disruption in energy balance was due to an iron dependent increase in energy expenditure caused by a heightened basal metabolic rate and activity level. Using a transgenic mouse model lacking adipose triglyceride lipase (ATGL) specifically in fat tissue, we found that to meet the increased energetic demands, dietary iron caused increased lipid utilization that required fat specific ATGL-mediated lipid mobilization and wasting of subcutaneous white adipose tissue deposits. When fed dietary iron, mice lacking fat-specific ATGL activity were protected from fat wasting, and developed a severe cachectic response that is necessary to meet the increased energetic demands caused by the dietary regimen. Our work highlights the multi-faceted role of iron regulation of organismal metabolism and provides a novel in vivo mechanism for micronutrient control of lipolysis that is necessary for regulating mammalian energy balance.

category: physiology


title: Integrating omics approaches to discover and prioritize candidate genes involved in oil biosynthesis in soybean

abstract: Soybean is one of the major sources of edible protein and oil. Oil content is a quantitative trait that is significantly determined by genetic and environmental factors. Over the past 30 years, a large volume of soybean genetic, genomic, and transcriptomic data have been accumulated. Nevertheless, integrative analyses of such data remain scarce, in spite of their importance for crop improvement. We hypothesized that the co-occurrence of genomic regions for oil-related traits in different studies may reveal more stable regions encompassing important genetic determinants of oil content and quality in soybean. We integrated publicly available data, obtained with distinct techniques, to discover and prioritize candidate genes involved in oil biosynthesis and regulation in soybean. We detected key fatty acid biosynthesis genes (e.g., BCCP and ACCase, FADs, KAS family proteins) and several transcripton factors, which are likely regulators of oil biosynthesis. In addition, we identified new candidates for seed oil accumulation and quality, such as Glyma.03G213300 and Glyma.19G160700, which encode a translocator protein and a histone acetyltransferase, respectively. Further, oil and protein genomic hotspots are strongly associated with breeding and not with domestication, suggesting that soybean domestication prioritized other traits. The genes identified here are promising targets for breeding programs and for the development of soybean lines with increased oil content and quality.

category: plant biology


title: Shading Reduces Water Deficit in Strawberry (Fragaria x ananassa Duch.) Plants during Vegetative Growth

abstract: Strawberry (Fragaria xananassa Duch.) is a commercially important crop with high water requirements, for which it is necessary to find strategies that mitigate the influence of water deficit on plant growth. This study was aimed to evaluate the effects of shading on the vegetative growth of strawberry cv. Sweet Ann under water deficit. The treatments consisted of the combination of two levels of shading (light intensity reduced on 47% vs. non-shaded plants) and two levels of water availability (water deficit vs. well-watered plants). The water deficit reduced the leaf water potential from -1.52 to -2.21 MPa, and diminished stomatal conductance, net photosynthetic rate (from 9.13 to 2.5 mol m-2 s-1), photosystem II photochemical efficiency (from 0.79 to 0.67), and biomass accumulation, while increased the electrolyte leakage. The shading allowed the water-deficient plants to maintain water potential (-1.58 MPa) and photosystem II efficiency (0.79) and to increase water use efficiency (from 14.80 to 86.90 mol CO2/mmol H2O), net photosynthetic rate (from 2.40 to 9.40 mol m-2 s-1) and biomass of leaves, crowns, and roots compared to non-shaded plants without water limitation. These results suggest that a reduction in incident light intensity attenuates the effects of stomatic and non-stomatic limitations caused by water deficit during vegetative growth of strawberry.


title: A Network-centric Framework for theEvaluation of Mutual Exclusivity Tests onCancer Drivers

abstract: One of the key concepts employed in cancer driver gene identification is that of mutual exclusivity (ME); a driver mutation is less likely to occur in case of an earlier mutation that has common functionality in the same molecular pathway. Several ME tests have been proposed recently, however the current protocols to evaluate ME tests have two main limitations. Firstly the evaluations are mostly with respect to simulated data and secondly the evaluation metrics lack a network-centric view. The latter is especially crucial as the notion of common functionality can be achieved through searching for interaction patterns in relevant networks. We propose a network-centric framework to evaluate the pairwise significances found by statistical ME tests. It has three main components. The first component consists of metrics employed in the network-centric ME evaluations. Such metrics are designed so that network knowledge and the reference set of known cancer genes are incorporated in ME evaluations under a careful definition of proper control groups. The other two components are designed as further mechanisms to avoid confounders inherent in ME detection on top of the network-centric view. To this end, our second objective is to dissect the side effects caused by mutation load artifacts where mutations driving tumor subtypes with low mutation load might be incorrectly diagnosed as mutually exclusive. Finally, as part of the third main component, the confounding issue stemming from the use of nonspecific interaction networks generated as combinations of interactions from different tissues is resolved through the creation and use of tissue-specific networks in the proposed framework. The data, the source code and useful scripts are available at: https://github.com/abu-compbio/NetCentric.


title: Is the genomics cart before the restoration ecology horse? Insights from qualitative interviews and trends from the literature

abstract: Harnessing new technologies is vital to achieve global imperatives to restore degraded ecosystems. We explored the potential of genomics as one such tool. We aimed to understand barriers hindering the uptake of genomics, and how to overcome them, via exploratory interviews with leading scholars in both restoration and its sister discipline of conservation - a discipline that has successfully leveraged genomics. We also conducted an examination of research trends to explore some insights that emerged from the interviews, including publication trends that have used genomics to address restoration and conservation questions. Our qualitative findings revealed varied perspectives in harnessing genomics. For example, scholars in restoration without genomics experience felt genomics was over-hyped. Scholars with genomics experience emphatically emphasised the need to proceed cautiously in using genomics in restoration. Both genomics-experienced and less-experienced scholars called for case studies to demonstrate the benefits of genomics in restoration. These qualitative data contrasted with our examination of research trends, which revealed 70 restoration genomics studies, particularly studies using environmental DNA as a monitoring tool. We provide a roadmap to facilitate the uptake of genomics into restoration, to help the restoration sector meet the monumental task of restoring huge areas to biodiverse and functional ecosystems.

category: scientific communication and education


title: Seipin forms a flexible cage at lipid droplet formation sites

abstract: Lipid droplets (LDs) form in the endoplasmic reticulum by phase separation of neutral lipids. This process is facilitated by the seipin protein complex, which consists of a ring of seipin monomers, with yet unclear function. Here, we report a structure of yeast seipin based on cryo-electron microscopy and structural modeling data. Seipin forms a decameric, cage-like structure with the lumenal domains forming a stable ring at the cage floor and transmembrane segments forming the cage sides and top. The transmembrane segments interact with adjacent monomers in two distinct, alternating conformations. These conformations result from changes in switch regions, located between the lumenal domains and the transmembrane segments, that are required for seipin function. Our data suggest a model for LD formation in which a closed seipin cage enables TG phase separation and subsequently switches to an open conformation to allow LD growth and budding.

category: molecular biology


title: Spatial organisation and homeostasis of epithelial lineages at the gastroesophageal junction is regulated by the divergent Wnt mucosal microenvironment

abstract: The gastroesophageal junction (GEJ), where squamous and columnar epithelia meet, is a hotspot for Barretts metaplasia development, dysbiosis and carcinogenesis. However, the mechanisms regulating GEJ homeostasis remain unclear. Here, by employing organoids, bulk and single-cell transcriptomics, single-molecule RNA in situ hybridisations and lineage tracing, we identified the spatial organisation of the epithelial, stromal compartment and the regulators that maintain the normal GEJ homeostasis. During development, common KRT8 progenitors generate committed unilineage p63/KRT5-squamous and KRT8-columnar stem cells responsible for the regeneration of postnatal esophagus and gastric epithelium that meet at GEJ. A unique spatial distribution of Wnt regulators in the underlying stromal compartment of these stem cells creates diverging Wnt microenvironments at GEJ and supports their differential regeneration. Further, we show that these tissue-resident stem cells do not possess the plasticity to transdifferentiate to the other lineage with the altered Wnt signals. Our study provides invaluable insights into the fundamental process of GEJ homeostasis and is crucial for understanding disease development.


title: The role of genetic diversity and arbuscular mycorrhizal fungal diversity in population recovery of the semi-natural grassland plant species Succisa pratensis

abstract:
BackgroundEcosystem restoration is as a critical tool to counteract the decline of biodiversity and recover vital ecosystem services. Restoration efforts, however, often fall short of meeting their goals. Although functionally important levels of biodiversity can significantly contribute to the outcome of ecosystem restoration, they are often overlooked. One such important facet of biodiversity is within-species genetic diversity, which is fundamental to population fitness and adaptation to environmental change. Also the diversity of arbuscular mycorrhizal fungi (AMF), obligate root symbionts that regulate nutrient and carbon cycles, potentially plays a vital role in mediating ecosystem restoration outcome. In this study, we investigated the relative contribution of intraspecific population genetic diversity, AMF diversity, and their interaction, to population recovery of Succisa pratensis, a key species of nutrient poor semi natural grasslands. We genotyped 180 individuals from 12 populations of S. pratensis and characterized AMF composition in their roots, using microsatellite markers and next generation amplicon sequencing, respectively. We also investigated whether the genetic makeup of the host plant species can structure the composition of root-inhabiting AMF communities.

ResultsOur analysis revealed that population allelic richness was strongly positively correlated to relative population growth, whereas AMF richness and its interaction with population genetic diversity did not significantly contribute. The variation partitioning analysis showed that, after accounting for soil and spatial variables, the plant genetic makeup explained a small but significant part of the unique variation in AMF communities.

ConclusionsOur results confirm that population genetic diversity can contribute to population recovery, highlighting the importance of within-species genetic diversity for the success of restoration. We could not find evidence, however, that population recovery benefits from the presence of more diverse AMF communities. Our analysis also showed that the genetic makeup of the host plant structured root-inhabiting AMF communities, suggesting that the plant genetic makeup may be linked to genes that control symbiosis development.


title: slimr: An R package for integrating data and tailor-made population genomic simulations over space and time

abstract: Software for realistically simulating complex population genomic processes is revolutionizing our understanding of evolutionary processes, and providing novel opportunities for integrating empirical data with simulations. However, the integration between simulation software and software designed for working with empirical data is currently not well developed. Here we present slimr, an R package designed to create a seamless link between standalone software SLiM 3.0, one of the most powerful population genomic simulation frameworks, and the R development environment, with its powerful data manipulation and analysis tools. We show how slimr facilitates smooth integration between genetic data, ecological data and simulation in a single environment. The package enables pipelines that begin with data reading, cleaning, and manipulation, proceed to constructing empirically-based parameters and initial conditions for simulations, then to running numerical simulations, and finally to retrieving simulation results in a format suitable for comparisons with empirical data - aided by advanced analysis and visualization tools provided by R. We demonstrate the use of slimr with an example from our own work on the landscape population genomics of desert mammals, highlighting the advantage of having a single integrated tool for both data analysis and simulation. slimr makes the powerful simulation ability of SliM 3.0 directly accessible to R users, allowing integrated simulation projects that incorporate empirical data without the need to switch between software environments. This should provide more opportunities for evolutionary biologists and ecologists to use realistic simulations to better understand the interplay between ecological and evolutionary processes.

category: evolutionary biology


title: The challenge of delimiting cryptic species, and a supervised machine learning solution

abstract: AO_SCPLOWBSTRACTC_SCPLOWThe diversity of biological and ecological characteristics of organisms, and the underlying genetic patterns and processes of speciation, makes the development of universally applicable genetic species delimitation methods challenging. Many approaches, like those incorporating the multispecies coalescent, sometimes delimit populations and overestimate species numbers. This issue is exacerbated in taxa with inherently high population structure due to low dispersal ability, and in cryptic species resulting from nonecological speciation. These taxa present a conundrum when delimiting species: analyses rely heavily, if not entirely, on genetic data which over split species, while other lines of evidence lump. We showcase this conundrum in the harvester Theromaster brunneus, a low dispersal taxon with a wide geographic distribution and high potential for cryptic species. Integrating morphology, mitochondrial, and sub-genomic (double-digest RADSeq and ultraconserved elements) data, we find high discordance across analyses and data types in the number of inferred species, with further evidence that multispecies coalescent approaches over split. We demonstrate the power of a supervised machine learning approach in effectively delimiting cryptic species by creating a "custom" training dataset derived from a well-studied lineage with similar biological characteristics as Theromaster. This novel approach uses known taxa with particular biological characteristics to inform unknown taxa with similar characteristics, and uses modern computational tools ideally suited for species delimitation while also considering the biology and natural history of organisms to make more biologically informed species delimitation decisions. In principle, this approach is universally applicable for species delimitation of any taxon with genetic data, particularly for cryptic species.


title: Differential use of multiple genetic sex determination systems in divergent ecomorphs of an African crater lake cichlid

abstract: African cichlid fishes not only exhibit remarkably high rates of speciation but also have some of the fastest evolving sex determination systems in vertebrates. However, little is known empirically in cichlids about the genetic mechanisms generating new sex-determining variants, what forces dictate their fate, the demographic scales at which they evolve, and whether they are related to speciation. To address these questions, we looked for sex-associated loci in full genome data from 647 individuals of Astatotilapia calliptera from Lake Masoko, a small isolated crater lake in Tanzania, which contains two distinct ecomorphs of the species. We identified three separate XY systems on recombining chromosomes. Two Y alleles derive from mutations that increase expression of the gonadal soma-derived factor gene (gsdf) on chromosome 7; the first is a tandem duplication of the entire gene observed throughout much of the Lake Malawi haplochromine cichlid radiation to which A. calliptera belongs, and the second is a 5 kb insertion directly upstream of gsdf. Both the latter variant and another 700 bp insertion on chromosome 19 responsible for the third Y allele arose from transposable element insertions. Males belonging to the Masoko deep-water benthic ecomorph are determined exclusively by the gsdf duplication, whereas all three Y alleles are used in the Masoko littoral ecomorph, in which they appear to act antagonistically among males with different amounts of benthic admixture. This antagonism in the face of ongoing admixture may be important for sustaining multifactorial sex determination in Lake Masoko. In addition to identifying the molecular basis of three coexisting sex determining alleles, these results demonstrate that genetic interactions between Y alleles and genetic background can potentially affect fitness and adaptive evolution.


title: Monomorphic ESS does not imply the stability of the corresponding polymorphic state in the replicator dynamics in matrix games under time constraints

abstract: One of the main result in the theory of classical evolutionary matrix games (Maynard Smith and Price 1973, Maynard Smith 1982) claims that monomorphic ESS condition implies the stability of the corresponding state of the polymorphic replicator dynamics (Hofbauer et al. 1979, Zeeman 1980). The picture was then refined by Cressman (1990) introducing the strong stability concept which says that if there is a monomorphic ESS then stable polymorphism is established in polymorphic populations. In this paper we demonstrate with examples that this relationship generally does not hold in three or higher dimensions if times related to the interactions vary with the strategies of the participants.


title: GC content but not nucleosome positioning directly contributes to intron-splicing efficiency in Paramecium

abstract: Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the >90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur co-transcriptionally and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We showed that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of [~]151 bp, amongst the smallest periodicities reported. Our analysis revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint to locate introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for the nonsense-mediated decay effectors, we showed that introns located at the edge of nucleosomes display higher splicing efficiency than those at the centre. However, multiple regression analysis indicated that the GC content, rather than nucleosome positioning, directly contributes to intron splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning and intron evolution in Paramecium.


title: Small-molecule ligands can inhibit -1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses

abstract: Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases since 2000 have high-lighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use -1 programmed ribosomal frameshifting (-1 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates -1 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored a group of 6 small-molecule ligands, evaluating their activity against the frameshift signals from a panel of representative bat CoVs--the most likely source of future zoonoses--as well as SARS-CoV-2 and MERS-CoV. We found that whereas some ligands had notable activity against only a few of the frameshift signals, the serine protease inhibitor nafamostat suppressed -1 PRF significantly in several of them, while having limited to no effect on -1 PRF caused by frameshift signals from other viruses used as negative controls. These results suggest it is possible to find small-molecule ligands that inhibit -1 PRF specifically in a broad spectrum of CoVs, establishing the frameshift signal as a viable target for developing pan-coronaviral therapeutics.


title: Expression of soluble methane monooxygenase in Escherichia coli enables methane conversion

abstract: Natural gas and biogas provide an opportunity to harness methane as an industrial feedstock. Bioconversion is a promising alternative to chemical catalysis, which requires extreme operating conditions and exhibits poor specificities. Though methanotrophs natively utilize methane, efforts have been focused on engineering platform organisms like Escherichia coli for synthetic methanotrophy. Here, a synthetic E. coli methanotroph was developed by engineering functional expression of the Methylococcus capsulatus soluble methane monooxygenase in vivo via expression of its cognate GroESL chaperone. Additional overexpression of E. coli GroESL further improved activity. Incorporation of an acetone formation pathway then enabled the conversion of methane to acetone in vivo, as validated via 13C tracing. This work provides the first reported demonstration of methane bioconversion to liquid chemicals in a synthetic methanotroph.

category: bioengineering


title: Identification of biomarkers, pathways and potential therapeutic targets for heart failure using bioinformatics analysis

abstract: Heart failure (HF) is a complex cardiovascular diseases associated with high mortality. To discover key molecular changes in HF, we analyzed next-generation sequencing (NGS) data of HF. In this investigation, differentially expressed genes (DEGs) were analyzed using limma in R package from GSE161472 of the Gene Expression Omnibus (GEO). Then, gene enrichment analysis, protein-protein interaction (PPI) network, miRNA-hub gene regulatory network and TF-hub gene regulatory network construction, and topological analysis were performed on the DEGs by the Gene Ontology (GO), REACTOME pathway, STRING, HiPPIE, miRNet, NetworkAnalyst and Cytoscape. Finally, we performed receiver operating characteristic curve (ROC) analysis of hub genes. A total of 930 DEGs 9464 up regulated genes and 466 down regulated genes) were identified in HF. GO and REACTOME pathway enrichment results showed that DEGs mainly enriched in localization, small molecule metabolic process, SARS-CoV infections and the citric acid (TCA) cycle and respiratory electron transport. Subsequently, the PPI network, miRNA-hub gene regulatory network and TF-hub gene regulatory network were constructed, and 10 hub genes in these network were focused on by centrality analysis and module analysis. Furthermore, data showed that HSP90AA1, ARRB2, MYH9, HSP90AB1, FLNA, EGFR, PIK3R1, CUL4A, YEATS4 and KAT2B were good diagnostic values. In summary, this study suggests that HSP90AA1, ARRB2, MYH9, HSP90AB1, FLNA, EGFR, PIK3R1, CUL4A, YEATS4 and KAT2B may act as the key genes in HF.


title: The Inherent Flexibility of Receptor Binding Domains in SARS-CoV-2 Spike Protein

abstract: Spike (S) protein is the primary antigenic target for neutralization and vaccine development for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It decorates the virus surface and undergoes large conformational changes of its receptor binding domain (RBD) to enter the host cell, as the abundant structural studies suggest. Here, we observe Down, one-Up, one-Open, and two-Up-like structures in enhanced molecular dynamics simulations without pre-defined reaction coordinates. The RBDA transition from Down to one-Up is supported by transient salt-bridges between RBDA and RBDC and by the glycan at N343B. Reduced interactions between RBDA and RBDB induce the RBDB motions toward two-Up. Glycan shielding for neutralizing antibodies is the weakest in one-Open. Cryptic pockets are revealed at the RBD interfaces in intermediate structures between Down and one-Up. The inherent flexibility in S-protein is, thus, essential for the structure transition and shall be considered for antiviral drug rational design or vaccine development.


title: Presenting the Compendium Isotoporum Medii Aevi (CIMA) and Bayesian Case Studies

abstract: AO_SCPLOWBSTRACTC_SCPLOWThe Compendium Isotoporum Medii Aevi (CIMA) gathers more than 50 000 isotopic measurements for bioarchaeological samples located within Europe and its margins dating between AD 500-1500. This volume of isotopic data, together with collected supporting information, offers multiple research opportunities. This is illustrated here using novel Bayesian modelling methods on selected case studies to reconstruct medieval human lifeways (i.e. human subsistence, spatial mobility), animal management practices, and paleo-environmental conditions. We also discuss how the integration of isotopic data with other types of archaeological and historical data can improve our knowledge of historical developments throughout medieval Europe.

category: paleontology


title: Agent-Based Model of Multicellular Spheroid Pattern Formation Driven by Synthetic Cell Adhesion Signaling Circuits

abstract: Differential adhesion within cell populations enables the emergence of unique patterns in heterogeneous multicellular systems. This process has been previously explored using synthetically engineered heterogenous multicell spheroid systems, in which cell subpopulations engage in bidirectional intercellular signaling to regulate the expression of different cadherins. While engineered cell systems provide excellent experimental tools to observe pattern formation in cell populations, computational models may be leveraged to more systematically explore the key parameters that drive the emergence of different patterns. We developed and validated two- and three-dimensional agent-based models (ABMs) of spheroid patterning for cells engineered with a bidirectional signaling circuit that regulates N- and P-cadherin expression. The model was used to predict how varying initial cell seedings, cadherin induction probabilities, or homotypic adhesion strengths between cells impact spheroid patterning, and unsupervised machine learning techniques were used to map system parameters to unique spheroid patterns. The model was then deployed to design new synthetic cell signaling circuits based on a desired final multicell pattern.

category: systems biology


title: A computational modeling approach for predicting multicell patterns based on signaling-induced differential adhesion


title: A population of descending neurons that regulate the flight motor of Drosophila

abstract: Like many insect species, Drosophila melanogaster are capable of maintaining a stable flight trajectory for periods lasting up to several hours(1, 2). Because aerodynamic torque is roughly proportional to the fifth power of wing length(3), even small asymmetries in wing size require the maintenance of subtle bilateral differences in flapping motion to maintain a stable path. Flies can even fly straight after losing half of a wing, a feat they accomplish via very large, sustained kinematic changes to the both damaged and intact wings(4). Thus, the neural network responsible for stable flight must be capable of sustaining fine-scaled control over wing motion across a large dynamic range. In this paper, we describe an unusual type of descending neurons (DNg02) that project directly from visual output regions of the brain to the dorsal flight neuropil of the ventral nerve cord. Unlike most descending neurons, which exist as single bilateral pairs with unique morphology, there is a population of at least 15 DNg02 cell pairs with nearly identical shape. By optogenetically activating different numbers of DNg02 cells, we demonstrate that these neurons regulate wingbeat amplitude over a wide dynamic range via a population code. Using 2-photon functional imaging, we show that DNg02 cells are responsive to visual motion during flight in a manner that would make them well suited to continuously regulate bilateral changes in wing kinematics. Collectively, we have identified a critical set of DNs that provide the sensitivity and dynamic range required for flight control.

category: neuroscience


title: Pupil correlates of decision variables in mice playing a competitive mixed-strategy game

abstract: In a competitive game involving an animal and an opponent, the outcome is contingent on the choices of both players. To succeed, the animal must continually adapt to competitive pressure, or else risk being exploited and lose out on rewards. In this study, we demonstrate that head-fixed mice can be trained to play the iterative competitive game  matching pennies against a virtual computer opponent. We find that the animals performance is well described by a hybrid computational model that includes Q-learning and choice kernels. Comparing between matching pennies and a non-competitive two-armed bandit task, we show that the tasks encourage animals to operate at different regimes of reinforcement learning. To understand the involvement of neuromodulatory mechanisms, we measure fluctuations in pupil size and use multiple linear regression to relate the trial-by-trial transient pupil responses to decision-related variables. The analysis reveals that pupil responses are modulated by observable variables, including choice and outcome, as well as latent variables for value updating, but not action selection. Collectively, these results establish a paradigm for studying competitive decision-making in head-fixed mice and provide insights into the role of arousal-linked neuromodulation in the decision process.


title: Voltage imaging in Drosophila using a hybrid chemical-genetic rhodamine voltage reporter

abstract:
We combine a chemically-synthesized, voltage-sensitive fluorophore with a genetically encoded, self-labeling enzyme to enable voltage imaging in Drosophila melanogaster. Previously, we showed that a rhodamine voltage reporter (RhoVR) combined with the HaloTag self-labeling enzyme could be used to monitor membrane potential changes from mammalian neurons in culture and brain slice. Here, we apply this hybrid RhoVR-Halo approach in vivo to achieve selective neuron labeling in intact fly brains. We generate a Drosophila UAS-HaloTag reporter line in which the HaloTag enzyme is expressed on the surface of cells. We validate the voltage sensitivity of this new construct in cell culture before driving expression of HaloTag in specific brain neurons in flies. We show that selective labeling of synapses, cells, and brain regions can be achieved with RhoVR-Halo in either larval neuromuscular junction (NMJ) or in whole adult brains. Finally, we validate the voltage sensitivity of RhoVR-Halo in fly tissue via dual-electrode/imaging at the NMJ, show the efficacy of this approach for measuring synaptic excitatory post-synaptic potentials (EPSPs) in muscle cells, and perform voltage imaging of carbachol-evoked depolarization and osmolarity-evoked hyperpolarization in projection neurons and in interoceptive subesophageal zone neurons in fly brain explants following in vivo labeling. We envision the turn-on response to depolarizations, fast response kinetics, and two-photon compatibility of chemical indicators, coupled with the cellular and synaptic specificity of genetically-encoded enzymes, will make RhoVR-Halo a powerful complement to neurobiological imaging in Drosophila.

Significance StatementVoltage imaging is a powerful method for interrogating neurobiology. Chemical indicators possess fast response kinetics, turn-on responses to membrane depolarization, and can be compatible with two-photon excitation. However, selective cell labeling in intact tissues and in vivo remains a challenge for completely synthetic fluorophores. Here, we show that a chemical - genetic hybrid approach in Drosophila enables cell-specific staining in vivo and voltage imaging in whole-brain explants and at neuromuscular junction synapses.


title: Contextual expectations shape cortical reinstatement of sensory representations

abstract: When making a turn at a familiar intersection, we know what items and landmarks will come into view. These perceptual expectations, or predictions, come from our knowledge of the context, however its unclear how memory and perceptual systems interact to support the prediction and reactivation of sensory details in cortex. To address this, human participants learned the spatial layout of animals positioned in a cross maze. During fMRI, participants navigated between animals to reach a target, and in the process saw a predictable sequence of five animal images. Critically, to isolate activity patterns related to item predictions, rather than bottom-up inputs, one quarter of trials ended early, with a blank screen presented instead. Using multivariate pattern similarity analysis, we reveal that activity patterns in early visual cortex, posterior medial regions, and the posterior hippocampus showed greater similarity when seeing the same item compared to different items. Further, item effects in posterior hippocampus were specific to the sequence context. Critically, activity patterns associated with seeing an item in visual cortex and posterior medial cortex, were also related to activity patterns when an item was expected, but omitted, suggesting sequence predictions were reinstated in these regions. Finally, multivariate connectivity showed that patterns in the posterior hippocampus at one position in the sequence were related to patterns in early visual cortex and posterior medial cortex at a later position. Together, our results support the idea that hippocampal representations facilitate sensory processing by modulating visual cortical activity in anticipation of expected items.


title: Transitions in the proteome and phospho-proteome during early embryonic development in Xenopus

abstract: Vertebrate development from an egg to a complex multi-tissue million-cell organism is supported by multiple phases of genome-scale remodeling of the repertoire of proteins and their post-translational modifications, yet so far we know little about these phases. In this paper we present comprehensive characterization of these processes reflected by eleven time points, approximately eleven thousand proteins, and six thousand phospho-forms in two replicates. We find that the most dramatic changes to the proteome occur during the transition to functional organ systems, which occurs as the embryo becomes a tadpole. At that time the absolute amount of non-yolk protein increases two-fold, and there is a shift in the balance of expression from proteins regulating gene expression to receptors, ligands, and proteins involved in cell-cell and cell-environment interactions. Between the early and late tadpole, the median increase of membrane and secreted proteins is substantially higher than that of nuclear proteins. For the first time, we have included quantitative phospho-proteomic data across the same developmental stages. In contrast to the significant protein changes that are concentrated at the end of the time series, the most significant phosphorylation changes are concentrated in the very early stages of development. A clear exception are phosphorylations of proteins involved in gene expression; these increase just after fertilization, with patterns that are highly correlated with the underlying protein changes. To improve our interpretation of this unique data set, we created a pipeline for identifying homologous human phosphorylations from the measured Xenopus phospho-proteome, and we estimated, where possible, the occupancy of phosphorylation sites. Overall, we detected many profound temporal transitions, which suggest concerted changes in developmental strategies in the embryo that are particularly pronounced once early patterning and specification are complete.


title: A general deep learning model for bird detection in high resolution airborne imagery

abstract: Advances in artificial intelligence for computer vision hold great promise for increasing the scales at which ecological systems can be studied. The distribution and behavior of individuals is central to ecology, and computer vision using deep neural networks can learn to detect individual objects in imagery. However, developing supervised models for ecological monitoring is challenging because it needs large amounts of human-labeled training data, requires advanced technical expertise and computational infrastructure, and is prone to overfitting. This limits application across space and time. One solution is developing generalized models that can be applied across species and ecosystems. Using over 250,000 annotations from 13 projects from around the world, we develop a general bird detection model that achieves over 65% recall and 50% precision on novel aerial data without any local training despite differences in species, habitat, and imaging methodology. Fine-tuning this model with only 1000 local annotations increase these values to an average of 84% recall and 69% precision by building on the general features learned from other data sources. Retraining from the general model improves local predictions even when moderately large annotation sets are available and makes model training faster and more stable. Our results demonstrate that general models for detecting broad classes of organisms using airborne imagery are achievable. These models can reduce the effort, expertise, and computational resources necessary for automating the detection of individual organisms across large scales, helping to transform the scale of data collection in ecology and the questions that can be addressed.

category: ecology


title: An introgression breakthrough left by an anthropogenic contact between two ascidians

abstract: Human-driven translocations of species have diverse evolutionary consequences such as promoting hybridization between previously geographically isolated taxa. This is well-illustrated by the solitary tunicate, Ciona robusta, native to the North East Pacific and introduced in the North East Atlantic. It is now co-occurring with its congener C. intestinalis in the English Channel, and C. roulei in the Mediterranean Sea. Despite their long allopatric divergence, first and second generation crosses showed a high hybridization success between the introduced and native taxa in the laboratory. However, previous genetic studies failed to provide evidence of recent hybridization between C. robusta and C. intestinalis in the wild. Using SNPs obtained from ddRAD-sequencing of 397 individuals from 26 populations, we further explored the genome-wide population structure of the native Ciona taxa. We first confirmed results documented in previous studies, notably i) a chaotic genetic structure at regional scale, and ii) a high genetic similarity between C. roulei and C. intestinalis, which is calling for further taxonomic investigation. More importantly, and unexpectedly, we also observed a genomic hotspot of long introgressed C. robusta tracts into C. intestinalis genomes at several locations of their contact zone. Both the genomic architecture of introgression, restricted to a 1.5 Mb region of chromosome 5, and its absence in allopatric populations suggest introgression is recent and occurred after the introduction of the non-indigenous species. Overall, our study shows that anthropogenic hybridization can be effective in promoting introgression breakthroughs between species at a late stage of the speciation continuum.


title: Adaptation of the autosomal part of the genome on the presence of dioecy

abstract: We have attempted to answer the question of whether the presence of sex chromosomes in the genome can affect the evolution of the autosomal part of the genome. As a model, we used dioecious plants from the section Otites of the genus Silene. We have observed a rise in adaptive evolution in the autosomal and pseudoautosomal parts of the genome, which are associated with the evolution of dioecy. This rise is caused neither by the accumulation of sexually antagonistic genes in the pseudoautosomal region nor by the co-evolution of genes acting in mitochondria (in spite of the fact that the dioecy evolved in this case most likely from cytoplasmic male sterility). Thus, this rise in the amount of positively selected codons is most likely caused by the adaptive evolution of genes involved in the specialization of the autosomal part of the genome on the dioecy as described in sex-allocation theory.


title: A graph-theoretical approach to DNA similarity analysis

abstract: One of the very active research areas in bioinformatics is DNA similarity analysis. There are several approaches using alignment-based or alignment-free methods to analyze similarities/dissimilarities between DNA sequences. In this work, we introduce a novel representation of DNA sequences, using n-ary Cartesian products of graphs for arbitrary positive integers n. Each of the component graphs in the representing Cartesian product of each DNA sequence contain combinatorial information of certain tuples of nucleotides appearing in the DNA sequence. We further introduce a metric space structure to the set of all Cartesian products of graphs that represent a given collection of DNA sequences in order to be able to compare different Cartesian products of graphs, which in turn signifies similarities/dissimilarities between DNA sequences. We test our proposed method on several datasets including Human Papillomavirus, Human rhinovirus, Influenza A virus, and Mammals. We compare our method to other methods in literature, which indicates that our analysis results are comparable in terms of time complexity and high accuracy, and in one dataset, our method performs the best in comparison with other methods.

category: genomics


title: Functionalized Biomimetic Hydrogels Enhance Salivary Stem/Progenitor Cell Organization

abstract: Complex branched salivary structures remain challenging to replicate within implant ready hydrogels. We showed previously that hyaluronic acid (HA)-based hydrogels enable growth and organization of primary salivary-derived human stem/progenitor cells (hS/PCs) into multicellular spheroids. Here, we systematically functionalized three components of migration-permissive hydrogels to foster salivary tissue morphogenesis. We separately analyzed contributions of an enzymatically degradable crosslinker, a pendant integrin-binding site, and hydrogel porosity to best support high viability, integrin-dependent cell adhesion and migration. Structure size, frequency, and morphology were all affected by hydrogel crosslink density and integration of biofunctional peptides. Viability and proliferation data suggested that integration of integrin binding sites had the greatest effect on hS/PCs behavior. A larger internal matrix space, created by increasing both crosslinker length and PEG content, was needed to form large multicellular hS/PC structures. Peptide-modified hydrogels with more internal space shifted hS/PC organization from spheroidal, surrounded by thick basement membrane, to an asymmetric arrangement with punctate matrix proteins defining a "wrinkled" perimeter. Integrin-binding peptides activated integrin {beta}1, with highest activation observed in hydrogels having both cleavable peptide and integrin ligand. The design parameters we prescribe allowed us to encapsulate hS/PCs in a humanized biomimetic hydrogel matrix able to support morphogenesis needed for salivary restoration.


title: Large field-of-view incoherent volumetric imaging in living human retina by confocal oblique scanning laser ophthalmoscopy

abstract: Three-dimensional (3D) volumetric imaging of the human retina is instrumental to monitor and diagnose blinding conditions. Although coherent retinal imaging is well established by optical coherence tomography, it is still a large void for incoherent volumetric imaging in the human retina. Here, we report confocal oblique scanning laser ophthalmoscopy (CoSLO), to fill that void and harness incoherent optical contrast in 3D. CoSLO uses oblique scanning laser and remote focusing to acquire depth signal in parallel, avoid the lengthy z-stacking, and image a large field of view (FOV). In addition, confocal gating is introduced by a linear sensor array to improve the contrast and resolution. For the first time, we achieved incoherent 3D human retinal imaging with >20{degrees} viewing angle within only 5 seconds. The depth resolution is [~]45 microns in vivo. We demonstrated label-free incoherent contrast by CoSLO, revealing unique features in the retina. CoSLO will be an important technique for clinical care of retinal conditions and fundamental vision science, by offering unique volumetric incoherent contrasts.


title: A Novel Mouse Wound Model for Scar Tissue Formation in Abdominal-Muscle Wall

abstract:
Scar tissue formation is a result of excess healing reactions after wounding. Hypertrophic scars scarcely develop in a mouse. In the present study, we established a novel experimental model of a scar-forming wound by resecting a small portion of the abdominal wall on the lower center of the abdomen, which exposed contractive forces by the surrounding muscle tissue. As a tension-less control, a back-skin excision model was used with a splint fixed onto the excised skin edge, and granulation tissue formed on the muscle facia supported by the back skeleton. One week after the resection, initial healing reactions such as fibroblast proliferation took place in both models.

However, after 21 days, lesions with collagen-rich granulation tissues forming multiple nodular/spherical-like structures developed only in the abdominal-wall model. The lesions are analogous to scar lesions in humans. Such lesions, however, did not develop in the back-skin excision model. Therefore, this animal model is unique in that fibrous scar tissues form under a physiological condition without using any artificial factors and is valuable for studying the pathogenesis and preclinical treatment of scar lesions.

Summary StatementScar lesions are hardly developed in animals. We thus developed a scar lesion in mice without using any artificial factors. The model is reliable, reproducible, and valuable.

category: pathology


title: Naked mole-rat and Damaraland mole-rat exhibit lower respiration in mitochondria, cellular and organismal levels

abstract: Naked mole-rats (NMR) and Damaraland mole-rats (DMR) are the only two eusocial mammals known. Both species exhibit extraordinary longevity for their body size, high tolerance to hypoxia and oxidative stress and high reproductive output; these collectively defy the concept that all life-history traits should be negatively correlated. However, when life-history traits share similar underpinning physiological mechanisms, these may be positively associated with each other. Here, we propose that the bioenergetic properties of mole-rats share a potential common mechanism. We adopted a top-down perspective measuring the bioenergetic properties at the organismal, cellular, and molecular level in both species and the biological significance of these properties were compared with the same measures in Siberian hamsters and C57BL/6 mice, chosen for their similar body size to the mole-rat species. We found mole-rats shared several bioenergetic properties that differed from their comparator species, including low basal metabolic rates, a high dependence on glycolysis rather than on oxidative phosphorylation for ATP production, and low proton conductance across the mitochondrial inner membrane. These shared mole-rat features could be a result of evolutionary adaptation to tolerating variable oxygen atmospheres, in particular hypoxia, and may in turn be one of the molecular mechanisms underlying their extremely long lifespans.


title: On the edge: Evolution of polarity protein BASL and the capacity for stomatal lineage asymmetric divisions

abstract:
Asymmetric and oriented stem cell divisions enable the continued production of patterned tissues. The molecules that guide these divisions include several "polarity proteins" that are localized to discrete plasma membrane domains, are differentially inherited during asymmetric divisions, and whose scaffolding activities can guide division plane orientation and subsequent cell fates. In the stomatal lineages on the surfaces of plant leaves, asymmetric and oriented divisions create distinct cell types in physiologically optimized patterns. The polarity protein BASL is a major regulator of stomatal lineage division and cell fate asymmetries in Arabidopsis, but its role in the stomatal lineages of other plants is unclear. Here, using phylogenetic and functional assays, we demonstrate that BASL is a eudicot-specific polarity protein. Among dicots, divergence in BASLs roles may reflect some intrinsic protein differences, but more likely reflects previously unappreciated differences in how asymmetric cell divisions are employed for pattern formation in different species. This multi-species analysis therefore provides insight into the evolution of a unique polarity regulator and into the developmental choices available to cells as they build and pattern tissues.

HIGHLIGHTSO_LIBASL is a eudicot-specific regulator of stomatal lineage asymmetric cell divisions
C_LIO_LIBASL protein evolution includes stepwise addition of polarity domains to an ancestral MAPK-binding chassis
C_LIO_LICellular quiescence and BASL-guided polarity generate proper stomatal spacing in tomato
C_LIO_LICell size and fate asymmetries are uncoupled in the tomato stomatal lineage
C_LI


title: Peptidergic modulation of fear responses by the Edinger-Westphal nucleus

abstract: Many neuronal populations that release fast-acting excitatory and inhibitory neurotransmitters in the brain also contain slower acting neuropeptides. These facultative peptidergic cell types are common, but it remains uncertain whether obligate peptidergic neurons exist. Our fluorescence in situ hybridization, genetically-targeted electron microscopy, and electrophysiological characterization data strongly suggest that neurons of the non-cholinergic, centrally-projecting Edinger-Westphal nucleus in mice are fundamentally obligately peptidergic. We further show, using fiber photometry, monosynaptic retrograde tracing, anterograde projection mapping, and a battery of behavioral assays, that this peptidergic population both promotes fear responses and analgesia and activates in response to loss of motor control and pain. Together, these findings elucidate an integrative, ethologically relevant function for the Edinger-Westphal nucleus and functionally align the nucleus with the periaqueductal gray, where it resides. This work advances our understanding of the peptidergic modulation of fear and provides a framework for future investigations of putative obligate peptidergic systems.


title: Differential mechanisms of cold sensitivity in mouse trigeminal and vagal ganglion neurons

abstract:
Thermal signals are critical elements in the operation of interoceptive and exteroceptive neural circuits, essential for triggering thermally-driven reflexes and conscious behaviors. A fraction of cutaneous and visceral sensory endings are activated by cold temperatures. Compared to somatic (DRG and TG) neurons, little is known about the mechanisms underlying cold sensitivity of visceral vagal neurons. We used pharmacological and genetic tools for a side-by-side characterization of cold-sensitive (CS) neurons in adult mouse trigeminal (TG) and vagal ganglia (VG).

We found that CS neurons are more abundant in VG than in TG. In both ganglia, sensitivity to cold varied widely and was enhanced by the potassium channel blocker 4-AP. The majority of CS neurons in VG co-express TRPA1 markers and cold-evoked responses are severely blunted in Trpa1 KO mice, with little impact of TRPM8 deletion or pharmacological TRPM8 blockade. Consistent with these findings, the expression of TRPM8-positive neurons was low in VG and restricted to the rostral jugular ganglion. In vivo retrograde labelling of airway-innervating vagal neurons demonstrated their enhanced cold sensitivity and a higher expression of TRPA1 compared to neurons innervating the stomach wall. In contrast, the majority of CS TG neurons co-express TRPM8 markers and their cold sensitivity is reduced after TRPM8 deletion or blockade. However, pharmacological or genetic reduction of TRPA1 showed that these channels contribute significantly to their cold sensitivity in TG. In both ganglia, a fraction of CS neuron respond to cooling by a mechanism independent of TRPA1 or TRPM8 yet to be characterized.


title: Neural manifolds for odor-driven innate and acquired appetitive preferences

abstract: Sensory stimuli evoke spiking neural responses that innately or after learning drive suitable behavioral outputs. How are these spiking activities intrinsically patterned to encode for innate preferences, and could the neural response organization impose constraints on learning? We examined this issue in the locust olfactory system. Using a diverse odor panel, we found that ensemble activities both during ( ON response) and after stimulus presentations ( OFF response) could be linearly mapped onto overall appetitive preference indices. Although diverse, ON and OFF response patterns generated by innately appetitive odorants were still limited to a low-dimensional subspace (a  neural manifold). Similarly, innately non-appetitive odorants evoked responses that were separable yet confined to another neural manifold. Notably, only odorants that evoked neural response excursions in the appetitive manifold were conducive for learning. In sum, these results provide insights on how encoding for innate preferences can also set limits on associative learning.


title: Interleukin-33 coordinates a microglial phagocytic response and limits corticothalamic excitability and seizure susceptibility

abstract: Microglia are key remodelers of neuronal synapses during brain development, but the mechanisms that regulate this process and its ultimate impact on neural circuit function are not well defined. We previously identified the IL-1 family cytokine Interleukin-33 (IL-33) as a novel mediator of microglial synapse remodeling. Here we define the phagocytic program induced in microglia in response to IL-33. We find that IL-33 markedly alters the microglial enhancer landscape and exposes AP-1 transcription factor sites that promote target gene expression. We identify the scavenger receptor MARCO and the pattern recognition receptor TLR2 as downstream mediators of IL-33 dependent synapse engulfment. Conditional deletion of IL-33 in the CNS or its receptor on microglia results in increased numbers of excitatory synapses in the corticothalamic circuit and spontaneous epileptiform activity as well as increased seizure susceptibility by early adulthood. These findings define novel mechanisms through which IL-33 coordinates experience-dependent synaptic refinement to restrict hyperexcitability in the developing brain.