title: Cheating the cheater: Suppressing false positive enrichment during biosensor-guided biocatalyst engineering

abstract: Transcription factor (TF)-based biosensors are very desirable reagents for high-throughput enzyme and strain engineering campaigns. Despite their potential, they are often difficult to deploy effectively as the small molecules being detected can leak out of high-producer cells, into low-producer cells, and activate the biosensor therein. This crosstalk leads to the overrepresentation of false positive/cheater cells in the enriched population. While the host cell can be engineered to minimize crosstalk (e.g., by deleting responsible transporters), this is not easily applicable to all molecules of interest, particularly those that can diffuse passively. One such biosensor recently reported for trans-cinnamic acid (tCA) suffers from crosstalk when used for phenylalanine ammonia-lyase (PAL) enzyme engineering by directed evolution. We report that desensitizing the biosensor (i.e., increasing the limit of detection, LOD) suppresses cheater population enrichment. Further we show that, if we couple the biosensor-based screen with an orthogonal pre-screen that eliminates a large fraction of true negatives, we can successfully reduce the cheater population during the fluorescence-activated cell sorting (FACS). Using the approach developed here, we were successfully able to isolate PAL variants with ~70% high kcat after a single sort. These mutants have tremendous potential in Phenylketonuria (PKU) treatment and flavonoid production.


title: Hearing Ability of Prairie Voles (Microtus ochrogaster)

abstract:
Hearing ability of mammals can be impacted by many factors including social cues, environment, and physical properties of animal morphology. Despite being used commonly to study social behaviors, hearing of the monogamous prairie vole (Microtus ochrogaster) has never been fully characterized. In this study, we measure morphological head and pinna features and use auditory brainstem responses to measure auditory capabilities of prairie voles characterizing monaural and binaural hearing and hearing range. Additionally, we measured unbonded male and female voles to characterize differences due to sex. We found that prairie voles show a hearing range with greatest sensitivity between 8 - 32 kHz, robust binaural hearing, and characteristic monaural ABRs. We show no differences between the sexes for binaural hearing or hearing range, however female voles have increased amplitude of peripheral ABR waves I and II and increased latency of wave IV. Our results confirm that prairie voles have both low and high frequency hearing, binaural hearing, and despite biparental care and monogamy, differences in processing of sound information between the sexes. These data further highlight the necessity to understand sex-specific differences in neural processing that may underly variability in behavioral responses between sexes.

HighlightsO_LIMonogamous prairie voles hear across both low and high frequencies.
C_LIO_LIFemale prairie voles show differences in monaural hearing compared to males.
C_LIO_LIThere is no difference in binaural hearing or pinna/head size morphology between the sexes.
C_LI

category: neuroscience


title: Automated skull stripping in mouse fMRI analysis using 3D U-Net

abstract: Skull stripping is an initial and critical step in the pipeline of mouse fMRI analysis. Manual labeling of the brain usually suffers from intra- and inter-rater variability and is highly time-consuming. Hence, an automatic and efficient skull-stripping method is in high demand for mouse fMRI studies. In this study, we investigated a 3D U-Net based method for automatic brain extraction in mouse fMRI studies. Two U-Net models were separately trained on T2-weighted anatomical images and T2*-weighted functional images. The trained models were tested on both interior and exterior datasets. The 3D U-Net models yielded a higher accuracy in brain extraction from both T2-weighted images (Dice > 0.984, Jaccard index > 0.968 and Hausdorff distance < 7.7) and T2*-weighted images (Dice > 0.964, Jaccard index > 0.931 and Hausdorff distance < 3.3), compared with the two widely used mouse skull-stripping methods (RATS and SHERM). The resting-state fMRI results using automatic segmentation with the 3D U-Net models are identical to those obtained by manual segmentation for both the seed-based and group independent component analysis. These results demonstrate that the 3D U-Net based method can replace manual brain extraction in mouse fMRI analysis.


title: A partial reduction of Drp1 enhances mitophagy, autophagy, mitochondrial biogenesis, dendritic spines and synaptic activity in a transgenic Tau mouse model of Alzheimer disease

abstract: The purpose of our study is to understand the impact of a partial dynamin-related protein 1 (Drp1) on cognitive behavior, mitophagy/autophagy, mitochondrial and synaptic activities in transgenic Tau mice in Alzheimers disease (AD). Our lab reported increased levels of A{beta} and P-Tau, and abnormal interactions between A{beta} and Drp1, P-Tau and Drp1 induced increased mitochondrial fragmentation and reduced fusion and synaptic activities in AD. These abnormal interactions, result in the proliferation of dysfunctional mitochondria in AD neurons. Recent research on mitochondria revealed that fission protein Drp1 is largely implicated in mitochondrial dynamics in AD. To determine the impact of reduced Drp1 in AD, we recently crossed transgenic Tau mice with Drp1 heterozygote knockout (Drp1+/-) mice and generated double mutant (Drp1+/- X Tau) mice. In the current study, we assessed cognitive behavior, mRNA and protein levels of mitophagy, autophagy, mitochondrial biogenesis, dynamics and synaptic genes, mitochondrial morphology & mitochondrial function, dendritic spines in Tau mice relative to double mutant mice. When compared to Tau mice, double mutant mice did better on Morris Maze (reduced latency to find hidden platform, increased swimming speed and time spent on quadrant) and rotarod (stayed a longer period of time) tests. Both mRNA and proteins levels autophagy, mitophagy, mitochondrial biogenesis and synaptic proteins were increased in double mutant mice compared to Tau mice. Dendritic spines were significantly increased; mitochondrial number is reduced and length is increased in double mutant mice. Based on these observations, we conclude that reduced Drp1 is beneficial in a symptomatic-transgenic Tau mice.


title: Variability is actively regulated in speech

abstract: Although movement variability is often attributed to unwanted noise in the motor system, recent work has demonstrated that variability may be actively controlled. To date, research on regulation of motor variability has relied on relatively simple, laboratory-specific reaching tasks. It is not clear how these results translate to complex, well-practiced and real-world tasks. Here, we test how variability is regulated during speech production, a complex, highly over-practiced and natural motor behavior that relies on auditory and somatosensory feedback. Specifically, in a series of four experiments, we assessed the effects of auditory feedback manipulations that modulate perceived speech variability, shifting every production either towards (inward-pushing) or away from (outward-pushing) the center of the distribution for each vowel. Participants exposed to the inward-pushing perturbation (Experiment 1) increased produced variability while the perturbation was applied as well as after it was removed. Unexpectedly, the outward-pushing perturbation (Experiment 2) also increased produced variability during exposure, but variability returned to near baseline levels when the perturbation was removed. Outward-pushing perturbations failed to reduce participants produced variability both with larger perturbation magnitude (Experiment 3) or after their variability had increased above baseline levels as a result of the inward-pushing perturbation (Experiment 4). Simulations of the applied perturbations using a state space model of motor behavior suggest that the increases in produced variability in response to the two types of perturbations may arise through distinct mechanisms: an increase in controlled variability in response to the inward-pushing perturbation, and an increase in sensitivity to auditory errors in response to the outward-pushing perturbation. Together, these results suggest that motor variability is actively regulated even in complex and well-practiced behaviors, such as speech.


title: BDNF signaling in Hebbian and Stentian structural plasticity in the developing visual system

abstract: During development, patterned neural activity in input neurons innervating their target, instructs topographic map refinement. Axons from adjacent neurons, firing with similar patterns of neural activity, converge onto target neurons and stabilize their synapses with these postsynaptic partners (Hebbian plasticity). On the other hand, non-correlated firing of inputs promotes synaptic weakening and exploratory axonal growth (Stentian plasticity). We used visual stimulation to control the visually-evoked correlation structure of neural activity in ectopic ipsilaterally projecting (ipsi) retinal ganglion cell axons with respect to their neighboring contralateral eye inputs in the optic tectum of albino Xenopus laevis tadpoles. Multiphoton imaging of the ipsi axons in the live tadpole, combined with manipulation of brain-derived neurotrophic factor (BDNF) signaling, revealed that presynaptic p75NTR and TrkB both promoted axonal branch addition during Stentian plasticity, whereas predominantly postsynaptic BDNF signaling mediated activity-dependent Hebbian suppression of axon branch addition. Additionally, we found that BDNF signaling is required for local suppression of branch loss induced by correlated firing.


title: Retrograde adenosine/A2A receptor signaling mediates presynaptic hippocampal LTP and facilitates epileptic seizures

abstract: A long-term change in neurotransmitter release is a widely expressed mechanism controlling neural circuits in the mammalian brain. This presynaptic plasticity is commonly mediated by retrograde signaling whereby a messenger released from the postsynaptic neuron upon activity modifies neurotransmitter release in a long-term manner by targeting a presynaptic receptor. In the dentate gyrus (DG), the main input area of the hippocampus, granule cells (GCs) and mossy cells (MCs) form a recurrent excitatory circuit that is critically involved in DG function and epilepsy. Here, we identified adenosine/A2A receptor (A2AR) as a novel retrograde signaling system that mediates presynaptic long-term potentiation (LTP) at MC-GC synapses. Using an adenosine sensor, we found that neuronal activity triggered phasic, postsynaptic TrkB-dependent release of adenosine. Additionally, epileptic seizures released adenosine in vivo, while removing A2ARs from DG decreased seizure susceptibility. Thus, adenosine/A2AR retrograde signaling mediates presynaptic LTP that may contribute to DG-dependent learning and promote epilepsy.


title: Longitudinal dynamics of human B-cell response at single-cell level in response to Tdap vaccination

abstract:
Adaptation of the immune system to mount an adequate immune response against pathogens is a crucial function of the adaptive immune system. To better characterize a successful vaccination response, we performed longitudinal (days 0, 5, 7, 10, and 14 after Boostrix vaccination) analysis of the single cell transcriptome as well as the B-cell receptor (BCR) repertoire (scBCR-rep) in plasma cells of an immunized donor and compared it with baseline B cell characteristics as well as flow cytometry findings.

Based on the flow cytometry knowledge and literature findings, we discriminated individual B cell subsets in the transcriptomics data and traced over-time maturation of plasmablasts/plasma cells (PB/PCs) and identified the pathways associated with the plasma cell maturation. We observed that the repertoire in PB/PCs differed from the baseline B cell repertoire e.g. regarding expansion of unique clones in post-vaccination visits, high usage of IGHG1 in expanded clones, increased class switching events post-vaccination represented by clonotypes spanning multiple IGHC classes and positive selection of CDR3 sequences over time. Importantly, the Variable gene family-based clustering of BCRs represented a similar measure as the gene-based clustering, however, certainly improved the clustering of BCRs, as BCRs from duplicated Variable gene families could be clustered together. Finally, we developed a query tool to dissect the immune response to the components of Boostrix vaccine. Using this tool, we could identify the BCRs related to anti-tetanus and anti-pertussis toxoid.

Collectively, we developed a workflow which allows description of key features of an ongoing immune response, such as activation of PB/PCs, Ig class switching, somatic hypermutation, and clonal expansion, all of which are hallmarks of antigen exposure.

category: immunology


title: Development of a highly sensitive luciferase-based reporter system to study two-step protein secretion in cyanobacteria

abstract:
Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multi-protein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however little is known regarding their function, regulation and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of non-pilin proteins, we developed a NanoLuc-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. PCC 6803. The NLuc reporter presented a wide dynamic range with at least one order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that two-step protein secretion in cyanobacteria is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to explore protein secretion in cyanobacteria further.

ImportanceProtein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here we introduce a NanoLuc (NLuc)-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge on cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.

category: microbiology


title: Amikacin potentiator activity of zinc complexed to a pyrithione derivative with enhanced solubility

abstract: Resistance to amikacin in Gram-negatives is usually mediated by the 6'-N-acetyltransferase type Ib [AAC(6')-Ib], which catalyzes the transfer of an acetyl group from acetyl CoA to the 6' position of the antibiotic molecule. A path to continue the effective use of amikacin against resistant infections is to combine it with inhibitors of the inactivating reaction. We have recently observed that addition of Zn2+ to in-vitro enzymatic reactions, obliterates acetylation of the acceptor antibiotic. Furthermore, when added to amikacin-containing culture medium in complex to ionophores such as pyrithione (ZnPT), it prevents the growth of resistant strains. An undesired property of ZnPT is its poor water-solubility, a problem that currently affects a large percentage of newly designed drugs. Water-solubility helps drugs to dissolve in body fluids and be transported to the target location. We tested a pyrithione derivative described previously (Magda et al. Cancer Res. 2008, 68:5318-5325) that contains the amphoteric group di(ethyleneglycol)-methyl ether at position 5 (compound 5002), a modification that enhances the solubility. Compound 5002 in complex with zinc (Zn5002) was tested to assess growth inhibition of amikacin-resistant Acinetobacter baumannii and Klebsiella pneumoniae strains in the presence of the antibiotic. Zn5002 complexes in combination with amikacin at different concentrations completely inhibited growth of the tested strains. However, the concentrations needed to achieve growth inhibition were higher than those required to achieve the same results using ZnPT. Time-kill assays showed that the effect of the combination amikacin/Zn5002 was bactericidal. These results indicate that derivatives of pyrithione with enhanced water-solubility, a property that would make them drugs with better bioavailability and absorption, are a viable option for designing inhibitors of the resistance to amikacin mediated by AAC(6')-Ib, an enzyme commonly found in the clinics.


title: NOD1 is super-activated through spatially-selective ubiquitination by the Salmonella effector SspH2

abstract:
As part of its pathogenesis, Salmonella enterica serovar Typhimurium delivers effector proteins into host cells. One effector is SspH2, a member of the novel E3 ubiquitin ligase family, interacts with, and enhances, NOD1 pro-inflammatory signaling, though the underlying mechanisms are unclear. Here, we report the novel discovery that SspH2 interacts with multiple members of the NLRC family to enhance pro-inflammatory signaling that results from targeted ubiquitination. We show that SspH2 modulates host innate immunity by interacting with both NOD1 and NOD2 in mammalian epithelial cell culture. We also show that SspH2 specifically interacts with the NBD and LRR domains of NOD1 and super-activates NOD1- and NOD2-mediated cytokine secretion via the NF-{kappa}B pathway. Mass spectrometry analyses identified lysine residues in NOD1 that were ubiquitinated after interaction with SspH2. Through NOD1 mutational analyses, we identified four key lysine residues that are required for NOD1 super-activation by SspH2, but not its basal activity. These critical lysine residues are positioned in the same region of NOD1 and define a surface on NOD1 that is targeted by SspH2. Overall, this work provides evidence for post-translational modification of NOD1 by ubiquitin, and uncovers a unique mechanism of spatially-selective ubiquitination to enhance the activation of an archetypal NLR.

SYNOPSISSspH2 is an E3 ubiquitin ligase injected by Salmonella Typhimurium into host cells that induces pro-inflammatory signaling. The immune receptor, NOD1, is ubiquitinated in the presence of SspH2, resulting in increased pro-inflammatory cytokine secretion.

O_LISspH2 super-activates NOD1 and NOD2 to increase pro-inflammatory cytokine secretion, in part, through the NF-{kappa}B pathway
C_LIO_LIUbiquitin modification of NOD1 were identified by mass spectrometry
C_LIO_LIA specific region of NOD1 is targeted by SspH2 to enhance NOD1 activity.
C_LI


title: The initiation of the wound healing program is regulated by the convergence of mechanical and epigenetic cues

abstract:
Wound healing in the skin is a complex physiological process that is a product of a cell state transition from homeostasis to repair. Mechanical cues are increasingly being recognized as important regulators of cellular reprogramming, but the mechanism by which it is translated to changes in gene expression and ultimately cellular behavior remains largely a mystery. To probe the molecular underpinnings of this phenomenon further, we used the downregulation of caspase-8 as a biomarker of a cell entering the wound-healing program. We found that the wound-induced release of tension within the epidermis leads to the alteration of gene expression via the nuclear translocation of the DNA methyltransferase 3A (DNMT3a). This enzyme then methylates promoters of genes that are known to be downregulated in response to wound stimuli as well as potentially novel players in the repair program. Overall, these findings illuminate the convergence of mechanical and epigenetic signaling modules that are important regulators of the transcriptome landscape required to initiate the tissue repair process in the differentiated layers of the epidermis.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/463764v2_ufig1.gif" ALT="Figure 1">
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category: cell biology


title: Pseudo-dynamic analysis of heart tube formation in the mouse reveals strong regional variability and early left-right asymmetry

abstract:
Understanding organ morphogenesis requires a precise geometrical description of the tissues involved in the process. In highly regulative embryos, like those of mammals, morphological variability hinders the quantitative analysis of morphogenesis. In particular, the study of early heart development in mammals remains a challenging problem, due to imaging limitations and innate complexity. Around embryonic day 7.5 (E7.5), the cardiac crescent folds in an intricate and coordinated manner to produce a pumping linear heart tube at E8.25, followed by heart looping at E8.5. In this work we provide a complete morphological description of this process based on detailed imaging of a temporally dense collection of embryonic heart morphologies. We apply new approaches for morphometric staging and quantification of local morphological variations between specimens at the same stage. We identify hot spots of regionalized variability and identify left-right asymmetry in the inflow region starting at the late cardiac crescent stage, which represents the earliest signs of organ left-right asymmetry in the mammalian embryo. Finally, we generate a 3D+t digital model that provides a framework suitable for co-representation of data from different sources and for the computer modelling of the process.

SUMMARY STATEMENTWe provide the first complete atlas for morphometric analysis and visualization of heart tube morphogenesis, reporting morphological variability and early emergence of left-right asymmetry patterns.

category: developmental biology


title: Development of transcriptomics-based eukaryotes growth rate indices

abstract:
O_LIGrowth rate estimation is important to understand the flow of energy and nutrient elements in an ecosystem, but it has remained challenging, especially on microscopic organisms.
C_LIO_LIIn this study, we propose four growth rate indices that use mRNA abundance ratios between nuclear and mitochondrial genes: (1) total nuclear and mitochondrial mRNA ratio (Nuc:Mito-TmRNA), (2) nuclear and mitochondrial ribosomal protein mRNA ratio (Nuc:Mito-RPmRNA), (3) gene ontology (GO) terms and total mitochondrial mRNA ratios and (4) nuclear and mitochondrial specific gene mRNA ratio. We examine these proposed ratios using RNA-Seq datasets of Daphnia magna and Saccharomyces cerevisiae.
C_LIO_LIThe results showed that both Nuc:Mito-TmRNA and Nuc:Mito-RPmRNA ratio indices showed significant correlations with the growth rate for both species. A large number of GO terms mRNA ratios showed significant correlations with the growth rate of S. cerevisiae. Lastly, we identified mRNA ratios of several specific nuclear and mitochondrial gene pairs that showed significant correlations.
C_LIO_LIWe foresee future implications for the proposed mRNA ratios used in metatranscriptome analyses to estimate the growth rate of communities and species.
C_LI

category: ecology


title: Trait-environment relationship in tadpoles of the southern Atlantic Forest

abstract:
O_LIEnvironmental predictors select individuals by their functional traits, shaping the anuran assembly patterns. Individuals respond to environmental filters that can be on a local or regional scale.
C_LIO_LIIn this study, we investigated the association between local (water and microhabitat) and landscape variables and the morphological traits of tadpoles of ponds and streams. The study was conducted in the southern region of the Brazilian Atlantic Forest. We sampled 28 waterbodies and recorded 22 anurans species. We performed RLQ and fourth-corner analyses to determine the patterns of trait-environment relationships and determine which environmental and landscape variables influence the morphological characteristics of tadpoles from streams and ponds.
C_LIO_LIWe found that the morphological traits of tadpoles are influenced mainly by physicochemical and microhabitat attributes, being distinct between ponds and streams. In ponds, water depth, pH, and the presence of vegetation influence the morphological traits of the tadpoles, while in the streams water pH, temperature, conductivity, total alkalinity, Alk HCO3, and microhabitat variables played a major role in defining the traits.
C_LIO_LIOur results indicate that local components of habitat (water characteristics and microhabitat) influence functional traits of tadpoles in both ponds and streams, especially those supposedly related to locomotory, foraging and prey-detection abilities.
C_LI


title: Early-life immune expression profiles predict later life health and fitness in a wild rodent

abstract: Individuals differ in the nature of the immune responses they produce, affecting disease susceptibility and ultimately health and fitness. These differences have been hypothesised to have an origin in events experienced early in life that then affect trajectories of immune development and responsiveness. Here we investigate early life influences on immune expression profiles using a natural population of field voles, Microtus agrestis, in which we are able to monitor variation between and within individuals though time by repeat (longitudinal) sampling of individually marked animals. We analysed the co-expression of 20 immune genes in early life to create a correlational network consisting of three main clusters, one of which (containing Gata3, Il10 and Il17) was associated with later life reproductive success and susceptibility to chronic bacterial (Bartonella) infection. More detailed analyses supported associations between early life expression of Il17 and reproductive success later in life, and of increased Il10 expression early in life and later infection with Bartonella. We also found significant association between an Il17 genotype and the early life expression of Il10. Our results demonstrate that immune expression profiles can be manifested during early life with effects that persist through adulthood and that shape the variability among individuals in susceptibility to infection and fitness widely seen in natural populations.


title: Can floral nectars reduce transmission of Leishmania?

abstract: Insect-vectored Leishmania are the second-most debilitating of human parasites worldwide. Elucidation of the environmental factors that affect parasite transmission by vectors is essential to develop sustainable methods of parasite control that do not have off-target effects on beneficial insects or environmental health. Many phytochemicals that inhibit growth of sand fly-vectored Leishmania--which have been exhaustively studied in the search for phytochemical-based drugs--are abundant in nectar, which provide sugar-based meals to infected sand flies. In a quantitative meta-analysis, we compare inhibitory phytochemical concentrations for Leishmania to concentrations present in floral nectar and pollen. We show that nectar concentrations of several flowering plant species exceed those that inhibit growth of Leishmania cell cultures, suggesting an unexplored, landscape ecology-based approach to reduce Leishmania transmission. Strategic planting of antiparasitic phytochemical-rich floral resources or phytochemically enriched baits could reduce Leishmania loads in vectors, providing an environmentally friendly complement to existing means of disease control.


title: Population genetic bottleneck has only marginal effects on fitness evolution and its repeatability in dioecious C. elegans

abstract: The predictability of evolution is expected to depend on the relative contribution of deterministic and stochastic processes. This ratio is modulated by effective population size. Smaller effective populations harbor less genetic diversity and stochastic processes are generally expected to play a larger role, leading to less repeatable evolutionary trajectories. Empirical insight into the relationship between effective population size and repeatability is limited and focused mostly on asexual organisms. Here, we tested whether fitness evolution was less repeatable after a population bottleneck in obligately outcrossing populations of Caenorhabditis elegans. Replicated populations founded by 500, 50, or 5 individuals (no/moderate/strong bottleneck) were exposed to a novel environment with a different bacterial prey. As a proxy for fitness, population size was measured after one week of growth before and after 15 weeks of evolution. Surprisingly, we found no significant differences among treatments in their fitness evolution. Even though the strong bottleneck reduced the relative contribution of selection to fitness variation, this did not translate to a significant reduction in the repeatability of fitness evolution. Thus, although a bottleneck reduced the contribution of deterministic processes, we conclude that the predictability of evolution may not universally depend on effective population size, especially in sexual organisms.

category: evolutionary biology


title: Population bottleneck has only marginal effect on fitness evolution and its repeatability in dioecious C. elegans


title: A genome-wide epistatic network underlies the molecular architecture of continuous color variation of body extremities: a rabbit model

abstract: Deciphering the molecular architecture of coat coloration for a better understanding of the biological mechanisms underlying pigmentation still remains a challenge. We took advantage of a rabbit French experimental population in which both a pattern and a gradient of coloration from white to brown segregated within the himalayan phenotype. The whole experimental design was genotyped using the high density Affymetrix(R) AxiomOrcun SNP Array and phenotyped into 6 different groups ordered from the lighter to the darker. Genome-wide association analyses pinpointed an oligogenic determinism, under recessive and additive inheritance, involving genes already known in melanogenesis (ASIP, KIT, MC1R, TYR), and likely processed pseudogenes linked to ribosomal function, RPS20 and RPS14. We also identified (i) gene-gene interactions through ASIP:MC1R affecting light cream/beige phenotypes while KIT:RPS responsible of dark chocolate/brown colors and (ii) a genome-wide epistatic network involving several others coloration genes such as POT1 or HPS5. Finally, we determined the recessive inheritance of the English spotting phenotype likely involving a copy number variation affecting at least the end of the coding sequence of the KIT gene. Our analyses of coloration as a continuous trait allowed us to go beyond much of the established knowledge through the detection of additional genes and gene-gene interactions that may contribute to the molecular architecture of the coloration phenotype. Moreover, the characterization of a network including genes that contribute to melanogenesis and pigmentation, two processes affected in various human disorders, shows the potential interest of our rabbit model for transversal studies.

category: genetics


title: A COMPARATIVE ANALAYSIS OF INTER-SITE GENE EXPRESSION HETEROGENICITY OF NORMAL HUMAN BUCCAL MUCOSA WITH NORMAL GINGIVAL MUCOSA

abstract:
BACKGROUNDDescription of heterogeneity of gene expression of various human intraoral sites are not adequate. The aim of this study was to explore the difference of gene expression profiles of whole tissue obtained from apparently normal human gingiva and buccal mucosa (HGM, HBM).

MATERIALS AND METHODSGene sets fulfilling inclusion and exclusion criteria of HGM and HBM in gene Expression Omnibus(GEO) database were identified, segregated, filtered and analysed using the ExAtlas online web tool using pre-determined cut-off. The differentially expressed genes were studied for epithelial keratinization related, housekeeping(HKG), extracellular matrix related(ECMRG) and epithelial-mesenchymal transition related genes(EMTRGs).

RESULTSIn all 40 HBM and 64 HGM formed the study group. In all there were 18012 significantly expressed genes. Of this, 1814 were over-expressed and 1862 under-expressed HBM genes as compared to HGM. One in five of all studied genes significantly differed between HBM and HGM. For the keratinization genes, 1 in 6 differed. One of every 5 HKG-proteomics genes differed between HBM and HGM, while this ratio was 1-in 4 for all ECMRGs and EMTRGs.

DISCUSSIONThis difference in the gene expression between the HBM and HGM could possibly influence a multitude of biological pathways. This result could explain partly the difference in clinicopathological features of oral lesions occurring in HBM and HGM. The innate genotypic difference between the two intra-oral niches could serve as confounding factor in genotypic studies. Hence studies that compare the HBM and HGM should factor-in these findings while evaluating their results.

category: genomics


title: Investigating lead removal at trace concentrations from water by inactive yeast cells

abstract:
Traces of heavy metals found in water resources, due to mining activities and e-waste discharge, pose a global threat. Conventional treatment processes fail to remove toxic heavy metals, such as lead, from drinking water in a resource-efficient manner when their initial concentrations are low. Here, we show that by using the yeast Saccharomyces cerevisiae we can effectively remove trace lead from water via a rapid mass transfer process, achieving an uptake of up to 12 mg lead per gram of biomass in solutions with initial lead concentrations below 1 part per million. We found that the yeast cell wall plays a crucial role in this process, with its mannoproteins and {beta}-glucans being the key potential lead adsorbents. Furthermore, we discovered that biosorption is linked to a significant increase in cell wall stiffness. These findings open new opportunities for using environmentally friendly and abundant biomaterials for advanced water treatment targeting emerging contaminants.

One-Sentence SummaryRemoving toxic heavy metals from water at challenging trace levels in an environmentally friendly, resource-efficient manner.

category: bioengineering


title: General structure-free energy relationships of hERG blocker binding under native cellular conditions

abstract: We proposed previously that aqueous non-covalent barriers arise from solute-induced perturbation of the H-bond network of solvating water ("the solvation field") relative to bulk solvent, where the association barrier equates to enthalpic losses incurred from incomplete replacement of the H- bonds of expelled H-bond enriched solvation by inter-partner H-bonds, and the dissociation barrier equates to enthalpic + entropic losses incurred during dissociation-induced resolvation of H-bond depleted positions of the free partners (where dynamic occupancy is powered largely by the expulsion of such solvation to bulk solvent during association). We analyzed blockade of the ether-a-go-go-related gene potassium channel (hERG) based on these principles, the results of which suggest that blockers: 1) project a single rod-shaped R-group (denoted as "BP") into the pore at a rate proportional to the desolvation cost of BP, with the largely solvated remainder (denoted as "BC") occupying the cytoplasmic "antechamber" of hERG; and 2) undergo second-order entry to the antechamber, followed by first-order association of BP to the pore. In this work, we used WATMD to qualitatively survey the solvation fields of the pore and a representative set of 16 blockers sampled from the Redfern dataset of marketed drugs spanning a range of pro-arrhythmicity. We show that the highly non-polar pore is solvated principally by H-bond depleted and bulk-like water (incurring zero desolvation cost), whereas blocker BP moieties are solvated by variable combinations of H-bond enriched and depleted water. With a few explainable exceptions, the blocker solvation fields (and implied desolvation/resolvation costs) are qualitatively well-correlated with blocker potency and Redfern safety classification.

category: pharmacology and toxicology


title: Stimulus-specific processing of the plasma membrane receptor-like kinase FERONIA in Arabidopsis thaliana

abstract: FERONIA (FER), a receptor-like kinase involved in plant immunity, cell expansion, and mechanical signal transduction, is known to be endocytosed and degraded in response to treatment with its peptide ligand RAPID ALKALINIZATION FACTOR 1 (RALF1). Using confocal fluorescence microscopy and biochemical assays, we have found that full length FER-eGFP abundance at the plasma membrane is also regulated by mechanical stimulation, but through a separate, cysteine protease-dependent pathway. Like RALF1 treatment, both mechanical bending and mechanical wounding trigger a reduction in plasma membrane-localized, native promoter-driven FER-eGFP in Arabidopsis roots, hypocotyls, and cotyledons. However, pharmacological inhibition of protein trafficking and degradation suggests that while RALF1 induces clathrin-mediated endocytosis and subsequent degradation of FER-eGFP, mechanical stimulation triggers cleavage and/or degradation of FER-eGFP in a cysteine protease-dependent, clathrin-independent manner. Despite the stimulus-dependent differences in these two pathways, we found that both require early FER signaling components, including Ca2+ signaling, FER kinase activity, and the presence of LLG1, a FER-interacting protein with an essential role in FER-dependent signal transduction.

category: plant biology


title: Genome-scale modeling specifies the metabolic capabilities of Rhizophagus irregularis

abstract:
Rhizophagus irregularis is one of the most extensively studied arbuscular mycorrhizal fungi (AMF) that forms symbioses with and improves the performance of many crops. Lack of transformation protocol for R. irregularis renders it challenging to investigate molecular mechanisms that shape the physiology and interactions of this AMF with plants. Here we used all published genomics, transcriptomics, and metabolomics resources to gain insights in the metabolic functionalities of R. irregularis by reconstructing its high-quality genome-scale metabolic network that considers enzyme constraints. Extensive validation tests with the enzyme-constrained metabolic model demonstrated that it can be used to: (1) accurately predict increased growth of R. irregularis on myristate with minimal medium; (2) integrate enzyme abundances and carbon source concentrations that yield growth predictions with high and significant Spearman correlation ({rho}S = 0.74) to measured hyphal dry weight; and (3) simulated growth rate increases with tighter association of this AMF with the host plant across three fungal structures. Based on the validated model and system-level analyses that integrate data from transcriptomics studies, we predicted that differences in flux distributions between intraradical mycelium and arbuscles are linked to changes in amino acid and cofactor biosynthesis. Therefore, our results demonstrated that the enzyme-constrained metabolic model can be employed to pinpoint mechanisms driving developmental and physiological responses of R. irregularis to different environmental cues. In conclusion, this model can serve as a template for other AMF and paves the way to identify metabolic engineering strategies to modulate fungal metabolic traits that directly affect plant performance.

ImportanceMounting evidence points at the benefits of the symbiotic interactions between the arbuscular mycorrhiza fungus Rhizophagus irregularis and crops; yet, the molecular mechanisms underlying the physiological responses of this fungus to different host plants and environments remain largely unknown. We present a manually curated, enzyme-constrained genome-scale metabolic model of R. irregularis that can accurately predict experimentally observed phenotypes. We show that this high-quality model provides an entry point into better understanding the metabolic and physiological responses of this fungus to changing environments due to the availability of different nutrients. The model can be used to design metabolic engineering strategies to tailor R. irregularis metabolism towards improving the performance of host plants.

category: systems biology


title: Morphodynamical cell-state description via live-cell imaging trajectory embedding

abstract: Time-lapse imaging provides powerful insight into the dynamical response of cells to perturbation, but the quantitative analysis of morphological changes over time is a challenge. Here, we exploit the concept of "morphodynamical trajectories" to analyze cellular behavior using morphological feature trajectory histories, rather than the common practice of examining morphological feature time courses in the space of single-timepoint (snapshot) morphological features. Our morphodynamical trajectory embedding analysis yielded quantitative and descriptive models of future time points based on the extended history information of MCF10A mammary epithelial cells treated with a panel of ligands. The trajectory analysis constructs a shared morphodynamical cell-state landscape, where the response of MCF10A cells induced by various extracellular signals is characterized by ligand-specific regulation of state transitions. Additionally, we show that including trajectories in single-cell morphological analysis enables (i) systematic characterization of cell state trajectories, and (ii) better separation of phenotypes and more descriptive models of ligand-induced differences as compared to snapshot-based analysis. This morphodynamical trajectory embedding is broadly applicable for the quantitative analysis of cell responses via live-cell imaging across many biological and biomedical applications.


title: Islet transplantation tolerance in animals with defined histocompatibility and diabetes

abstract: Advances in organ transplantation benefit from development of genetically inbred animal strains with defined histocompatibility and cell-specific markers to distinguish donor and host cell subsets. For studies of pancreatic islet transplantation tolerance in diabetes, an invariant method to ablate host {beta} cells and induce diabetes would provide an immense additional advantage. Here we detail development and use of B6 RIP-DTR mice, an immunocompetent line permitting diabetes induction with 100% penetrance. This inbred line is homozygous for the C57BL/6J major histocompatibility complex (MHC) haplotype and expresses the mutant CD45.1 allele in the hematopoietic lineage. {beta} cell-specific expression of a high-affinity receptor for diphtheria toxin (DT) permits experimental {beta} cell ablation and diabetes induction after DT administration. Diabetes reversal for over one year was achieved after transplantation with congenic C57BL/6J islets, but not with MHC-mismatched BALB/c islets, which were rapidly rejected. In summary, the generation of a C57BL/6J congenic line harboring the CD45.1 allele and Ins2-HBEGF transgene should advance studies of islet transplantation tolerance and mechanisms to improve islet engraftment and function, thereby optimizing development of cell replacement strategies for diabetes mellitus.


title: Cloacimonadota metabolisms include adaptations for engineered environments that are reflected in the evolutionary history of the phylum

abstract:
Phylum Cloacimonadota (previously Cloacimonetes, WWE1) is an understudied bacterial lineage frequently associated with engineered and wastewater systems. Cloacimonadota members were abundant and diverse in metagenomic datasets from a municipal landfill, prompting an examination of phylogenetic relationships, metabolic diversity, and pangenomic dynamics across the phylum, based on 22 publicly available genomes and 24 from landfill samples. Cloacimonadota formed two discrete clades, with one clades genomes principally deriving from engineered systems. A few more-divergent genomes were placed basal in the tree, and not associated with either clade. Metabolic reconstructions for metagenome-assembled genomes predict an anaerobic, acetogenic, and fermentative lifestyle for the majority of Cloacimonadota surveyed. Genomes from engineered ecosystems (first clade) encode a unique suite of genes not typically found in genomes from natural environments (second clade). These encoded functions include acetate kinase, the enzyme responsible for the formation of acetate from acetyl phosphate, and carbon utilization enzymes, suggesting different substrate degradation and energy generation strategies in these ecologically and phylogenetically distinct lineages.

Originality/Significance StatementCloacimonadota is a bacterial phylum that is under-described compared to its members prevalence in genome repositories. Cloacimonadota are frequently associated with engineered systems, including being identified as abundant and diverse in the municipal landfill site surveyed in this study. We reconstructed twenty-four landfill-associated Cloacimonadota metagenome assembled genomes (MAGs), more than doubling the number of publicly available Cloacimonadota genomes. We combined these MAGs with available reference genomes to predict major metabolic pathways and to describe the conserved features in the lifestyle of phylum Cloacimonadota. We found that Cloacimonadota have distinct evolutionary histories associated with engineered versus natural environments. Prior studies have evaluated metabolism from individual Cloacimonadota genomes - this work is the first to examine trait distribution across a more-complete representation of the phylum, including identification of genomic features and metabolic strategies that correlate to habitat of origin.


title: On the relation between input and output distributions of scRNA-seq experiments

abstract:
Structured AbstractO_ST_ABSMotivationC_ST_ABSSingle-cell RNA sequencing determines RNA copy numbers per cell for a given gene. However, technical noise poses the question how observed distributions (output) are connected to their cellular distributions (input).

ResultsWe model a single-cell RNA sequencing setup consisting of PCR amplification and sequencing, and derive probability distribution functions for the output distribution given an input distribution. We provide copy number distributions arising from single transcripts during PCR amplification with exact expressions for mean and variance. We prove that the coefficient of variation of the output of sequencing is always larger than that of the input distribution. Experimental data reveals the variance and mean of the input distribution to obey characteristic relations, which we specifically determine for a HeLa data set. We can calculate as many moments of the input distribution as are known of the output distribution (up to all). This, in principle, completely determines the input from the output distribution.

Contactmartin.falcke@mdc-berlin.de


title: Broad-scale puma connectivity could restore genomic diversity to fine-scale coastal populations

abstract: Urbanization is decreasing wildlife habitat and connectivity worldwide, including for apex predators, such as the puma (Puma concolor). Puma populations along Californias central and southern coastal habitats have experienced rapid fragmentation from development, leading to calls for demographic and genetic management. To address urgent conservation genomic concerns, we used double-digest restriction-site associated DNA (ddRAD) sequencing to analyze 16,285 genome-wide single-nucleotide polymorphisms (SNPs) from 401 broadly sampled pumas. Our analyses indicated support for 4-10 geographically nested, broad- to fine-scale genetic clusters. At the broadest scale, the 4 genetic clusters had high genetic diversity and exhibited low linkage disequilibrium, indicating pumas have retained statewide genomic diversity. However, multiple lines of evidence indicated substructure, including 10 fine-scale genetic clusters, some of which exhibited allelic fixation and linkage disequilibrium. Fragmented populations along the Southern Coast and Central Coast had particularly low genetic diversity and strong linkage disequilibrium, indicating genetic drift and close inbreeding. Our results demonstrate that genetically at-risk populations are typically nested within a broader-scale group of interconnected populations that collectively retains high genetic diversity and heterogeneous fixations. Thus, extant variation at the broader scale has potential to restore diversity to local populations if management actions can enhance vital gene flow and recombine locally sequestered genetic diversity. These state- and genome-wide results are critically important for science-based conservation and management practices. Our broad- and fine-scale population genomic analysis highlights the information that can be gained from population genomic studies aiming to provide guidance for fragmented population conservation management.

category: zoology


title: Evolving in the darkness: phylogenomics of Sinocyclocheilus cavefishes highlights recent diversification and cryptic diversity

abstract: Troglomorphism-- any morphological adaptation enabling life to the constant darkness of caves, such as loss of pigment, reduced eyesight or blindness, over-developed tactile and olfactory organs--has long intrigued biologists. However, inferring the proximate and ultimate mechanisms driving the evolution of troglomorphism in freshwater fish requires a sound understanding of the evolutionary relationships between surface and troglomorphic lineages. We use Restriction Site Associated DNA Sequencing (RADseq) to better understand the evolution of the Sinocyclocheilus fishes of China. With a remarkable array of derived troglomorphic traits, they comprise the largest cavefish diversification in the world, emerging as a multi-species model system to study evolutionary novelty. We sequenced a total of 120 individuals throughout the Sinocyclocheilus distribution. The data comprised a total of 646,497bp per individual, including 4378 loci and 67,983 SNPs shared across a minimum of 114 individuals at a given locus. Phylogenetic analyses using either the concatenated RAD loci (RAxML) or the SNPs under a coalescent model (SVDquartets, SNAPP) showed a high degree of congruence with similar topologies and high node support (> 95 for most nodes in the phylogeny). The major clades recovered conform to a pattern previously established using Sanger-based mt-DNA sequences, with a few notable exceptions. We now recognize six major clades in this group, elevating the blind cavefish S. tianlinensis and the micro-eyed S. microphthalmus as two new distinct clades due to their deep divergence from other clades. PCA plots of the SNP data also supports the recognition of six major clusters of species congruent with the identified clades based on the spatial arrangement and overlap of the species in the PC space. A Bayes factor delimitation (BFD) analysis showed support for 21 species, recognizing 19 previously described species and two putative new cryptic ones. Two species whose identities were previously disputed, S. furcodorsalis and S. tianeensis, are supported here as distinct species. In addition, our multi-species calibrated tree in SNAPP suggests that the genus Sinocyclocheilus originated around 10.5 Mya, with most speciation events occurring in the last 2 Mya, likely favored by the uplift of the Qinghai-Tibetan Plateau and cave occupation induced by climate-driven aridification during this period. These results provide a firm basis for future comparative studies on the evolution of Sinocyclocheilus and its adaptations to cave life.


title: Inferring the mode and strength of ongoing selection

abstract: Genome sequence data is no longer scarce. The UK Biobank alone comprises 200,000 individual genomes, with more on the way, leading the field of human genetics towards sequencing entire populations. Within the next decades, other model organisms will follow suit, especially domesticated species such as crops and livestock. Having sequences from most individuals in a population will present new challenges for using these data to improve health and agriculture in the pursuit of a sustainable future. Existing population genetic methods are designed to model hundreds of randomly sampled sequences, but are not optimized for extracting the information contained in the larger and richer datasets that are beginning to emerge, with thousands of closely related individuals. Here we develop a new method called TIDES (Trio-based Inference of Dominance and Selection) that uses data from tens of thousands of family trios to make inferences about natural selection acting in a single generation. TIDES further improves on the state-of-the-art by making no assumptions regarding demography, linkage or dominance. We discuss how our method paves the way for studying natural selection from new angles.


title: TGFβR Inhibition Represses TGF-β1 Initiated Keratin-7 Expression in Human Salivary Gland Progenitor Cells

abstract: Towards the goal of engineering an implantable salivary gland for the treatment of xerostomia, we culture primary human salivary gland stem/progenitor cells (hS/PCs) in hyaluronic acid (HA)-based hydrogels containing a covalently conjugate integrin-binding peptide (RGDSP). We characterize how RGDSP affects hS/PC phenotype and discover the presence of cells expressing both amylase and keratin-7 (K7) in our 3D cultures. Typically, amylase is expressed by acinar cells, and K7 is found in ducts. After assaying an array of transforming growth factor-{beta} (TGF-{beta}) superfamily members, we find increased expression of TGF-{beta}1 and growth/differentiation factor-15 (GDF-15) in RGDSP cultures. However, 2D model studies confirm that only TGF-{beta}1 is required to induce K7 expression in hS/PCs. We then demonstrate that with pharmacological inhibition of TGF-{beta} signaling, K7 expression is repressed while amylase expression is maintained in RGDSP cultures. Thus, TGF-{beta} signaling regulates K7 expression in hS/PCs, and modulation of TGF-{beta} signaling is essential for the regeneration of salivary gland function.


title: Identification of Iron-Sulfur (Fe-S) and Zn-binding Sites Within Proteomes Predicted by DeepMind's AlphaFold2 Program Dramatically Expands the Metalloproteome

abstract: DeepMinds AlphaFold2 software has ushered in a revolution in high quality, 3D protein structure prediction. In very recent work by the DeepMind team, structure predictions have been made for entire proteomes of twenty-one organisms, with >360,000 structures made available for download. Here we show that thousands of novel binding sites for iron-sulfur (Fe-S) clusters and zinc ions can be identified within these predicted structures by exhaustive enumeration of all potential ligand-binding orientations. We demonstrate that AlphaFold2 routinely makes highly specific predictions of ligand binding sites: for example, binding sites that are comprised exclusively of four cysteine sidechains fall into three clusters, representing binding sites for 4Fe-4S clusters, 2Fe-2S clusters, or individual Zn ions. We show further: (a) that the majority of known Fe-S cluster and Zn-binding sites documented in UniProt are recovered by the AlphaFold2 structures, (b) that there are occasional disputes between AlphaFold2 and UniProt with AlphaFold2 predicting highly plausible alternative binding sites, (c) that the Fe-S cluster binding sites that we identify in E. coli agree well with previous bioinformatics predictions, (d) that cysteines predicted here to be part of Fe-S cluster or Zn-binding sites show little overlap with those shown via chemoproteomics techniques to be highly reactive, and (e) that AlphaFold2 occasionally appears to build erroneous disulfide bonds between cysteines that should instead coordinate a ligand. These results suggest that AlphaFold2 could be an important tool for the functional annotation of proteomes, and the methodology presented here is likely to be useful for predicting other ligand-binding sites.

category: bioinformatics


title: Intermolecular reorganisation of single-componentcondensates during ageing promotes multiphasearchitectures.

abstract:
Phase-separated biomolecular condensates that contain multiple coexisting phases are widespread in vitro and in cells. Multiphase condensates emerge readily within multi-component mixtures of biomolecules (e.g. proteins and nucleic acids) when the different components present sufficient physicochemical diversity (e.g. in inter-molecular forces, structure, and chemical composition) to sustain separate coexisting phases. Because such diversity is highly coupled to the solution conditions (e.g. temperature, pH, salt, composition), it can manifest itself immediately from the nucleation and growth stages of condensate formation, develop spontaneously due to external stimuli, or progressively as the condensates age. Here, we investigate thermodynamic factors that can explain the intrinsic transformation of single-component condensates into multiphase architectures during the nonequilibrium process of ageing. We develop a multiscale model that integrates atomistic simulations of proteins, sequence-dependent coarse-grained simulations of condensates, and a minimal model of dynamically ageing condensates with non-conservative inter-molecular forces. Our nonequilibrium simulations of condensate ageing predict that single-component condensates that are initially homogeneous and liquid-like can transform into gel-core/liquid-shell or liquid-core/gel-shell multiphase condensates as they age, due to gradual and irreversible enhancement of inter-protein interactions. The type of multiphase architecture is determined by the ageing mechanism, the molecular organization of the gel and liquid phases, and the chemical make up of the protein. Notably, we predict that inter-protein disorder-to-order transitions within the prion-like domains of intracellular proteins could lead to the required non-conservative enhancement of inter-molecular interactions. Our study, therefore, predicts a potential mechanism

Significance StatementBiomolecular condensates are highly diverse systems spanning not only homogeneous liquid droplets, but also gels, glasses, and even multiphase architectures that contain various coexisting liquid-like and/or gel-like inner phases. Multiphase architectures form when the different biomolecular components in a multi-component condensate establish sufficiently imbalanced inter-molecular forces to sustain different coexisting phases. While such a requirement seems, at first glance, impossible to fulfil for a condensate formed exclusively of chemically-identical proteins (i.e., single-component), our simulations predict conditions under which this may be possible. During condensate ageing, a sufficiently large imbalance in inter-molecular interactions can emerge intrinsically from the accumulation of protein structural transitions--driving even single-component condensates into nonequilibrium liquid-core/gel-shell or gel-core/liquid-shell multiphase architectures.

category: biophysics


title: Can single-component protein condensates form multiphase architectures?


title: Mistic: an open-source multiplexed image t-SNE viewer

abstract: Understanding the complex ecology of a tumor tissue and the spatio-temporal relationships between its cellular and microenvironment components is becoming a key component of translational research, especially in immune-oncology. The generation and analysis of multiplexed images from patient samples is of paramount importance to facilitate this understanding. In this work, we present Mistic, an open-source multiplexed image t-SNE viewer that enables the simultaneous viewing of multiple 2D images rendered using multiple layout options to provide an overall visual preview of the entire dataset. In particular, the positions of the images can be taken from t-SNE or UMAP coordinates. This grouped view of all the images further aids an exploratory understanding of the specific expression pattern of a given biomarker or collection of biomarkers across all images, helps to identify images expressing a particular phenotype or to select images for subsequent downstream analysis. Currently there is no freely available tool to generate such image t-SNEs. Mistic is open-source and can be downloaded at: https://github.com/MathOnco/Mistic.

category: cancer biology


title: Self-grooming promotes social interaction in mice via chemosensory communication

abstract: Self-grooming is a stereotyped behavior displayed by nearly all animals. Among other established functions, self-grooming is implicated in social communication in some animals. However, whether self-grooming specifically influences behaviors of nearby individuals has not been directly tested, partly due to the technical challenge of inducing self-grooming in a reliable and temporally controllable manner. We recently found that optogenetic activation of dopamine D3 receptor expressing neurons in the ventral striatal islands of Calleja robustly induces orofacial grooming in mice. Using this optogenetic manipulation, here we demonstrate that observer mice exhibit social preference for mice that groom more regardless of biological sex. Moreover, grooming-induced social attraction depends on volatile chemosensory cues broadcasted from grooming mice. Collectively, our study establishes self-grooming as a means of promoting social attraction among mice via volatile cues, suggesting an additional benefit for animals to allocate a significant amount of time to this behavior.

category: neuroscience


title: Self-grooming promotes social attraction in mice via chemosensory communication


title: Neural mechanisms underlying anxiety-related deficits of attentional inhibition: direct ERP evidence from the Pd component

abstract: Behavioral evidence shows that anxious individuals tend to be distracted by irrelevant stimulation not only for threat-related stimuli but also for non-emotional neutral stimuli. These findings suggest that anxious individuals may have a general impairment of attentional control, especially inhibition function. However, the neural mechanism underlying the anxiety-related impairment in attentional control is unclear. Here, in a visual search task with geometric stimuli, we examined attentional processing of the non-emotional neutral distractor on participants with different levels of anxiety, using the event-related-potential (ERP) indices of attentional selection (N2 posterior contralateral [N2pc]) and top-down inhibition (distractor positivity [Pd]). We found that distractor-evoked Pd amplitudes were negatively correlated with trait-anxiety scores, i.e., the higher the level of anxiety, the worse the ability of attentional inhibition. In contrast, the amplitudes of distractor-evoked N2pc did not vary with anxiety levels, suggesting that trait-anxiety level does not affect stimulus-driven attentional capture. We also observed attentional processing of target stimuli and found that the peak latency of target-evoked N2pc was delayed as anxiety levels rise, suggesting that anxiety impairs the efficiency of top-down attentional selection of the target. The present study provides direct neurophysiological evidence for general anxiety-related impairment of attentional control.


title: Efficient Suppression of Endogenous CFTR Nonsense Mutations Using Anticodon Engineered Transfer RNAs

abstract:
Nonsense mutations or premature termination codons (PTCs) comprise [~]11% of all genetic lesions, which result in over 7,000 distinct genetic diseases. Due to their outsized impact on human health, considerable effort has been made to find therapies for nonsense-associated diseases. Suppressor tRNAs have long been identified as a possible therapeutic for nonsense-associated diseases, however their ability to inhibit nonsense-mediated mRNA decay (NMD) and support significant protein translation from endogenous transcripts has not been determined in mammalian cells. Here we investigated the ability of anticodon edited (ACE)-tRNAs to suppress cystic fibrosis (CF) causing PTCs in the cystic fibrosis transmembrane regulator (CFTR) gene in gene-edited immortalized human bronchial epithelial (16HBEge) cells. Delivery of ACE-tRNAs to 16HBEge cells harboring three common CF mutations G542X-, R1162X- and W1282X-CFTR PTCs significantly inhibited NMD and rescued endogenous mRNA expression. Furthermore, delivery of our highly active leucine encoding ACE-tRNA resulted in rescue of W1282X-CFTR channel function to levels that significantly exceed the necessary CFTR channel function for therapeutic relevance. This study establishes the ACE-tRNA approach as a potential stand-alone therapeutic for nonsense-associated diseases due to its ability to rescue both mRNA and full-length protein expression from PTC containing endogenous genes.

One Sentence SummarySuppression of endogenous CFTR nonsense mutations by anticodon engineered tRNAs significantly increases mRNA expression and channel function.

category: molecular biology


title: Disorder or a new order: how climate change affects phenological variability

abstract: Advancing spring phenology is a well-documented consequence of anthropogenic climate change, but it is not well understood how climate change will affect the variability of phenology year-to-year. Species phenological timings reflect adaptation to a broad suite of abiotic needs (e.g. thermal energy) and biotic interactions (e.g. predation and pollination), and changes in patterns of variability may disrupt those adaptations and interactions. Here, we present a geographically and taxonomically broad analysis of phenological shifts, temperature sensitivity, and changes in inter-annual variance encompassing nearly 10,000 long-term phenology time-series representing over 1,000 species across much of the northern hemisphere. We show that early-season species in colder and less seasonal regions were the most sensitive to temperature change and had the least variable phenologies. The timings of leaf-out, flowering, insect first-occurrence, and bird arrival have all shifted earlier and tend to be less variable in warmer years. This has led leaf-out and flower phenology to become moderately but significantly less variable over time. These simultaneous changes in phenological averages and the variation around them have the potential to influence mismatches among interacting species that are difficult to anticipate if shifts in average are studied in isolation.


title: Trade-off between competition ability and invulnerability to predation in marine microbes; protist grazing versus viral lysis effects

abstract: Trade-offs between competition ability and invulnerability to predation are important mechanisms explaining how predation promotes bacterial diversity. However, existence of these trade-offs has apparently not been investigated in natural marine bacterial communities. Here, we address this question with growth-based measurements for each marine bacterial taxon by conducting on-board dilution experiments to manipulate predation pressure and using high-throughput sequencing to assess the response of bacterial communities. We determined that bacterial taxa with a higher predation-free growth rate were accompanied with higher predation-caused mortality, supporting existence of competitiveness-invulnerability trade-off. This trade-off was stronger and more consistent under viral lysis than protist grazing. In addition, predation generally flattened out the rank-abundance distribution and increased the evenness and richness of the bacterial community. These findings supported the "Kill-the-Winner" hypothesis. All experiments supported a significant competitiveness-invulnerability trade-off, but there was substantial variation among bacterial communities in response to predation across experiments conducted in various sites and seasons. Therefore, we inferred that the Kill-the-Winner hypothesis is important but likely not the only deterministic mechanism explaining how predation shapes bacterial assemblages in natural marine systems.


title: Chronic caffeine treatment disrupts the circadian rhythm in Drosophila

abstract:
The circadian clock governs the timing of sleep-wake cycles as well as of other behavioural, physiological and metabolic processes. While the endogenous circadian clock mediates the timing of sleep, homeostatic mechanisms modulate the amount and depth of sleep. Evidence from previous studies showed that caffeine intake promotes wakefulness, whereas adult-stage specific caffeine treatment not only suppresses sleep but also delays the phase of circadian rhythm in Drosophila. In humans, caffeine is consumed on a daily basis and hence it is important to understand the effect of prolonged caffeine intake on circadian and homeostatic regulation of sleep. In the present study we examined the differential effect of acute and chronic caffeine treatment on sleep ontogeny as well as on circadian and homeostatic regulation of sleep in Drosophila. The results of our study showed that acute caffeine treatment reduces day and night sleep in mature flies through the homeostatic pathway whereas it reduced only the day sleep in young flies. Chronic caffeine treatment did not exert any significant effect on sleep in young flies. On the other hand, it delayed the timing of sleep in mature flies and in addition flies under higher caffeine concentration reduced the morning and evening anticipatory activity under 12 hour: 12 hour light: dark cycles. These flies also exhibited either a longer free running period or arrhythmicity under constant darkness. The results of our study showed that acute caffeine treatment suppresses sleep through the homeostatic pathway whereas prolonged caffeine treatment disrupts the circadian rhythm in mature flies.

Statement of significanceIn the present study, we assessed the effect of caffeine on sleep ontogeny and circadian rhythm by using Drosophila as a model organism. We found that acute treatment of caffeine reduced sleep in both young and mature flies. Young flies were tolerant to chronic caffeine treatment whereas it affected the timing of sleep in mature flies. As in mammals, caffeine slows down the circadian clock in flies. Most importantly, prolonged caffeine treatment disrupted the circadian rhythm in Drosophila. Our studies provide new insights into the effect of prolonged daily caffeine intake on circadian clock and sleep.

category: animal behavior and cognition


title: Deep-Learning Super-Resolution Microscopy Reveals Nanometer-Scale Intracellular Dynamics at the Millisecond Temporal Resolution

abstract: Single-molecule localization microscopy (SMLM) can be used to resolve subcellular structures and achieve a tenfold improvement in spatial resolution compared to that obtained by conventional fluorescence microscopy. However, the separation of single-molecule fluorescence events in thousands of frames dramatically increases the image acquisition time and phototoxicity, impeding the observation of instantaneous intracellular dynamics. Based on deep learning networks, we develop a single-frame super-resolution microscopy (SFSRM) approach that reconstructs a super-resolution image from a single frame of a diffraction-limited image to support live-cell super-resolution imaging at a [~]20 nm spatial resolution and a temporal resolution of up to 10 ms over thousands of time points. We demonstrate that our SFSRM method enables the visualization of the dynamics of vesicle transport at a millisecond temporal resolution in the dense and vibrant microtubule network in live cells. Moreover, the well-trained network model can be used with different live-cell imaging systems, such as confocal and light-sheet microscopes, making super-resolution microscopy accessible to nonexperts.


title: Remote targeted electrical stimulation

abstract: The ability to generate electric fields in deep tissues remotely, without surgically implanting electrodes, could transform diagnoses and treatments of nervous system disorders. Here, we show that focal electrostimulation effects can be elicited remotely by combining two noninvasive forms of energies--magnetic and focused ultrasonic fields. The approach, based in the Lorentz equation and referred to as Lstim, electrically stimulates specified tissue targets with the precision of deep brain or spinal cord stimulation, but does not require electrode implantation. Lstim potentiated the responses of human nerves, enhancing the neuromodulatory effects of ultrasound by 74% on average. The effects showed a double dissociation--a significant and substantial increase in nociceptive responses, yet a significant reduction in tactile responses. In line with the Lorentz equation, Lstim was only observed when nerves were oriented perpendicularly to the magnetic and ultrasonic fields. A sham condition showed no effects. Both the ultrasonic and the induced electric fields were well below the respective safety indices, and no detrimental effects were detected. Lstim uniquely integrates noninvasiveness, sharp focus, and the efficacy of electrical stimulation. The approach has the potential to provide a noninvasive tool to dissect brain function in humans and to diagnose the neural circuits involved in nervous system disorders. Moreover, this effect should be taken into account when ultrasound is applied inside MRI.


title: A protocol for the reduction of applied torque on parent vessel duringelastase-induced aneurysm formation using rabbit animal models

abstract: Endovascular therapies for intracranial aneurysms requires animal models for testing the safety and effectiveness prior to translation to the clinic. Rabbits combined with the elastase and right common carotid artery (RCCA) ligation methods is currently a widely used animal model for endovascular device testing. However, the injection of elastase utilizing angiocatheters may potentially exerts adverse torque to the parent vessel and the optimal aneurysm creation period has not been well investigated. In this study, we present a modification to the elastase/RCCA-ligation method by replacing the angio-catheter with a butterfly catheter. Formation of saccular aneurysms was introduced in New Zealand white rabbits (n = 6), and were maintained for 2, 4 and 6 weeks. The formed aneurysms exhibited an elongated geometry and were stable during the study period. We found that the modification in the animal surgery procedure provides improved manipulation of the surgical area, prolonged injection of elastase, and effective degradation of the vascular elastic lamina. Compared to the traditional elastase/RCCA-ligation method, the present technique can more effectively reduce unwanted injury to the parent vessel and, therefore, improved stability of the vasculature for testing the efficacy of newly developed endovascular embolization devices.


title: Mutagenesis-visualization: analysis of site saturation mutagenesis datasets in Python

abstract:
SummarySite-saturation mutagenesis experiments have been transformative in our study of protein function. Despite the rich data generated from such experiments, current tools for processing, analyzing, and visualizing the data offer only a limited set of static visualization tools that are difficult to customize. Furthermore, usage of the tools requires extensive experience and programming knowledge, slowing the research process for those in the biological field who are unfamiliar with programming. Here, we introduce mutagenesis-visualization, a Python package for creating publication-quality figures for site-saturation mutagenesis datasets without the need for prior Python or statistics experience, where each of the graphs is generated with a one-line command. The plots can be rendered as native Matplotlib objects (easy to stylize) or Plotly objects (interactive graphs). Additionally, the software offers the possibility to visualize the datasets on Pymol.

Availability and implementationThe software can be installed from PyPI or GitHub using the pip package manager and is compatible with Python [≥] 3.8. The documentation can be found at readthedocs and the source code on GitHub.


title: Cetylpyridinium chloride (CPC) reduces zebrafish mortality from influenza infection: Super-resolution microscopy reveals CPC interference with multiple protein interactions with phosphatidylinositol 4,5-bisphosphate in immune function

abstract: The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 M, 1 hour) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5-10 {micro}M) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2 is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.


title: Quantitative multiplex immunohistochemistry reveals inter- and intra-patient lymphovascular and immune heterogeneity in primary cutaneous melanoma

abstract:
PurposeQuantitative, multiplexed imaging is revealing complex spatial relationships between phenotypically diverse tumor infiltrating leukocyte populations and their prognostic implications. The underlying mechanisms and tissue structures that determine leukocyte distribution within and around tumor nests, however, remain poorly understood. While presumed players in metastatic dissemination, new preclinical data demonstrates that blood and lymphatic vessels (lymphovasculature) also dictate leukocyte trafficking within tumor microenvironments and thereby impact anti-tumor immunity. Here we interrogate these relationships in primary human cutaneous melanoma.

Experimental DesignWe established a quantitative, multiplexed imaging platform to simultaneously detect immune infiltrates and tumor-associated vessels in formalin-fixed paraffin embedded patient samples. We performed a discovery, retrospective analysis of 28 treatment-naive, primary cutaneous melanomas.

ResultsHere we find that the lymphvasculature and immune infiltrate is heterogenous across patients in treatment naive, primary melanoma. We categorized five lymphovascular subtypes that differ by functionality and morphology and mapped their localization in and around primary tumors. Interestingly, the localization of specific vessel subtypes, but not overall vessel density, significantly associated with the presence of lymphoid aggregates, regional progression, and intratumoral T cell infiltrates.

ConclusionsWe describe a quantitative platform to enable simultaneous lymphovascular and immune infiltrate analysis and map their spatial relationships in primary melanoma. Our data indicate that tumor-associated vessels exist in different states and that their localization may determine potential for tumor cell exit (metastasis) or leukocyte trafficking (immune response).

This platform will support future efforts to map tumor-associated lymphovascular evolution across stage, assess its prognostic value, and stratify patients for adjuvant therapy.

TRANSLATIONAL RELEVANCEThis report describes a quantitative, image-based method to investigate the relationship between the tumor-associated lymphovasculature and immune landscape in treatment naive, primary human melanoma. The research shows that melanoma-associated blood and lymphatic vessels display context-dependent phenotypes that associate both with the risk of regional progression and immune infiltration. These findings indicate that stromal/vascular heterogeneity may underlie regional differences in immunogenicity and thus present opportunities for future biomarker development and therapeutic intervention.


title: SBMate: A Framework for Evaluating Quality of Annotations in Systems Biology Models

abstract: The interests in repurposing and reusing systems biology models have been growing in recent years. Semantic annotations play an important role for this, as they provide crucial information on the meanings and functions of models. However, there are a limited number of tools that evaluate the existence or quality of such annotations. In this paper, we introduce SBMate, a python package that would serve as a framework for evaluating the quality of annotations in systems biology models. Three default metrics are provided: coverage, consistency, and specificity. Coverage checks whether annotations exist in a model. Consistency tests if the annotations are appropriate for the given model element. Finally, specificity represents how detailed the annotations are. We analyzed 1,000 curated models from the BioModels repository using the three metrics and discussed the results. Additional metrics can be easily added to extend the current version of SBMate.


title: Evaluating Network Threshold Selection for Structural and Functional Brain Connectomes

abstract: Structural and functional brain connectomes represent macroscale neurophysical data collected through methods such as magnetic resonance imaging (MRI). Such data may contain noise that contribute to false positive edges, which motivates the use of edge-wise thresholding. Thresholding procedures are useful for reducing network density in graphs to retain only the most informative, non-noisy edges. Nevertheless, limited consensus exists on selecting appropriate threshold levels. We compare existing thresholding methods and introduce a novel thresholding approach in the context of MRI-derived and simulated brain connectomes. Performance is measured using normalized mutual information (NMI), a quantity robust to arbitrary changes in partition labeling, and describes the similarity of community structure between two node-matched networks. We found that the percolation-based threshold and our newly proposed "objective function"-based threshold exhibited the best performance in terms of NMI. We show an application of these two thresholding methods to real data that showed that both percolation-based and objective function-based thresholding provide statistically similar NMI values between real world FC networks and structural connectivity (SC) counterparts, where shared modular structure is assumed. The two thresholding methods, however, achieve these NMI values at significantly different thresholds (p<0.0001) in both simulated and real networks. Moreover, the threshold obtained from the objective function gives a more accurate estimate of the number of modules present in the network and includes more flexibility in threshold selection, suggesting that this method may represent a useful option for graph thresholding.


title: Reliability and Stability Challenges in ABCD Task fMRI Data

abstract: Trait stability of measures is an essential requirement for individual differences research. Functional MRI has been increasingly used in studies that rely on the assumption of trait stability, such as attempts to relate task related brain activation to individual differences in behavior and psychopathology. However, recent research using adult samples has questioned the trait stability of task-fMRI measures, as assessed by test-retest correlations. To date, little is known about trait stability of task fMRI in children. Here, we examined within-session reliability and longitudinal stability of task-fMRI using data from the Adolescent Brain Cognitive Development (ABCD) Study using its tasks focused on reward processing, response inhibition, and working memory. We also evaluated the effects of factors potentially affecting reliability and stability. Reliability and stability [quantified via an intraclass correlation (ICC) that focuses on rank consistency] was poor in virtually all brain regions, with an average ICC of .078 and .054 for short (within-session) and long-term (between-session) ICCs, respectively, in regions of interest (ROIs) historically-recruited by the tasks. ICC values in ROIs did not exceed the  poor cut-off of .4, and in fact rarely exceeded .2 (only 5.9%). Motion had a pronounced effect on estimated ICCs, with the lowest motion quartile of participants having a mean reliability/stability three times higher (albeit still  poor) than the highest motion quartile. Regions with stronger activation tended to show higher ICCs, with the absolute value of activity and reliability/stability correlating at .53. Across regions, the magnitude of age-related longitudinal (between-session) changes positively correlated with the longitudinal stability of individual differences, which suggests developmental change was not necessarily responsible for poor stability. Poor reliability and stability of task-fMRI, particularly in children, diminishes potential utility of fMRI data due to a drastic reduction of effect sizes and, consequently, statistical power for the detection of brain-behavior associations. This essential issue needs to be addressed through optimization of preprocessing pipelines and data denoising methods.


title: Convergent evolution of polyploid genomes from across the eukaryotic tree of life

abstract: By modeling the homoeologous gene losses that occurred in fifty genomes deriving from ten distinct polyploidy events, we show that the evolutionary forces acting on polyploids are remarkably similar, regardless of whether they occur in flowering plants, ciliates, fishes or yeasts. The models suggest these events were nearly all allopolyploidies, with two distinct progenitors contributing to the modern species. We show that many of the events show a relative rate of duplicate gene loss prior to the first post-polyploidy speciation that is significantly higher than in later phases of their evolution. The relatively low selective constraint seen for the single-copy genes these losses produced lead us to suggest that most of the purely selectively neutral duplicate gene losses occur in the immediate post-polyploid period. We also find ongoing and extensive reciprocal gene losses (RGL; alternative losses of duplicated ancestral genes) between these genomes. With the exception of a handful of closely related taxa, all of these polyploid organisms are separated from each other by tens to thousands of reciprocal gene losses. As a result, it is very unlikely that viable diploid hybrid species could form between these taxa, since matings between such hybrids would tend to produce offspring lacking essential genes. It is therefore possible that the relatively high frequency of recurrent polyploidies in some lineages may be due to the ability of new polyploidies to bypass RGL barriers.


title: Wind and route choice affect performance of bees flying above versus within a cluttered obstacle field

abstract: Bees flying through natural landscapes encounter physical challenges, such as wind and cluttered vegetation. The influence of these factors on the flight performance of bees remains unknown. We analyzed 548 videos of wild-caught honeybees (Apis mellifera) flying through an enclosure containing a field of vertical obstacles that bees could fly within (through open corridors, without maneuvering) or above. We examined how obstacle field height, wind presence and direction (headwinds or tailwinds) affected altitude, ground speed, and side-to-side casting (lateral excursions) of bees. When obstacle fields were short, bees flew at altitudes near the midpoint between the tunnel floor and ceiling. When obstacle fields approached or exceeded this midpoint, bees typically, but not always, increased their altitudes to fly over the obstacles. Bees that flew above the obstacle fields exhibited 40% faster ground speeds and 36% larger lateral excursions than bees that flew within the obstacle fields, likely due to the visual feedback from obstacles and narrow space available within the obstacle field. Wind had a strong effect on ground speed and lateral excursions, but not altitude. Bees flew 12-19% faster in tailwinds than in the other wind conditions, but their lateral excursions were 19% larger in any wind, regardless of its direction, than in still air. Our results show that bees flying through complex environments display flexible flight behaviors (e.g., flying above versus within obstacles), which affect flight performance. Similar choices in natural landscapes could have broad implications for foraging efficiency, pollination, and mortality in wild bees.


title: Functional Effects of Four or Fewer Critical Genes Linked to Lung Cancers and New Subtypes Detected by A New Machine Learning Classifier

abstract: Finding genes biologically directly or indirectly related to lung cancer has been drawing much attention, and many genes directly related to lung cancer have been reported. However, it has not been confirmed whether those published 'key' genes are truly critical to lung cancer formation, i.e., they may be with very limited useful information. As a result, finding essential genes remains a challenging lung cancer research problem. Using a recently developed competing linear factor analysis method in differentially expressed gene detection, we advance the study of lung cancer critical genes detection to a uniformly informative level. A set of common four genes and their functional effects are detected to be differentially expressed in tumor and non-tumor samples with 100% sensitivity and 100% specificity in one study of lung adenocarcinoma (LUAD) and one study of squamous cell lung cancers (LUSC) (two North American cohorts with 20429 genes, 576 and 552 samples respectively). Two additional analyses also gain accuracy of 97.8% sensitivity and 100% specificity in one study of non-small cell lung carcinomas (NSCLC, a European cohort with 20356 genes and 156 samples), and an accuracy of 100% sensitivity and 95% specificity (1 out of 20 non-tumor samples) in one study of ALK-positive and EGFR/KRAS/ALK-negative lung adenocarcinomas (LUAD, a Japanese cohort with 20356 genes and 224 samples). There are some common genes, but different functional effects, within each set of four genes among two North American cohorts and a European cohort and among North American cohorts and the Japanese cohort. These results show the four-gene-based classifiers are robust with different types of lung cancers and different race cohorts and accurate. The functional effects of four genes disclose significantly other mechanisms (mysteries) between LUAD and LUSC. These sets of four genes and their functional effects are considered to be essential for lung cancer studies and practice. These genes' functional effects naturally classify patients into different groups (more than seven subtypes). Subtype information is useful for personalized therapies. The new findings can motivate new lung cancer research in more focused and targeted directions to save lives, protect people, and reduce enormous economic costs in research and lung cancer treatments.


title: Annotation of Putative Circadian Rhythm-Associated Genes in Diaphorina citri (Hemiptera : Liviidae)

abstract: The circadian rhythm is a process involving multiple genes that generates an internal molecular clock, allowing organisms to anticipate environmental conditions produced by the earths rotation on its axis. This report presents the results of the manual curation of twenty-seven genes likely associated with circadian rhythm in the genome of Diaphorina citri, the Asian citrus psyllid. This insect acts as the vector of the bacterial pathogen Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease (Huanglongbing). This disease is the most severe detriment to citrus industries and has drastically decreased crop yields worldwide. Based on the genes identified in the psyllid genome, namely cry1 and cry2, D. citri likely possesses a circadian model similar to that of the lepidopteran butterfly, Danaus plexippus. Manual annotation of these genes will allow future molecular therapeutics to be developed that can disrupt the psyllid biology.


title: TSCDA: A novel greedy approach for community discovery in networks

abstract:
In this paper, we introduce a new approach for detecting community structures in networks. The approach is subject to modifying one of the connectivity-based community quality functions based on considering the impact that each communitys most influential node has on the other vertices. Utilizing the proposed quality measure, we devise an algorithm that aims to detect high-quality communities of a given network based on two stages: finding a promising initial solution using greedy methods and then refining the solutions in a local search manner.

The performance of our algorithm has been evaluated on some standard real-world networks as well as on some artificial networks. The experimental results of the algorithm are reported and compared with several state-of-the-art algorithms. The experiments show that our approach is competitive with the other well-known techniques in the literature and even outperforms them. This approach can be used as a new community detection method in network analysis.


title: Deep learning-based tumor microenvironment segmentation is predictive of tumor mutations and patient survival in non-small-cell lung cancer

abstract: Despite the fact that tumor microenvironment (TME) and gene mutations are the main determinants of progression of the deadliest cancer in the world - lung cancer - their interrelations are not well understood. Digital pathology data provide a unique insight into the spatial composition of the TME. Various spatial metrics and machine learning approaches were proposed for prediction of either patient survival or gene mutations from these data. Still, these approaches are limited in the scope of analyzed features and in their explainability and as such fail to transfer to clinical practice. Here, we generated 23,199 image patches from 55 hematoxylin-and-eosin (H&E)-stained lung cancer tissue sections and annotated them into 9 different tissue classes. Using this dataset, we trained a deep neural network ARA-CNN, achieving per-class AUC ranging from 0.72 to 0.99. We applied the trained network to segment 467 lung cancer H&E images downloaded from The Cancer Genome Atlas (TCGA) database. We used the segmented images to compute human interpretable features reflecting the heterogeneous composition of the TME, and successfully utilized them to predict patient survival (c-index 0.723) and cancer gene mutations (largest AUC 73.5% for PDGFRB). Our approach can be generalized to different cancer types to inform precision medicine strategies.


title: Annotation of Hox cluster and Hox cofactor genes in the Asian citrus psyllid, Diaphorina citri, reveals novel features

abstract: Hox genes and their cofactors are essential developmental genes that specify regional identity in animals, including insects. A particularly interesting feature of Hox genes is their conserved arrangement in clusters in the same order in which they specify identity along the anterior-posterior axis. Among insects, breaks in the cluster have been reported in a few species, but these seem to be the exception rather than the rule. We have annotated the ten Hox genes of the Asian citrus psyllid, Diaphorina citri, and determined that there is a split in its Hox cluster between the Deformed and Sex combs reduced genes. This is the first time a break at this position has been observed in an insect Hox cluster. We have also annotated the D. citri orthologs of the Hox cofactor genes homothorax, PKNOX and extradenticle. Interestingly, we found an additional copy of extradenticle in D. citri that appears to be a retrogene. Expression data and sequence conservation suggest that the extradenticle retrogene may have retained the original extradenticle function and allowed the parental extradenticle gene to diverge.