title: The Foster method: Rapid and non-invasive detection of clinically relevant American Foulbrood disease levels using eDNA sampling and a dual-target qPCR assay, with its potential for other hive pathogens.

abstract:
Clinical signs of American Foulbrood can be difficult to diagnose and thus disease is missed and leads to further spread. Diagnosis is centred on the beekeepers skill in recognising clinical symptoms - a highly subjective and time-consuming activity. Previous testing methods have relied on sampling that necessitates dismantling the hive and/or requires multiple visits to retrieve passive samples. The Foster method is a novel environmental DNA sampling method using entrance swabs together with a dual-target qPCR for AFB. The quantification data generated can be used to detect hives with clinically significant infections, even before visual symptoms are apparent. Such a method will be applicable to other bee pathogens and incursion pests.

ImportanceDiscovery of the disease American foulbrood typically means the destruction of the bees and hive by burning. This discovery is typically by visual examination of capped brood by the beekeeper - a subjective skill that means the disease is being missed or not recognised. It is a time-consuming and exacting process to inspect hives for AFB. Here we present a rapid sampling method that does not require opening/dismantling the hive, in conjunction with a dual target quantitative PCR assay for the bacteria responsible, Paenibacillus larvae. Using the resulting quantitative data, hives presenting clinical symptoms or likely to become clinical visually can be determined and the hives dealt with appropriately before further spread occurs.


title: The Foster method: Rapid and non-invasive detection of clinically significant American Foulbrood disease levels using eDNA sampling and a dual-target qPCR assay, with its potential for other hive pathogens.

category: microbiology


title: Sequencing using a two-steps strategy reveals high genetic diversity in the S gene of SARS-CoV-2 after a high transmission period in Tunis, Tunisia.

abstract:
Recent efforts have reported numerous variants that influence SARS-CoV-2 viral characteristics including pathogenicity, transmission rate and ability of detection by molecular tests. Whole genome sequencing based on NGS technologies is the method of choice to identify all viral variants; however, the resources needed to use these techniques for a representative number of specimens remain limited in many low and middle income countries. To decrease sequencing cost, we developed a couple of primers allowing to generate partial sequences in the viral S gene allowing rapid detection of numerous variants of concern (VOCs) and variants of interest (VOIs); whole genome sequencing is then performed on a selection of viruses based on partial sequencing results. Two hundred and one nasopharyngeal specimens collected during the decreasing phase of a high transmission COVID-19 wave in T unisia were analyzed. The results reveal high genetic variability within the sequenced fragment and allowed the detection of first introduction in the country of already known VOCs and VOIs as well as others variants that have interesting genomic mutations and need to be kept under surveillance.

ImportanceThe method of choice for SARS-CoV-2 variants detection is whole genome sequencing using NGS technologies. Resources for this technology remain limited in many low and middle income countries where it is not possible to perform whole genome sequencing for representative number of SARS-CoV-2 positive cases. In the present work, we developed a novel strategy based on a first partial sanger screening in the S gene including key mutations of the already known VOCs and VOIs for rapid identification of these VOCs and VOIs and helps to better select specimens that need to be sequenced by NGS technologies. The second step consisting in whole genome sequencing allowed to have a holistic view of all variants within the selected viral strains and confirmed the initial classification of the strains based on partial S gene sequencing.


title: Mutations within the Open Reading Frame (ORF) including Ochre stop codon of the Surface Glycoprotein gene of SARS-CoV-2 virus erase potential seed location motifs of human non-coding microRNAs.

abstract: MicroRNA are short and non-coding RNA, 18-25 nucleotides in length. They are produced at the early stage of viral infection. The roles played by cellular miRNAs and miRNA-mediated gene-silencing in the COVID-19 epidemic period is critical in order to develop novel therapeutics. We analysed SARS-CoV-2 Surface Glycoprotein (S) nucleotide sequence originating from India as well as Iran, Australia, Germany, Italy, Russia, China, Japan and Turkey and identified mutation in potential seed location of several human miRNA. Seventy single nucleotide polymorphisms (SNP) were detected in the S gene out of which, 36, 32 and 2 were cases of transitions, transversions and deletions respectively. Eleven human miRNA targets were identified on the reference S gene sequence with a score >80 in the miRDB database. Mutation A845S erased a common binding site of 7 human miRNA (miR-195-5p, miR-16-5p, miR-15b-5p, miR-15a-5p, miR-497-5p, miR-424-5p and miR-6838-5p). A synonymous mutation altered the wild type Ochre stop codon within the S gene sequence (Italy) to Opal thereby changing the seed sequence of miR-511-3p. Similar (synonymous) mutations were detected at amino acid position 659 and 1116 of the S gene where amino acids serine and threonine were retained, abolishing potential seed location for miR-219a-1-3p and miR-20b-3p respectively. The significance of this finding in reference to the strategy to use synthetic miRNA combinations as a novel therapeutic tool is discussed.

category: molecular biology


title: PTT-quant - a new method for direct identification and absolute quantification of premature transcription termination events, following the example of bacterial riboswitches.

abstract: Regulation of gene expression by premature termination of transcription has been well described in all domains of life, including metazoans, yeast, plants and bacteria. Although methods for identification of such regulatory events by sequencing are available, the focused biochemical studies of the mechanism is hampered by lack of highly sensitive and accurate experimental methods. Here we propose a new method for absolute quantification of premature transcription termination events, PTT-quant. It is based on highly sensitive two-step digital droplet PCR protocol, coupled with normalized cDNA synthesis attained by site-specific pre-cleavage of investigated transcripts with RNase H. As a consequence, our method enables the reliable and sensitive quantification of both, prematurely terminated and full-length transcripts. By application of our method to investigation of transcriptional riboswitches in Bacillus subtilis, we were able to precisely measure the dynamics of SAM riboswitch induction, which turned to be [~]23% higher in comparison the results obtained without cDNA synthesis normalization.


title: A method to study extracellular vesicles secreted in vitro by cultured cells with minimum sample processing and extracellular vesicle loss

abstract: Extracellular vesicles (EVs) are involved in a multitude of physiological functions and play important roles in health and disease. The study of EV secretion and EV characterization remains challenging due to the small size of these particles, a lack of universal EV markers, and sample loss or technical artifacts that are often associated with EV separation techniques. We developed a method for in-cell EV labeling with fluorescent lipids (DiI), followed by DiI-labelled EV characterization in the conditioned medium by imaging flow cytometry (IFC). Direct IFC analysis of EVs in the conditioned medium, after removal of apoptotic bodies and cellular debris, significantly reduces sample processing and loss compared to established methods for EV separation, resulting in improved detection of quantitative changes in EV secretion and subpopulations compared to protocols that rely on EV separation by ultracentrifugation. In conclusion, our optimized protocol for EV labeling and analysis reduces EV sample processing and loss, and is well suited for cell biology studies that focus on modulation of EV secretion by cells in culture.

category: cell biology


title: Recycling of nutrient transporters from yeast endosomes is regulated by ubiquitinated Ist1

abstract: Trafficking of cell surface membrane proteins to and from the plasma membrane impinges on myriad biological processes and ensures correct cellular function. Upon internalisation, most surface proteins are either sent to the lysosome for degradation or recycled back to the plasma membrane. Although these endosomal trafficking pathways control surface protein activity, the precise regulatory features and division of labour between interconnected pathways is poorly defined. Furthermore, how well endosomal trafficking mechanisms are conserved across eukaryotes is unclear. In yeast, we show cargo recycling back to the plasma membrane occurs through distinct pathways. In addition to the established retrograde recycling pathways via the late Golgi, which are used by synaptobrevins and driven by cargo ubiquitination, we find nutrient transporter recycling bypasses the Golgi in a pathway driven by cargo deubiquitination. Nutrient transporters rapidly internalise to, and recycle from, endosomes marked by Vps4 and the ESCRT-III associated factor Ist1. This endosome population serve as both  early and  recycling endosomes, implying these features of endosomal organisation are evolutionarily conserved. Ist1 has previously been implicated in recycling in yeast and other eukaryotes. We reveal Ist1 ubiquitination regulates its endosomal recruitment and is required for cargo recycling. Additionally, the ubiquitin-binding adaptor Npl4 and the essential ATPase Cdc48 regulate endosomal Ist1 and are required for cargo recycling, suggesting mechanistic features of recycling from endosomes to the plasma membrane are also conserved.


title: Recycling of cell surface membrane proteins from yeast endosomes is regulated by ubiquitinated Ist1


title: Statistics of eigenvalue dispersion indices: quantifying the magnitude of phenotypic integration

abstract: Quantification of the magnitude of trait covariation plays a pivotal role in the study of phenotypic evolution, for which statistics based on dispersion of eigenvalues of a covariance or correlation matrix--eigenvalue dispersion indices--are commonly used. This study remedies major issues over the use of these statistics, namely, a lack of clear understandings on their statistical justifications and sampling properties. The relative eigenvalue variance of a covariance matrix is known in the statistical literature a test statistic for sphericity, thus is an appropriate measure of eccentricity of variation. The same of a correlation matrix is equal to the average squared correlation, which has a straightforward interpretation as a measure of integration. Expressions for the mean and variance of these statistics are analytically derived under multivariate normality, clarifying the effects of sample size N, number of variables p, and parameters on sampling bias and error. Simulations confirmed that approximations involved are reasonably accurate with a moderate sample size (N [≥] 16-64). Importantly, sampling properties of these indices are not adversely affected by a high p:N ratio, promising their utility in high-dimensional phenotypic analyses. They can furthermore be applied to shape variables and phylogenetically structured data with appropriate modifications.

category: evolutionary biology


title: Impact of genomic preselection on subsequent genetic evaluations with ssGBLUP - using real data from pigs

abstract:
BackgroundEmpirically assessing the impact of preselection on subsequent genetic evaluations of preselected animals requires comparison of scenarios taking into account different approaches, including scenarios without preselection. However, preselection almost always takes place in animal breeding programs, so it is difficult to have a dataset without preselection. Hence most studies on preselection used simulated datasets, concluding that genomic estimated breeding values (GEBV) from subsequent single-step genomic best linear unbiased prediction (ssGBLUP) evaluations are unbiased. The aim of this study was to investigate the impact of genomic preselection (GPS) on accuracy and bias in subsequent ssGBLUP evaluations, using data from a commercial pig breeding program.

MethodsWe used data on four pig production traits from one sire line and one dam line. The traits are average daily gain during performance testing, average daily gain throughout life, backfat thickness, and loin depth. As these traits had different weights in the breeding goals of the two lines, we analyzed the two lines separately. Per line, we had a reference GPS scenario which kept all available data, against which the next two scenarios were compared. We then implemented two other scenarios with additional layers of GPS by removing all animals without progeny either i) only in the validation generation, or ii) in all generations. We conducted subsequent ssGBLUP evaluations per GPS scenario, utilizing all the data remaining after implementing the GPS scenario. In computing accuracy and bias, we compared GEBV against progeny yield deviations of validation animals.

ResultsResults for all traits in both lines showed marginal loss in accuracy due to the additional layers of GPS. Average accuracy across all GPS scenarios in both lines was 0.39, 0.47, 0.56, and 0.60 respectively for the four traits considered in this study. Bias was largely absent, and when present did not differ greatly among corresponding GPS scenarios.

ConclusionAs preselection generally has the same effect in animal breeding programs, we concluded that impact of preselection is generally minimal on accuracy and bias in subsequent ssGBLUP evaluations of selection candidates in pigs and in other animal breeding programs.

category: genetics


title: Mapping the host protein interactome of non-coding regions in SARS-CoV-2 genome

abstract: A deep understanding of SARS-CoV-2-host interactions is crucial to the development of effective therapeutics. The role of non-coding regions of viral RNA (ncrRNAs) has not been scrutinized. We developed a method using MS2 affinity purification coupled with liquid chromatography-mass spectrometry (MAMS) to systematically map the interactome of SARS-CoV-2 ncrRNA in different human cell lines. Integration of the results defined the core and cell-type-specific ncrRNA-host protein interactomes. The majority of ncrRNA-binding proteins were involved in RNA biogenesis, protein translation, viral infection, and stress response. The 5' UTR interactome is enriched with proteins in the snRNP family and is a target for the regulation of viral replication and transcription. The 3' UTR interactome is enriched with proteins involved in the cytoplasmic RNP granule (stress granule) and translation regulation. We show that the ORF10 is likely to be a part of 3' UTR. Intriguingly, the interactions between negative-sense ncrRNAs and host proteins, such as translation initiation factors and antiviral factors, suggest a pathological role of negative-sense ncrRNAs. Moreover, the cell-type-specific interactions between ncrRNAs and mitochondria may explain the differences of cell lines in viral susceptibility. Our study unveils a comprehensive landscape of the functional SARS-CoV-2 ncrRNA-host protein interactome, providing a new perspective on virus-host interactions and the design of future therapeutics.

category: genomics


title: Discovery of SARS-CoV-2 Mpro Peptide Inhibitors from Modelling Substrate and Ligand Binding

abstract: The main protease (Mpro) of SARS-CoV-2 is central to its viral lifecycle and is a promising drug target, but little is known concerning structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of classical molecular mechanics and quantum mechanical techniques, including automated docking, molecular dynamics (MD) simulations, linear-scaling DFT, QM/MM, and interactive MD in virtual reality, to investigate the molecular features underlying recognition of the natural Mpro substrates. Analyses of the subsite interactions of modelled 11-residue cleavage site peptides, ligands from high-throughput crystallography, and designed covalently binding inhibitors were performed. Modelling studies reveal remarkable conservation of hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular at the P2/S2 sites. The binding modes of the natural substrates, together with extensive interaction analyses of inhibitor and fragment binding to Mpro, reveal new opportunities for inhibition. Building on our initial Mpro-substrate models, computational mutagenesis scanning was employed to design peptides with improved affinity and which inhibit Mpro competitively. The combined results provide new insight useful for the development of Mpro inhibitors.

category: bioinformatics


title: Structural basis for the interaction of SARS-CoV-2 virulence factor nsp1 with Pol α - Primase

abstract:
The molecular mechanisms that drive the infection by the SARS-CoV-2 coronavirus - the causative agent of the COVID-19 (Coronavirus disease-2019) pandemic - are under intense current scrutiny, to understand how the virus operates and to uncover ways in which the disease can be prevented or alleviated.

Recent cell-based analyses of SARS-CoV-2 protein - protein interactions have mapped the human proteins targeted by the virus. The DNA polymerase  - primase complex or primosome - responsible for initiating DNA synthesis in genomic duplication - was identified as a target of nsp1 (non structural protein 1), a major virulence factor in the SARS-CoV-2 infection.

Here, we report the biochemical characterisation of the interaction between nsp1 and the primosome and the cryoEM structure of the primosome - nsp1 complex. Our data provide a structural basis for the reported interaction between the primosome and nsp1. They suggest that Pol  - primase plays a part in the immune response to the viral infection, and that its targeting by SARS-CoV-2 aims to interfere with such function.

category: biophysics


title: On electrical spiking of Ganoderma resinaceum

abstract: Fungi exhibit action-potential like spiking activity. Up to date most electrical activity of oyster fungi has been characterised in sufficient detail. It remains unclear if there are any patterns of electrical activity specific only for a certain set of species or if all fungi share the same  language of electrical signalling. We use pairs of differential electrodes to record extracellular electrical activity of the antler-like sporocarps of the polypore fungus Ganoderma resinaceum. The patterns of the electrical activity are analysed in terms of frequency of spiking and parameters of the spikes. The indicators of the propagation of electrical activity are also highlighted.


title: WWP1 deficiency protects from cardiac remodeling induced by simulated microgravity

abstract: Cardiac muscle is extremely sensitive to changes in loading conditions, the microgravity during space flight can cause cardiac remodeling and function decline. At present, the mechanism of microgravity-induced cardiac remodeling remains to be revealed. WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) is an important activator of pressure-overload induced cardiac remodeling by stabilizing disheveled segment polarity proteins 2 (DVL2) and activating CaMKII/HDAC4/MEF2C axis. However, the role of WWP1 in the cardiac remodeling induced by microgravity is unknown. The purpose of this study was to determine whether WWP1 was also involved in the regulation of cardiac remodeling caused by microgravity. Firstly, we detected the expression of WWP1 and DVL2 in the heart from mice and monkeys after simulated microgravity using western blotting and Immunohistochemistry. Secondly, WWP1 knockout (KO) and wild type mice were subjected to hindlimb unloading (HU) to simulate microgravity effect. We assessed the cardiac remodeling in morphology and function through histological analysis and echocardiography. Finally, we detected the phosphorylation level of CaMKII and HDAC4 in the heart from WT and WWP1 KO mice after HU. The results revealed the increased expression of WWP1 and DVL2 in the heart both from mice and monkey after simulated microgravity. WWP1 deficiency protected against simulated microgravity-induced cardiac atrophy and function decline. Histological analysis demonstrated WWP1 KO inhibited the decreases in the size of individual cardiomyocytes of mice after hindlimb unloading. WWP1 KO can inhibit the activation of DVL2/CaMKII/HDAC4 pathway in heart of mice induced by simulated microgravity. These results demonstrated WWP1 as a potential therapeutic target for cardiac remodeling and function decline induced by simulated microgravity.

category: physiology


title: EcoEvoApps: Interactive Apps for Teaching Theoretical Models in Ecology and Evolutionary Biology

abstract: Theoretical models are fundamental in Ecology and Evolutionary Biology (EEB), but they can be difficult to teach in undergraduate classrooms because students come in with inconsistent mathematical training and varying attitudes towards mathematics. A promising way to make these models more approachable is to use active learning exercises that allow model exploration and simulation. Thus, we present EcoEvoApps, a collection of free, open-source interactive web apps that simulate various theoretical EEB models. By surveying students who used these apps in two remote undergraduate Ecology courses in the U.S.A., we found that using the apps led to considerable gains in students confidence and understanding of the focal models and underlying concepts. We envision EcoEvoApps as a widely available, equitable tool for students around the globe. Consequently, we have developed EcoEvoApps as a fundamentally collaborative platform, with an aim to build an international community of EEB researchers, educators, and students.

category: scientific communication and education


title: Tracking the Brain's Intrinsic Connectivity Networks in EEG

abstract: Functional magnetic resonance imaging (fMRI) has identified dysfunctional network dynamics underlying a number of psychopathologies, including post-traumatic stress disorder, depression and schizophrenia. There is tremendous potential for the development of network-based clinical biomarkers to better characterize these disorders. However, to realize this potential requires the ability to track brain networks using a more affordable imaging modality, such as Electroencephalography (EEG). Here we present a novel analysis pipeline capable of tracking brain networks from EEG alone, after training on supervisory signals derived from data simultaneously recorded in EEG and fMRI, while people engaged in various cognitive tasks. EEG-based features were then used to classify three cognitively-relevant brain networks with up to 75% accuracy. These findings could lead to affordable and non-invasive methods to objectively diagnose brain disorders involving dysfunctional network dynamics, and to track and even predict treatment responses.

category: neuroscience


title: Differences in IgG antibody responses following BNT162b2 and mRNA-1273 Vaccines

abstract: Studies examining antibody responses by vaccine brand are lacking and may be informative for optimizing vaccine selection, dosage, and regimens. The purpose of this study is to assess IgG antibody responses following immunization with BNT162b2 (30 g S protein) and mRNA-1273 (100 g S protein) vaccines. A cohort of clinicians at a non-for-profit organization is being assessed clinically and serologically following immunization with BNT162b2 or mRNA-1273. IgG responses were measured at the Remington Laboratory by an IgG against the SARS-CoV-2 spike protein-receptor binding domain. Mixed-effect linear (MEL) regression modeling was used to examine whether the SARS-CoV-2 IgG level differed by vaccine brand, dosage, or days since vaccination. Among 532 SARS-CoV-2 seronegative participants, 530 (99.6%) seroconverted with either vaccine. After adjustments for age and gender MEL regression modeling revealed that the average IgG increased after the second dose compared to the first dose (p<0.001). Overall, titers peaked at week six for both vaccines. Titers were significantly higher for mRNA-1273 vaccine on days 14-20 (p < 0.05), 42-48 (p < 0.01), 70-76 (p < 0.05), 77-83 (p < 0.05), and higher for BNT162b2 vaccine on days 28-34 (p < 0.001). In two participants taking immunosuppressive drugs SARS-CoV-2 IgG remained negative. mRNA-1273 elicited both earlier and higher IgG antibody responses than BNT162b2, possibly due to the higher S-protein delivery. Prospective clinical and serological follow-up of defined cohorts such as this may prove useful in determining antibody protection and whether differences in antibody kinetics between the vaccines have manufacturing relevance and clinical significance.

category: immunology


title: The percentage of Monocytes CD39+ is higher in Pregnant COVID-19 than in Non-Pregnant COVID-19 patients

abstract: Current medical guidelines consider COVID-19 pregnant women a high-risk group. Physiological gestation down regulates the immunological response to maintain "maternal-fetal tolerance"; hence, a SARS-CoV-2 infection constitutes a potentially threatening condition to both the mother and the fetus. To establish the immune profile in pregnant COVID-19+ patients a cross-sectional study was conducted. Leukocyte immunophenotype, mononuclear leukocyte response to polyclonal stimulus and cytokine/chemokine serum concentration were analyzed in pregnant fifteen COVID-19+ and control groups (fifteen non-pregnant COVID-19+, and thirteen pregnant COVID-19-women). Pregnant COVID-19+ patients exhibit lower percentages of monocytes HLA-DR+ compared with control groups. Nevertheless, pregnant COVID-19+ women show a higher percentage of monocytes CD39+ than controls. Furthermore, a higher concentration of TNF-, IL-6, MIP1b and IL-4 was observed within the pregnant COVID-19+ group. Our result shows that pregnant women express immunological characteristics that potentially mediate the immune response in COVID-19.


title: Cell culture evolution of a HSV-1/VZV UL34/ORF24 chimeric virus reveals novel functions for HSV genes in capsid nuclear egress

abstract:
HSV and VZV are both members of the alphaherpesvirus subfamily, but are of different genera. Substitution of the HSV-1 UL34 coding sequence with that of its VZV homolog, ORF24, results in a virus that has defects in viral growth, spread, capsid egress, and nuclear lamina disruption very similar to those seen in a UL34-null virus despite normal interaction between ORF24 protein and HSV pUL31 and proper localization of the NEC at the nuclear envelope. Minimal selection for growth in cell culture resulted in viruses that grew and spread much more efficiently that the parental chimeric virus. These viruses varied in their ability to support nuclear lamina disruption, normal NEC localization and capsid de-envelopment. Single mutations that suppress the growth defect were mapped to the coding sequences of ORF24, ICP22 and ICP4 and one virus carried single mutations in each of the ICP22 and US3 coding sequences. The phenotypes of these viruses support a role for ICP22 in nuclear lamina disruption and a completely unexpected role for the major transcriptional regulator, ICP4, in capsid nuclear egress.

ImportanceInteractions among virus proteins are critical for assembly and egress of virus particles, and such interactions are attractive targets for antiviral therapy. Identification of critical functional interactions can be slow and tedious. Capsid nuclear egress of herpesviruses is a critical event in the assembly and egress pathway and is mediated by two proteins that are conserved among herpesviruses, pUL31 and pUL34. Here we describe a cell culture evolution approach to identify other viral gene products that functionally interact with pUL34.


title: Phylogenetic study of thermophilic genera Anoxybacillus, Geobacillus, Parageobacillus, and proposal of a new classification Quasigeobacillus gen. nov.

abstract: A phylogenetic study of Anoxybacillus, Geobacillus and Parageobacillus was performed using publicly available whole genome sequences. A total of 113 genomes were selected for phylogenomic metrics including calculation of Average Nucleotide Identity (ANI) and Average Amino acid Identity (AAI), and a maximum likelihood tree was built from alignment of a set of 662 orthologous core genes. The combined results from the core gene tree and ANI and AAI dendrograms show that the genomes split into two main clades, clade I containing all Geobacillus, all Parageobacillus and some species of Anoxybacillus, and clade II, containing the majority of Anoxybacillus species. Clade I is further partitioned into three clades, consisting separately of Geobacillus, Parageobacillus, and a third clade which we suggest should be elevated to a new genus Quasigeobacillus gen. nov. Two species of Anoxybacillus showed inconsistent positioning among the trees produced by differing methods and could not be clearly resolved into any of the three existing genera or the new genus. This research shows the importance of considering closely related genera together when studying phylogeny or assigning genomic affinities.


title: Release of Notch activity coordinated by IL-1β signalling confers differentiation plasticity of airway progenitors via Fosl2 during alveolar regeneration

abstract: While the acquisition of cellular plasticity in adult stem cells is essential for rapid regeneration after tissue injury, little is known about the underlying molecular mechanisms governing this process. Our data reveal the coordination of airway progenitor differentiation plasticity by inflammatory signals during alveolar regeneration. Upon damage, IL-1{beta} signalling-dependent modulation of Jag1/2 expression in ciliated cells results in the inhibition of Notch signalling in secretory cells, which drives reprogramming and acquisition of differentiation plasticity. We identify a core role for the transcription factor Fosl2/Fra2 in secretory cell fate conversion to alveolar type 2 (AT2) cells retaining the distinct genetic and epigenetic signatures of secretory lineages. We furthermore reveal that KDR/FLK-1+ human secretory cells display a conserved capacity to generate AT2 cells via Notch inhibition. Our results demonstrate the functional role of a IL-1{beta}-Notch-Fosl2 axis for the fate decision of secretory cells during injury repair, proposing a new potential therapeutic target for human lung alveolar regeneration.


title: The Cx43 Carboxyl-Terminal Mimetic Peptide alphaCT1 Protects Endothelial Barrier Function in a ZO1 Binding-Competent Manner

abstract: The Cx43 CT mimetic peptide, CT1, originally designed to bind to ZO1 and thereby inhibit Cx43/ZO1 interaction, was used as a tool to probe the role of Cx43/ZO1 association in regulation of epithelial/endothelial barrier function. Using both in vitro and ex vivo methods of barrier function measurement, including Electric Cell-Substrate Impedance Sensing(ECIS), a FITC-dextran transwell permeability assay, and a FITC-dextran cardiovascular leakage protocol involving Langendorff-perfused mouse hearts, CT1 was found to protect the endothelium from thrombin-induced breakdown in cell-cell contacts. Barrier protection was accompanied by significant remodeling of the F-actin cytoskeleton, characterized by a redistribution of F-actin away from the cytoplasmic and nuclear regions of the cell, towards the endothelial cell periphery, in association with alterations in cellular orientation distribution. In line with observations of increased cortical F-actin, CT1 upregulated cell-cell border localization of endothelial VE-cadherin, the Tight Junction protein Zonula Occludens 1 (ZO1), and the Gap Junction Protein (GJ) Connexin43 (Cx43). A ZO1-binding-incompetent variant of CT1, CT1-I, indicated that these effects on barrier function and barrier-associated proteins, were likely associated with Cx43 CT sequences retaining ability to interact with ZO1. These results implicate the Cx43 CT and its interaction with ZO1, in the regulation of endothelial barrier function, while revealing the therapeutic potential of CT1 in the treatment of vascular edema.


title: Ensuring that fundamentals of quantitative microbiology are reflected in microbial diversity analyses based on next-generation sequencing

abstract:
Diversity analysis of amplicon sequencing data is mainly limited to plug-in estimates calculated using normalized data to obtain a single value of an alpha diversity metric or a single point on a beta diversity ordination plot for each sample. As recognized for count data generated using classical microbiological methods, read counts obtained from a sample are random data linked to source properties by a probabilistic process. Thus, diversity analysis has focused on diversity of (normalized) samples rather than probabilistic inference about source diversity. This study applies fundamentals of statistical analysis for quantitative microbiology (e.g., microscopy, plating, most probable number methods) to sample collection and processing procedures of amplicon sequencing methods to facilitate inference reflecting the probabilistic nature of such data and evaluation of uncertainty in diversity metrics. Types of random error are described and clustering of microorganisms in the source, differential analytical recovery during sample processing, and amplification are found to invalidate a multinomial relative abundance model. The zeros often abounding in amplicon sequencing data and their implications are addressed, and Bayesian analysis is applied to estimate the source Shannon index given unnormalized data (both simulated and real). Inference about source diversity is found to require knowledge of the exact number of unique variants in the source, which is practically unknowable due to library size limitations and the inability to differentiate zeros corresponding to variants that are actually absent in the source from zeros corresponding to variants that were merely not detected. Given these problems with estimation of diversity in the source even when the basic multinomial model is valid, sample-level diversity analysis approaches are discussed.

HighlightsO_LIRandom error in amplicon sequencing method should be considered in diversity analysis
C_LIO_LIClustering, amplification, and differential recovery distort sample diversity
C_LIO_LIThe multinomial model for compositional count data is compromised by amplification
C_LIO_LIThere are three types of zeros in amplicon sequencing data, including missing zeros
C_LIO_LISource alpha diversity estimates are biased by unknown number of unique variants
C_LI


title: A framework for evaluating the performance of SMLM cluster analysis algorithms

abstract: Single molecule localisation microscopy (SMLM) generates data in the form of Cartesian coordinates of localised fluorophores. Cluster analysis is an attractive route for extracting biologically meaningful information from such data and has been widely applied. Despite the range of developed cluster analysis algorithms, there exists no consensus framework for the evaluation of their performance. Here, we use a systematic approach based on two metrics, the Adjusted Rand Index (ARI) and Intersection over Union (IoU), to score the success of clustering algorithms in diverse simulated clustering scenarios mimicking experimental data. We demonstrate the framework using three analysis algorithms: DBSCAN, ToMATo and KDE, show how to deduce optimal analysis parameters and how they are affected by fluorophore multiple blinking. We propose that these standard conditions and metrics become the basis for future analysis algorithm development and evaluation.


title: In situ X-ray assisted electron microscopy staining for large biological samples

abstract:
Electron microscopy of biological tissue has recently seen an unprecedented increase in imaging throughput moving the ultrastructural analysis of large tissue blocks such as whole brains into the realm of the feasible. However, homogeneous, high quality electron microscopy staining of large biological samples is still a major challenge. To date, assessing the staining quality in electron microscopy requires running a sample through the entire staining protocol end-to-end, which can take weeks or even months for large samples, rendering protocol optimization for such samples to be inefficient.

Here we present an in situ time-lapsed X-ray assisted staining procedure that opens the "black box" of electron microscopy staining and allows observation of individual staining steps in real time. Using this novel method we measured the accumulation of heavy metals in large tissue samples immersed in different staining solutions. We show that the measured accumulation of osmium in fixed tissue obeys empirically a quadratic dependence between the incubation time and sample size. We found that potassium ferrocyanide, a classic reducing agent for osmium tetroxide, clears the tissue after osmium staining and that the tissue expands in osmium tetroxide solution, but shrinks in reduced osmium solution.

X-ray assisted staining gave access to the in situ staining kinetics and allowed us to develop a diffusion-reaction-advection model that accurately simulates the measured accumulation of osmium in tissue. These are first steps towards in silico staining experiments and simulation-guided optimization of staining protocols for large samples. Hence, X-ray assisted staining will be a useful tool for the development of reliable staining procedures for large samples such as entire brains of mice, monkeys or humans.


title: Purinoreceptors and ectonucleotidases control ATP-induced calcium waveforms and calcium-dependent responses in microglia

abstract: 1Adenosine triphosphate (ATP) drives microglia motility and cytokine production by activating P2X- and P2Y- class purinergic receptors with extracellular ATP and its metabolites. Purinergic receptor activation gives rise to diverse intracellular Ca2+ signals, or waveforms, that differ in amplitude, duration, and frequency. Whether and how these diverse waveforms influence microglia function is not well established. We developed a computational model trained with published primary murine microglia studies. We simulate how purinoreceptors influence Ca2+ signaling and migration and how purinoreceptor expression modifies these processes. Our simulation confirmed that P2 receptors encode the amplitude and duration of the ATP-induced calcium waveforms. Our simulations also implicate CD39, an ectonucleotidase that rapidly degrades ATP, as a regulator of purinergic receptor-induced Ca2+ responses. We, therefore, next evaluated how purinoreceptors and ectonucleotidase work in tandem. Our modeling results indicate that small transients are sufficient to promote motility, while large and sustained transients are needed for cytokine responses. Lastly, we predict how these phenotypical responses vary in a BV2 microglia cell line using published P2 receptor mRNA data to illustrate how our computer model can be extrapolated to diverse microglia subtypes. These findings provide important insights into how differences in purinergic receptor expression influence the microglias responses to ATP.


title: Yet another mitochondrial genome of the Pacific cupped oyster: the published mitogenome of Alectryonella plicatula (Ostreinae) is based on a misidentified Magallana gigas (Crassostreinae)

abstract: The recently published mitochondrial genome of the fingerprint oyster Alectryonella plicatula (Gmelin, 1791) with GenBank accession number MW143047 was resolved in an unexpected phylogenetic position, as sister to the Pacific cupped oyster Magallana gigas (Thunberg, 1793) and share with this species three typical gene duplications that represent robust synapomorphies of the Magallana clade. In this study, we verified the identity of MW143047 using direct comparisons of single gene sequences, DNA barcoding and phylogenetic analyses. BLAST searches using each of the 12 protein coding genes and rRNA genes extracted from MW143047 as query retrieved M. gigas as best hit with 100% sequence identity. MW143047 is nested within the clade formed by M. gigas sequences, with virtually no difference between their terminal branch lengths, both in the cox1 gene tree (based on 3639 sequences) and in the 16S gene tree (based on 1839 sequences), as well as in the Maximum Likelihood mitogenomic tree based on concatenated sequence of 12 PCGs. Our findings suggest that the original specimen used for mitogenome sequencing was misidentified and represents an individual of M. gigas. This study reinforces the notion that morphological shell analysis alone is not sufficient for oyster identification, not even at high taxonomic ranks such as subfamilies. While it is well established that morphological identification of oysters should be validated by molecular data, this study emphasizes that also molecular data should be taxonomically validated by means of DNA barcoding and phylogenetic analyses. The implications of the publication of taxonomically misidentified sequences and mitogenomes are discussed.

category: zoology


title: Do we really understand the role of the prefrontal cortex in placebo analgesia?

abstract:
Several reviews have strongly implicated prefrontal cortical engagement in expectation-based placebo analgesia. We recently found a robust placebo analgesic response and associated decreases in pain-related cortical activations, without observable prefrontal engagement. We hypothesized our substantial conditioning and weak verbal instructions diminished expectation-related prefrontal activation. To test this, we examined the same subjects during a conditioning procedure, in which expectancy of pain relief was high. In two conditioning sessions, noxious heat was applied to a leg region treated with an "analgesic" cream and another treated with a "moisturizing" cream. In reality, both creams were inert, but the temperature applied to the moisturizing-cream area was 2{degrees}C higher than that applied to the analgesic-cream area.

Functional MRI was acquired during the second conditioning session. Pain ratings were lower for the low heat than the high heat, with corresponding reduced activations in pain-related regions. Similar to previous studies with strong expectation for pain relief, we observed more prefrontal activations during the "analgesic" than the control condition. Nevertheless, contrary to the idea of active prefrontal engagement, the relative activation was based on differences in negative BOLD signals. A literature review revealed that only a few studies conclusively showed active engagement of prefrontal cortex, i.e. increased positive BOLD signal during high expectation compared to a control, with variable timing and spatial-specificity. We suggest that this variability is due to the heterogeneous influence of cognitive, emotional and motivational factors. Future studies should attempt to unravel the multiple contributions to placebo responsiveness in the prefrontal cortex.


title: Engaging Biological Oscillators through Second Messenger Pathways Permits Emergence of a Robust Gastric Slow-Wave during Peristalsis

abstract:
Peristalsis, the coordinated contraction - relaxation of the muscles of the stomach is important for normal gastric motility and is impaired in motility disorders. Coordinated electrical depolarizations that originate and propagate within a network of interconnected layers of interstitial cells of Cajal (ICC) and smooth muscle (SM) cells of the stomach wall as a slow-wave, underly peristalsis. Normally, the gastric slow-wave oscillates with a single period and uniform rostrocaudal lag, exhibiting network entrainment. Understanding of the integrative role of neurotransmission and intercellular coupling in the propagation of an entrained gastric slow-wave, important for understanding motility disorders, however, remains incomplete. Using a computational framework constituted of a novel gastric motility network (GMN) model we address the hypothesis that engaging biological oscillators (i.e., ICCs) by constitutive gap junction coupling mechanisms and enteric neural stimulus activated signals can confer a robust entrained gastric slow-wave. We demonstrate that while a decreasing enteric neural stimulus gradient that modulates the intracellular IP3 concentration in the ICCs can guide the aboral slow-wave propagation essential for peristalsis, engaging ICCs by recruiting the exchange of second messengers (inositol trisphosphate (IP3) and Ca2+) ensures a robust entrained longitudinal slow-wave, even in the presence of biological variability in coupling strengths. Our GMN with the distinct intercellular coupling in conjunction with the intracellular feedback pathways and a rostrocaudal enteric neural stimulus gradient allows gastric slow waves to oscillate with a moderate range of frequencies and to propagate with a broad range of velocities, thus preventing decoupling observed in motility disorders. Overall, the findings provide a mechanistic explanation for the emergence of decoupled slow waves associated with motility impairments of the stomach, offer directions for future experiments and theoretical work, and can potentially aid in the design of new interventional pharmacological and neuromodulation device treatments for addressing gastric motility disorders.

Author SummaryThe coordinated contraction and relaxation of the muscles of the stomach, known as peristalsis is important for normal gastric motility and primarily governed by electrical depolarizations that originate and propagate within a network of interconnected layers of interstitial cells of Cajal (ICCs) and smooth muscle cells of the stomach wall as a slow-wave. Under normal conditions, a gastric slow-wave oscillates with a single period and uniform rostrocaudal lag, exhibiting network entrainment. However, the understanding of intrinsic and extrinsic mechanisms that ensure propagation of a robust entrained slow-wave remains incomplete. Here, using a computational framework, we show that in conjunction with an enteric neural stimulus gradient along the rostrocaudal ICC chain, and intercellular electrical coupling, the intercellular exchange of inositol trisphosphate between ICCs prevents decoupling by extending the longitudinal entrainment range along the stomach wall, even when variability in intercellular coupling exists. The findings from our study indicate ways that ensure the rostrocaudal spread of a robust gastric slow-wave and provide a mechanistic explanation for the emergence of decoupled slow waves associated with motility impairments of the stomach.


title: Development of opioid-induced hyperalgesia depends on reactive astrocytes controlled by Wnt5a signaling

abstract:
Opioid analgesics are the frontline pain medicine for managing various types of pain. Paradoxically, repeated use of opioid analgesics may cause an exacerbated pain state known as opioid-induced hyperalgesia (OIH). OIH significantly contributes to dose escalation and consequently opioid overdose. In addition to neuronal malplasticity, emerging evidence suggests a critical role of reactive glia in OIH development. A potential astrocytic underpinning of OIH pathogenesis is indicated by their prominently activation in OIH animal models. However, this hypothesis has not been conclusively tested and the mechanism underlying the astrocyte activation remains unclear. Here, we show that reactive astrocytes (a.k.a. astrogliosis) are critical for OIH development in mice. Genetic ablation of astrogliosis inhibited the expression of OIH and morphine-induced neural circuit polarization (NCP) in the spinal dorsal horn (SDH). We also found that Wnt5a is a neuron-to-astrocyte signal that is required for morphine-induced astrogliosis. Conditional knock-out of Wnt5a in neurons or its co-receptor ROR2 in astrocytes blocked not only morphine-induced astrogliosis but also OIH and NCP. Furthermore, we showed that the Wnt5a-ROR2 signaling-dependent astrogliosis contributes to OIH via inflammasome-regulated IL-1{beta}. Our results reveal an important role of morphine-induced astrogliosis in OIH pathogenesis and elucidate a neuron-to-astrocyte intercellular Wnt signaling pathway that controls the astrogliosis.

One sentence summaryNeuron-to-astrocyte Wnt5a signaling controls the pathogenesis of opioid-induced hyperalgesia via astrogliosis


title: MRP5 and MRP9 Play a Concerted Role in Male Reproduction and Mitochondrial Function

abstract:
Multidrug Resistance Proteins (MRPs) are transporters that play critical roles in cancer even though the physiological substrates of these enigmatic transporters are poorly elucidated. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter as mrp-5 mutants are unviable due to their inability to export heme from the intestine to extra-intestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. Correspondingly, cell biological studies show that MRP5 regulates heme levels in the mammalian secretory pathway even though MRP5 knockout (KO) mice do not show reproductive phenotypes. The closest homolog of MRP5 is MRP9/ABCC12, which is absent in C. elegans raising the possibility that MRP9 may genetically compensate for MRP5. Here, we show that MRP5 and MRP9 double KO mice are viable but reveal significant male reproductive deficits. Although MRP9 is highly expressed in sperm, MRP9 KO mice show reproductive phenotypes only when MRP5 is absent. Both ABCC transporters localize to mitochondrial-associated membranes (MAMs), dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, DKO mice reveal abnormal sperm mitochondria with reduced mitochondrial membrane potential and fertilization rates. Metabolomics show striking differences in metabolite profiles in the DKO testes and RNA-seq show significant alterations in genes related to mitochondrial function and retinoic acid metabolism. Targeted functional metabolomics reveal lower retinoic acid levels in the DKO testes and higher levels of triglycerides in the mitochondria. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating male reproductive functions and mitochondrial sufficiency.

Significance StatementMRPs are typically implicated in cancer biology. Here, we show that MRP9 and MRP5 localize to mitochondrial-associated membranes and play a concerted role in maintaining mitochondrial homeostasis and male reproductive fitness. Our work fills in significant gaps in our understanding of MRP9 and MRR5 with wider implications in male fertility. It is plausible that variants in these transporters are associated with male reproductive dysfunction.


title: A compendium of novel genomics technologies provides a chromosome-scale assembly and insights into the sex determining system of the Greenland Halibut

abstract: Despite the commercial importance of Greenland Halibut (Reinhardtius hippoglossoides), important gaps still persist in our knowledge of this species, including its reproductive biology and sex determination mechanism. In this study, we combined single molecule sequencing of long reads (Pacific Sciences) with Chromatin Conformation Capture sequencing (Hi-C) data to provide the first chromosome-level genome reference for this species. The high-quality assembly encompassed more than 598 Megabases (Mb) assigned to 1 594 scaffolds (scaffold N50 = 25 Mb) with 96 % of its total length distributed among 24 chromosomes. The investigation of its syntenic relationships with other economically important flatfish species revealed a high conservation of synteny blocks among members of this phylogenetic clade. Sex determination analysis revealed that flatfishes do not escape the rule applied to other teleost fish and exhibit a high level of plasticity and turnover in sex-determination mechanisms. A whole-genome sequence analysis of 198 individuals allowed us to draw a full picture of the molecular sex determination (SD) system for Greenland Halibut, revealing that this species possesses a very nascent male heterogametic XY system, with a putative major effect of the sox2 gene, also described as the main SD driver in two other flatfishes. Interestingly, our study also suggested for the first time in flatfishes that a putative Y-autosomal fusion could be associated with a reduction of recombination typical of early steps of sex chromosome evolution.


title: A Novel Membrane Protein in the Rhodobacter sphaeroides LH1-RC Photocomplex

abstract: We present a cryo-EM structure of the monomeric light-harvesting-reaction center (LH1-RC) core complex from photosynthetic purple bacterium Rhodobacter (Rba.) sphaeroides at 2.9 [A] resolution. The LH1 complex forms a C-shaped structure composed of 14 {beta}-polypeptides around the RC with a large ring opening. From the cryo-EM density map, a previously unrecognized integral membrane protein, referred to as protein-U, was identified. Protein-U has a U-shaped conformation near the LH1-ring opening and was annotated as a hypothetical protein in the Rba. sphaeroides genome. Deletion of protein-U resulted in a mutant strain that expressed a much-reduced amount of the dimeric LH1-RC, indicating an important role for protein-U in dimerization of the LH1-RC complex. PufX was located opposite protein-U on the LH1-ring opening, and both its position and conformation differed from that of previous reports of dimeric LH1-RC structures obtained at low-resolution. Twenty-six molecules of the carotenoid spheroidene arranged in two distinct configurations were resolved in the Rba. sphaeroides LH1 and were positioned within the complex to block its pores. Our findings offer a new view of the core photocomplex of Rba. sphaeroides and the connections between structure and function in bacterial photocomplexes in general.

category: biochemistry


title: Supervised Learning Model Predicts Protein Adsorption to Nanotubes

abstract:
Engineered nanoparticles are advantageous for numerous biotechnology applications, including biomolecular sensing and delivery. However, testing the compatibility and function of nanotechnologies in biological systems requires a heuristic approach, where unpredictable biofouling via protein corona formation often prevents effective implementation. Moreover, rational design of biomolecule-nanoparticle conjugates requires prior knowledge of such interactions or extensive experimental testing. Toward better applying engineered nanoparticles in biological systems, herein, we develop a random forest classifier (RFC) trained with proteomic mass spectrometry data that identifies proteins that adsorb to nanoparticles, based solely on the proteins amino acid sequence. We model proteins that populate the corona of a single-walled carbon nanotube (SWCNT)-based optical nanosensor and study whether there is a relationship between the proteins amino acid-based properties and the proteins adsorption to SWCNTs. We optimize the classifier and characterize the classifier performance against other models. To evaluate the predictive power of our model, we apply the classifier to rapidly identify proteins with high binding affinity to SWCNTs, followed by experimental validation. We further determine protein features associated with increased likelihood of SWCNT binding: high content of solvent-exposed glycine residues and non-secondary structure-associated amino acids. Conversely, proteins with high content of leucine residues and beta-sheet-associated amino acids are less likely to form the SWCNT protein corona. The classifier presented herein provides a step toward undertaking the otherwise intractable problem of predicting protein-nanoparticle interactions, which is needed for more rapid and effective translation of nanobiotechnologies from in vitro synthesis to in vivo use.

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category: bioengineering


title: Supervised Learning Model Predicts Protein Adsorption to Carbon Nanotubes


title: Stem Cell Microarrays for Assessing Growth Factor Signaling in Engineered Glycan Microenvironments

abstract:
Extracellular glycans, such as glycosaminoglycans (GAGs), provide an essential regulatory component during the development and maintenance of tissues. GAGs, which harbor binding sites for a range of growth factors and other morphogens, help establish gradients of these molecules in the extracellular matrix (ECM) and promote the formation of active signaling complexes when presented at the cell surface. As such, GAGs have been pursued as biologically active components for the development of biomaterials for cell-based regenerative therapies. However, their structural complexity and compositional heterogeneity make establishing structure-function relationships for this class of glycans difficult. Here, we describe a stem cell array platform, in which GAG polysaccharides are conjugated to adhesion proteins and introduced into a polyacrylamide hydrogel network to directly measure their contributions to the activation of growth factor signaling pathways in cells. With the recent emergence of powerful synthetic and recombinant technologies to produce well-defined GAG structures, a platform for analyzing both growth factor binding and signaling in response to the presence of these biomolecules will provide a powerful tool for integrating glycans into biomaterials to advance their biological properties and applications.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/448747v2_ufig1.gif" ALT="Figure 1">
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title: Photoreactive hydrogel stiffness influences volumetric muscle loss repair

abstract: Volumetric muscle loss (VML) injuries are characterized by permanent loss of muscle mass, structure, and function. Hydrogel biomaterials provide an attractive platform for skeletal muscle tissue engineering due to the ability to easily modulate their biophysical and biochemical properties to match a range of tissue characteristics. In this work we successfully developed a mechanically tunable hyaluronic acid (HA) hydrogel system to investigate the influence of hydrogel stiffness on VML repair. HA was functionalized with photoreactive norbornene groups to create hydrogel networks that rapidly crosslink via thiol-ene click chemistry with tailored mechanics. Mechanical properties were controlled by modulating the amount of matrix metalloproteinase (MMP)-degradable peptide crosslinker to produce hydrogels with increasing elastic moduli of 1.1 {+/-} 0.002, 3.0 {+/-} 0.002, and 10.6 {+/-} 0.006 kPa mimicking a relevant range of developing and mature muscle stiffnesses. Functional muscle recovery was assessed following implantation of the HA hydrogels by in situ photopolymerization into rat latissimus dorsi (LD) VML defects at 12 and 24 weeks post-injury. After 12 weeks, muscles treated with medium stiffness (3.0 kPa) hydrogels produced maximum isometric forces most similar to contralateral healthy LD muscles. This trend persisted at 24 weeks post-injury, suggestive of sustained functional recovery. Histological analysis revealed a significantly larger zone of regeneration with more de novo muscle fibers following implantation of medium stiffness hydrogels in VML-injured muscles compared to other experimental groups. Lower (low and medium) stiffness hydrogels also appeared to attenuate the chronic inflammatory response characteristic of VML injuries, displaying similar levels of macrophage infiltration and polarization to healthy muscle. Together these findings illustrate the importance of hydrogel mechanical properties in supporting functional repair of VML injuries.


title: Functional network motifs defined through integrationof protein-protein and genetic interactions

abstract: Cells are enticingly complex systems. The identification of feedback regulation is critically important for understanding this complexity. Network motifs defined as small graphlets that occur more frequently than expected by chance have revolutionized our understanding of feed-back circuits in cellular networks. However, with their definition solely based on statistical over-representation, network motifs often lack biological context, which limits their usefulness. Here, we define functional network motifs (FNMs) through the systematic integration of genetic interaction data that directly informs on functional relationships between genes and encoded proteins. Occurring two orders of magnitude less frequently than conventional network motifs, we found FNMs significantly enriched in genes known to be functionally related. Moreover, our comprehensive analyses of FNMs in yeast showed that they are powerful at capturing both known and putative novel regulatory interactions, thus suggesting a promising strategy towards the systematic identification of feedback regulation in biological networks. Many FNMs appeared as excellent candidates for the prioritization of follow-up biochemical characterization, which is a recurring bottleneck in the targeting of complex diseases. More generally, our work highlights a fruitful avenue for integrating and harnessing genomic network data.


title: Detecting global influence of transcription factor interactions on gene expression in lymphoblastoid cells using neural network models

abstract:
BackgroundTranscription factor(TF) interactions are known to regulate target gene(TG) expression in eukaryotes via TF regulatory modules(TRMs). Such interactions can be formed due to co-localizing TFs binding proximally to each other in the DNA sequence or over long distances between distally binding TFs via chromatin looping. While the former type of interaction has been characterized extensively, long distance TF interactions are still largely understudied. Furthermore, most prior approaches have focused on characterizing physical TF interactions without accounting for their effects on TG expression regulation. Understanding TRM based TG expression regulation could aid in understanding diseases caused by disruptions to these mechanisms. In this paper, we present a novel neural network based TRM detection approach that consists of using multi-omics TF based regulatory mechanism information to generate features for building non-linear multilayer perceptron TG expression prediction models in the GM12878 immortalized lymphoblastoid cells.

ResultsWe estimated main effects of 149 individual TFs and interaction effects of 48 distinct combinations of TFs forming TRMs based on their influence on TG expression. We identified several well-known and discovered multiple previously uncharacterized TF interactions within our detected set of TRMs. We further characterized the pairwise TRMs using long distance chromatin looping and motif co-occurrence data. We found that nearly all the TFs constituting TRMs detected by our approach interacted via chromatin looping, and that these TFs further interacted with promoters to influence TG expression through one of four possible regulatory configurations.

ConclusionHere, we have provided a framework for detecting TRMs using neural network models containing multi-omics TF based regulatory features. We have also described these TRMs based on their regulatory potential along with presenting evidence for the possibility of TF interactions forming the TRMs occurring via chromatin looping.


title: SPOT-Contact-Single: Improving Single-Sequence-Based Prediction of Protein Contact Map using a Transformer Language Model, Large Training Set and Ensembled Deep Learning

abstract:
MotivationAccurate prediction of protein contact-map is essential for accurate protein structure and function prediction. As a result, many methods have been developed for protein contact map prediction. However, most methods rely on protein-sequence-evolutionary information, which may not exist for many proteins due to lack of naturally occurring homologous sequences. Moreover, generating evolutionary profiles is computationally intensive. Here, we developed a contact-map predictor utilizing the output of a pre-trained language model ESM-1b as an input along with a large training set and an ensemble of residual neural networks.

ResultsWe showed that the proposed method makes a significant improvement over a single-sequence-based predictor SSCpred with 15% improvement in the F1-score for the independent CASP14-FM test set. It also outperforms evolutionary-profile-based methods TrRosetta and SPOT-Contact with 48.7% and 48.5% respective improvement in the F1-score on the proteins without homologs (Neff=1) in the independent SPOT-2018 set. The new method provides a much faster and reasonably accurate alternative to evolution-based methods, useful for large-scale prediction.

AvailabilityStand-alone-version of SPOT-Contact-Single is available at https://github.com/jas-preet/SPOT-Contact-Single. Direct prediction can also be made at https://sparks-lab.org/server/spot-contact-single. The datasets used in this research can also be downloaded from the GitHub.

Contactjaspreetsingh2@griffithuni.edu.au, k.paliwal@griffith.edu.au, and zhouyq@szbl.ac.cn

Supplementary informationSupplementary data are available at Bioinformatics online.


title: SPOT-Contact-Single: Improving Single-Sequence-Based Prediction of Protein Contact Map using a Transformer Language Model


title: Faster short-read mapping with strobemer seeds in syncmer space

abstract:
Read alignment to genomes is a fundamental computational step used in many bioinformatic analyses, and often, it is the computational bottleneck. Therefore, it is desirable to perform the alignment step as fast as possible without compromising accuracy. Most alignment algorithms consider a seed-and-extend approach, where the time-consuming seeding step identifies and decides on candidate mapping locations. Recently, several advances have been made on seeding methods for fast sequence comparison.

We combine two such methods, syncmers and strobemers, in a novel seeding approach for constructing dynamic-sized fuzzy seeds and implement the method in a short-read aligner, strobealign. We benchmark strobealign to other traditional and recently proposed aligners on simulated and biological data and show that strobealign is an order of magnitude faster than traditional aligners such as BWA and Bowtie2 while being both faster and more accurate than more recently proposed aligners. Our aligner can free up substantial time and computing resources needed for read alignment in many pipelines.

Availabilityhttps://github.com/ksahlin/strobealign.


title: Faster short-read mapping with strobemer seeds constructed from syncmers


title: Flexible seed size enables ultra-fast and accurate read alignment


title: In silico model for predicting IL-2 inducing peptides in human

abstract: Interleukin-2 (IL-2) based immunotherapy has been already approved to treat certain type of cancers as it plays vital role in immune system. Thus it is important to discover new peptides or epitopes that can induce IL-2 with high efficiency. We analyzed experimentally validated IL-2 inducing and non-inducing peptides and observed differ in average amino acid composition, motifs, length, and positional preference of amino acid residues at the N- and C-terminus. In this study, 2528 IL-2 inducing and 2104 non-IL-2 inducing peptides have been used for traning, testing, traing and validation of our models. A large number of machine learning techniques and around 10,000 peptide features have been used for developing prediction models. The Random Forest-based model using hybrid features achieved a maximum accuracy of 73.25%, with AUC of 0.73 on the training set; accuracy of 72.89% with AUC of 0.72 on validation dataset. A web-server IL2pred has been developed for predicting IL-2 inducing peptides, scanning IL-inducing regions in a protein and designing IL-2 specific epitopes by ranking peptide analogs (https://webs.iiitd.edu.in/raghava/il2pred/).


title: Wnt/β-catenin signaling and p68 conjointly regulate CHIP in colorectal cancer

abstract: The differential expression pattern of Carboxy terminus of Hsc70 Interacting Protein (CHIP, alias STIP1 Homology and U-box Containing Protein 1 or STUB1) in cancers is associated with ubiquitination mediated degradation of its client proteins. Emerging evidences suggest its abundant expression of CHIP in colorectal cancer compared to normal tissues, but the mechanistic detail of this augmented expression pattern is unclear. The signature driver of canonical Wnt pathway, {beta}-catenin, and its co-activator RNA helicase p68, are also overexpressed in colorectal cancer. In this study, we describe a novel mechanism of Wnt/{beta}-catenin and p68 mediated transcriptional activation of CHIP gene leading to enhanced proliferation of colorectal cancer cells. Wnt3A treatment and pharmacological activation of canonical Wnt signaling pathway resulted in increased nuclear translocation of {beta}-catenin and elevated expression of CHIP. Likewise, overexpression and knockdown of {beta}-catenin and p68 upregulated and downregulated CHIP expression, respectively, at both mRNA and protein levels. After cloning CHIP promoter, the increased and decreased promoter activities of CHIP induced by overexpression and knockdown of either {beta}-catenin or p68 further confirmed transcriptional regulation of CHIP gene by Wnt/{beta}-catenin signaling cascade. p68 along with {beta}-catenin were found to occupy Transcription Factor 4 (TCF4) binding sites on endogenous CHIP promoter and regulate its transcription. Finally, enhanced cellular propagation and migration of colorectal cancer cells induced by  Wnt/{beta}-catenin-p68-CHIP axis established the significance of this pathway in oncogenesis. To the best of our knowledge, this is the first report elucidating the mechanistic details of transcriptional regulation of CHIP (STUB1) gene expression.

category: cancer biology


title: Pan-Cancer Analysis of DNA Methylation Identifies Genes and Biological Functions Associated with Overall Survival

abstract: DNA methylation influences gene expression and is altered in many cancers, but the relationship between DNA methylation and cancer outcomes is not yet fully understood. If methylation of specific genes is associated with better or worse outcomes, it could implicate genes in driving cancer and suggest therapeutic strategies. To advance our understanding of DNA methylation in cancer biology, we conducted a pan-cancer analysis of the relationship between methylation and overall survival. Using data on 28 tumor types from The Cancer Genome Atlas (TCGA), we identified genes and genomic regions whose methylation was recurrently associated with survival across multiple cancer types. While global DNA methylation levels are associated with outcome in some cancers, we found that the gene-specific associations were largely independent of these global effects. Genes with recurrent associations across cancer types were enriched for certain biological functions, such as immunity and cell-cell adhesion. While these recurrently associated genes were found throughout the genome, they were enriched in certain genomic regions, which may further implicate certain gene families and gene clusters in affecting survival. By finding common features across cancer types, our results link DNA methylation to patient outcomes, identify biological mechanisms that could explain survival differences, and support the potential value of treatments that modulate the methylation of tumor DNA.


title: Taxonomic Monograph of Saxicolella (Podostemaceae), African waterfall plants highly threatened by Hydro-Electric projects, with five new species.

abstract:
The genus Saxicolella Engl. (Podostemaceae) are African rheophytes, restricted to rapids and waterfalls as are all members of the family. Previously, Saxicolella sensu lato was shown to be polyphyletic with two separate clades in the molecular phylogenetic study of Koi et al. (2012). The name Pohliella Engl. was recently resurrected for one clade that is sister to the American genera Ceratolacis (Tul.)Wedd., Podostemum Michx. and all Old World Podostemoideae (podostemoids) (Cheek 2020). Pohliella has distichous phyllotaxy, bilocular ovaries, filiform roots with paired holdfasts, and rootcaps. The second clade, Saxicolella sensu stricto, including the type of the generic name, has spiral phyllotaxy, unilocular ovaries, ribbon-like or crustose roots that lack both holdfasts and rootcaps. Saxicolella sensu stricto, sampled from the type species, S. nana Engl. of Cameroon, is embedded within and near the base of the major clade of African podostemoids and is sister to all other African genera apart from Inversodicraea R.E.Fr. and Monandriella Engl. Recently reduced to three species in Cameroon and S.E. Nigeria by the resurrection of Pohliella (3 - 4 species in Ghana and Nigeria-Cameroon), Saxicolella sensu stricto is expanded to eight species in this monograph by description of five new taxa. Saxicolella futa Cheek and S. deniseae Cheek are newly described from Guinea, S. ijim Cheek from Cameroon, the informally named S. sp. A from Gabon, and S. angola Cheek from Angola. The known geographic range of the genus is thus expanded c. 2,500 km westwards to Guinea from eastern Nigeria and c.1,500 km southeastwards from near Yaounde to Cuanza do Sul, Angola. The greatest concentration of species occurs in the Cross-Sanaga interval of western Cameroon and eastern Nigeria, with three species. Cameroon (3 species) followed by Nigeria and Guinea (2 species each) are the countries with highest species diversity. The genus can be expected to be found in Sierra Leone, Liberia, Ivory Coast and Congo Republic. A classification is proposed grouping the species into three subgenera (Saxicolella, Butumia (G.Taylor) Cheek comb. et. stat. nov. and Kinkonia Cheek subgen. nov.) based on root morphology and shoot position and morphology.

The discovery, morphology, circumscription, distribution, and ecology of Saxicolella is reviewed, an identification key to the species is presented, together with descriptions, synonymy, links to illustrations, and extinction risk assessments for each of the eight species now recognised. All of the species are provisionally assessed as either Endangered or Critically Endangered using the IUCN 2012 standard, making this genus among the most threatened of its size globally. The major threats, above all, are hydro-electric projects. Saxicolella deniseae may already be globally extinct, and two of the four known locations of S. angola appear lost, S. sp. A of Gabon is threatened at at least one of its three locations, while Saxicolella futa is threatened at all three locations, all due to incipient or active hydro-electric projects. Contamination of watercourses by increased turbidity from silt-load due anthropic changes and by eutrophication from pollution are also threats for the majority of the species.

category: plant biology


title: Robust Brain State Decoding using Bidirectional Long Short Term Memory Networks in functional MRI

abstract: Decoding brain states of the underlying cognitive processes via learning discriminative feature representations has recently gained a lot of interest in brain imaging studies. Particularly, there has been an impetus to encode the dynamics of brain functioning by analyzing temporal information available in the fMRI data. Long-short term memory (LSTM), a class of machine learning model possessing a "memory" component, is increasingly being observed to perform well in various applications with dynamic temporal behavior, including brain state decoding. Because of the dynamics and inherent latency in fMRI BOLD responses, future temporal context is crucial. However, it is neither encoded nor captured by the conventional LSTM model. This paper performs robust brain state decoding via information encapsulation from both the past and future instances of fMRI data via bi-directional LSTM. This allows for explicitly modeling the dynamics of BOLD response without any delay adjustment. The two hidden activations of forward and reverse directions in bi-LSTM are collated to build the "memory" of the model and are used to robustly predict the brain states at every time instance. Working memory data from the Human Connectome Project (HCP) is utilized for validation and was observed to perform 18% better than its unidirectional counterpart in terms of accuracy in predicting the brain states.


title: Disruption of VGLUT1 in cholinergic medial habenula projections increases nicotine self-administration

abstract: Cholinergic projections from the medial habenula (MHb) to the interpeduncular nucleus (IPN) have been studied for their complex contributions to nicotine addiction and have been implicated in nicotine reinforcement, aversion, and withdrawal. While it has been established that MHb cholinergic projections co-release glutamate, no direct evidence has demonstrated a role for this specific glutamate projection in nicotine consumption. In the present study, a novel floxed Slc17a7 (VGLUT1) mouse was generated and used to create conditional knockout (cKO) mice that lack VGLUT1 in MHb cholinergic neurons. Histochemical approaches and optogenetics-assisted electrophysiology were used to validate the disruption of VGLUT1 from cholinergic MHb to IPN projections. The mice displayed no gross phenotypic abnormalities and exhibited normal exploratory and locomotor behavior in the open-field assay. However, the loss of VGLUT1-mediated glutamate co-release led to increased nicotine self-administration. These findings indicate that glutamate co-release from ventral MHb cholinergic neurons opposes nicotine consumption and provide additional support for targeting this synapse to develop potential treatments to nicotine addiction.


title: Economic Choices under Simultaneous or Sequential Offers Rely on the Same Neural Circuit

abstract:
A series of studies in which monkeys chose between two juices offered in variable amounts identified in the orbitofrontal cortex (OFC) different groups of neurons encoding the value of individual options (offer value), the binary choice outcome (chosen juice) and the chosen value. These variables capture both the input and the output of the choice process, suggesting that the cell groups identified in OFC constitute the building blocks of a decision circuit. Several lines of evidence support this hypothesis. However, in previous experiments offers were presented simultaneously, raising the question of whether current notions generalize to when goods are presented or are examined in sequence. Recently, Ballesta and Padoa-Schioppa (2019) examined OFC activity under sequential offers. An analysis of neuronal responses across time windows revealed that a small number of cell groups encoded specific sequences of variables. These sequences appeared analogous to the variables identified under simultaneous offers, but the correspondence remained tentative. Thus in the present study we examined the relation between cell groups found under sequential versus simultaneous offers. We recorded from the OFC while monkeys chose between different juices. Trials with simultaneous and sequential offers were randomly interleaved in each session. We classified cells in each choice modality and we examined the relation between the two classifications. We found a strong correspondence - in other words, the cell groups measured under simultaneous offers and under sequential offers were one and the same. This result indicates that economic choices under simultaneous or sequential offers rely on the same neural circuit.

Significance StatementResearch in the past 20 years has shed light on the neuronal underpinnings of economic choices. A large number of results indicates that decisions between goods are formed in a neural circuit within the orbitofrontal cortex (OFC). In most previous studies, subjects chose between two goods offered simultaneously. Yet, in daily situations, goods available for choice are often presented or examined in sequence. Here we recorded neuronal activity in the primate OFC alternating trials under simultaneous and under sequential offers. Our analyses demonstrate that the same neural circuit supports choices in the two modalities. Hence current notions on the neuronal mechanisms underlying economic decisions generalize to choices under sequential offers.


title: Improvement of association between confidence and accuracy after integration of discrete evidence over time

abstract: When making decisions in real-life, we may receive discrete evidence during a time period. Although participants can integrate information from separate cues to improve their accuracy, it is still debatable how confidence changes after receiving discrete information. Nevertheless, based on the strong positive relationship between accuracy and confidence, we predicted that similar to what is observed in accuracy, confidence would improve following the integration of separate pieces of information. We used a Random-dot-motion discrimination task in which one or two brief stimuli (i.e., pulse[s]) were presented, and participants had to indicate the predominant direction of dot motions by saccadic eye movement. Two pulses intervals (up to 1s) were randomly selected, where color-coded targets facilitated indicating confidence simultaneously. Using behavioral data, computational models, pupillometry, and EEG methodology, our data revealed that compared to single-pulse trials, in double-pulse trials, participants improve their confidence resolution rather than reporting higher confidence and that the EEG signal (centro-parietal-positivity) and pupil dynamics appear to follow confidence levels but only in double-pulse trials. Overall, our findings suggest that evidence integration helps to improve the associations between confidence and accuracy.


title: Improvement of association between confidence and accuracy after integration of separate evidence over time


title: The effect of self- vs. externally generated actions on timing, duration and amplitude of BOLD response for visual feedback processing

abstract: It has been widely assumed that internal forward models use efference copies to create predictions about the sensory consequences of our own actions. While these predictions had been frequently associated with reduced neural processing in sensory cortices, the timing and duration of the hemodynamic response of self-generated as opposed to externally generated movements is poorly investigated. In the present study we tested the hypothesis that predictive mechanisms for self-generated actions lead to early and shorter neural processing compared with externally generated movements. Using a first and second-order Taylor approximation in terms of the temporal (TD) and dispersion (DD) derivatives of a canonical hemodynamic response function, we investigated the timing and duration of activation for self-generated and externally generated movements using a custom-made fMRI-compatible movement device. Visual video feedback of the active and passive hand movements were presented in real time or with variable delays (0 - 417 ms). Participants had to judge, whether the feedback was delayed. We found earlier feedback processing for self-generated compared to externally generated movements in several regions including the supplementary motor area, cerebellum, subcortical structures such as the putamen and visual cortices. Shorter processing was found in areas, which show also lower blood oxygen level dependent (BOLD) amplitudes, such as the SMA, occipital and parietal cortex. Specifically, earlier activation in the putamen, of self-generated movements was associated with worse performance in detecting delays. These findings support our hypothesis, that efference copy based predictive mechanisms enable earlier processing of action feedback, as potential source for behavioral effects.


title: Resting-state functional connectivity relates to interindividual variations in positive memory

abstract: Individual differences in positive memory recollection are of interest in mental health, as positive memories can help protect people against stress and depression. However, it is unclear how individual differences in positive memory recollection are reflected in brain activity in the resting state. Here, we investigate the resting-state functional connectivity (FC) associated with interindividual variations in positive memory by employing cluster-level inferences based on randomization/permutation region of interest (ROI)-to-ROI analyses. We identified a cluster of FCs that was positively associated with positive memory performance, including the frontal operculum, central operculum, parietal operculum, Heschls gyrus, and planum temporale. The current results suggest that positive memory is innervated by frontotemporal network connectivity, which may have implications for future investigations of vulnerability to stress and depression.


title: Dynamical differential covariance recovers directional network structure in multiscale neural systems

abstract:
Investigating neural interactions is essential to understanding the neural basis of behavior. Many statistical methods have been used for analyzing neural activity, but estimating the direction of network interactions correctly and efficiently remains a difficult problem (1). Here, we derive dynamical differential covariance (DDC), a new method based on dynamical network models that detects directional interactions with low bias and high noise tolerance under nonstationarity conditions. The method was first validated and compared with other methods on networks with false positive motifs and multiscale neural simulations where the ground truth connectivity was known. When applied to recordings of resting-state functional magnetic resonance imaging (rs-fMRI) recordings, DDC consistently detected regional interactions with strong structural connectivity in over 1,000 individual subjects obtained by diffusion MRI (dMRI). DDC is a promising new family of methods for estimating functional connectivity that can be generalized to a wide range of dynamical models and recording techniques and to other applications where system identification from sparse data is needed.

Significance StatementThrough dynamical interactions with each other, neurons make it possible for us to sense, move and think. It is now possible to simultaneously record from many individual neurons and brain regions. Methods for analyzing these large-scale recordings are needed that can reveal how the patterns of activity give rise to behavior. We developed an efficient, intuitive and robust way to analyze these recordings and validated it on simulations of model neural networks where the ground truth was known. We called this method dynamical differential covariance (DDC) because it can estimate not only the presence of a connection but also which direction the information is flowing in a network between neurons or cortical areas. We also successfully applied DDC to brain imaging data from functional Magnetic Resonance Imaging.


title: Visual stimulation induces distinct forms of sensitization of On-Off direction-selective ganglion cell responses in the dorsal and ventral retina

abstract: Experience-dependent modulation of neuronal responses is a key attribute in sensory processing. In the mammalian retina, the On-Off direction-selective ganglion cell (On-Off DSGC) is well known for its robust direction selectivity. However, how the On-Off DSGC light responsiveness dynamically adjusts to the changing visual environment is underexplored. Here, we report that the On-Off DSGC can be transiently sensitized by prior stimuli. Notably, distinct sensitization patterns are found in dorsal and ventral DSGCs that receive visual inputs from lower and upper visual fields respectively. Although responses of both dorsal and ventral DSGCs to dark stimuli (Off responses) are sensitized, only dorsal cells show sensitization of responses to bright stimuli (On responses). Visual stimulation to the dorsal retina potentiates a sustained excitatory input from Off bipolar cells, leading to tonic depolarization of dorsal DSGCs. Such tonic depolarization propagates from the Off to the On dendritic arbor of the DSGC to sensitize its On response. We also identified a previously overlooked feature of DSGC dendritic architecture that can support direct electrotonic propagation between On and Off dendritic layers. By contrast, ventral DSGCs lack a sensitized tonic depolarization and thus do not exhibit sensitization of their On responses. Our results highlight a topographic difference in Off bipolar cell inputs underlying divergent sensitization patterns of dorsal and ventral On-Off DSGCs. Moreover, substantial crossovers between dendritic layers of On-Off DSGCs suggest an interactive dendritic algorithm for processing On and Off signals before they reach the soma.


title: Nutrient-sensitive reinforcement learning in monkeys

abstract: Animals make adaptive food choices to acquire nutrients that are essential for survival. In reinforcement learning (RL), animals choose by assigning values to options and update these values with new experiences. This framework has been instrumental for identifying fundamental learning and decision variables, and their neural substrates. However, canonical RL models do not explain how learning depends on biologically critical intrinsic reward components, such as nutrients, and related homeostatic regulation. Here, we investigated this question in monkeys making choices for nutrient-defined food rewards under varying reward probabilities. We found that the nutrient composition of rewards strongly influenced monkeys choices and learning. The animals preferred rewards high in nutrient content and showed individual preferences for specific nutrients (sugar, fat). These nutrient preferences affected how the animals adapted to changing reward probabilities: the monkeys learned faster from preferred nutrient rewards and chose them frequently even when they were associated with lower reward probability. Although more recently experienced rewards generally had a stronger influence on monkeys choices, the impact of reward history depended on the rewards specific nutrient composition. A nutrient-sensitive RL model captured these processes. It updated the value of individual sugar and fat components of expected rewards from experience and integrated them into scalar values that explained the monkeys choices. Our findings indicate that nutrients constitute important reward components that influence subjective valuation, learning and choice. Incorporating nutrient-value functions into RL models may enhance their biological validity and help reveal unrecognized nutrient-specific learning and decision computations.


title: BehaviorDEPOT: a tool for automated behavior classification and analysis in rodents

abstract: Quantitative descriptions of animal behavior are essential to understand the underlying neural substrates. Many behavioral analyses are performed by hand or with expensive and inflexible commercial software that often fail on animals with attached head implants, such as those used for in vivo optogenetics and calcium imaging. With the development of machine learning algorithms that can estimate animal positions across time and space, it is becoming easier for users with no prior coding experience to perform automated animal tracking in behavioral video recordings. Yet classifying discrete behaviors based on positional tracking data remains a significant challenge. To achieve this, we must start with reliable ground truth definitions of behavior, a process that is hindered by unreliable human annotations. To overcome these barriers, we developed BehaviorDEPOT (DEcoding behavior based on POsitional Tracking), a MATLAB-based application comprising six independent modules and a graphical user interface. In the Analysis Module we provide hard-coded classifiers for freezing and rearing. Optionally applied spatiotemporal filters allow users to analyze behaviors in varied experimental designs (e.g. cued tasks or optogenetic manipulations). Even inexperienced users can generate organized behavioral data arrays that can be seamlessly aligned with neurophysiological recordings for detailed analyses of the neural substrates. Four additional modules create an easy-to-use pipeline for establishing reliable ground-truth definitions of behaviors as well as custom behavioral classifiers. Finally, our Experiment Module runs fear conditioning experiments using an Arduino-based design that interfaces with commercialhardware and significantly reduces associated costs. We demonstrate the utility and flexibility of BehaviorDEPOT in widely used behavioral assays including fear conditioning, avoidance, and decision-making tasks. We also demonstrate the robustness of the BehaviorDEPOT freezing classifier across multiple camera types and in mice and rats wearing optogenetic patch cables and head-mounted Miniscopes. BehaviorDEPOT provides a simple, flexible, automated pipeline to move from pose tracking to reliably quantifying a wide variety of task-relevant behaviors.


title: Linking spatial self-organization to community assembly and biodiversity

abstract: Drier climates impose environmental stresses on plant communities that may result in community reassembly and threatened ecosystem services, but also may trigger self-organization in spatial patterns of biota and resources, which act to relax these stresses. The complex relationships between these counteracting processes - community reassembly and spatial self-organization - have hardly been studied. Using a spatio-temporal model of dryland plant communities and a trait-based approach, we study the response of such communities to imposed water stress of increasing degrees. We first show that spatial patterning acts to reverse shifts from fast-growing species to stress-tolerant species, as well as to reverse functional-diversity loss. We then show that spatial re-patterning buffers the impact of further stress on community structure. Finally, we identify multistability ranges of uniform and patterned community states and use them to propose forms of non-uniform ecosystem management that integrate the need for provisioning ecosystem services with the need to preserve community structure.

category: ecology


title: Autogenous and anautogenous Culex pipiens bioforms exhibit insulin-like peptide signaling pathway gene expression differences that are not dependent upon larval nutrition

abstract: Culex pipiens form pipiens and Cx. pipiens form molestus differ in their ability to produce eggs without a bloodmeal. Autogenous mosquitoes, such as the molestus bioform of Cx. pipiens, depend on nutrition acquired as larvae instead of a bloodmeal to fuel the energy intensive process of vitellogenesis, which requires abundant production of yolk proteins. In anautogenous mosquito systems, ovary ecdysteroidogenic hormone (OEH) and insulin-like peptides (ILPs) transduce nutritional signals and trigger egg maturation in response to a bloodmeal. It is unclear to what extent the process is conserved in autogenous mosquitoes and how the bloodmeal trigger has been replaced by teneral reserves. Here, we measured the effects of a series of nutritional regimens on autogeny, time to pupation, and survival in Cx. pipiens form molestus and form pipiens. We find that abundant nutrients never result in autogenous form pipiens and extremely poor food availability rarely eliminates autogeny from form molestus. However, the number of autogenous eggs generated increases with nutrient availability. Similarly, using qPCR to quantify gene expression, we find several differences in the expression levels of ilps between bioforms that are reduced and delayed by poor nutrition, but not extinguished. Changes in OEH expression do not explain bioform-specific differences in autogeny. Surprisingly, the source of most of the gene expression differences correlated with autogeny is the abdomen, not the brain. Overall, our results suggest that autogeny is modulated by nutritional availability, but the trait is encoded by genetic differences between forms and these impact the expression of ILPs.