title: Deep learning applications in single-cell omics data analysis

abstract:
Traditional bulk sequencing methods are limited to measuring the average signal in a group of cells, potentially masking heterogeneity, and rare populations. The single-cell resolution, however, enhances our understanding of complex biological systems and diseases, such as cancer, the immune system, and chronic diseases. However, the single-cell technologies generate massive amounts of data that are often high-dimensional, sparse, and complex, thus making analysis with traditional computational approaches difficult and unfeasible. To tackle these challenges, many are turning to deep learning (DL) methods as potential alternatives to the conventional machine learning (ML) algorithms for single-cell studies. DL is a branch of ML capable of extracting high-level features from raw inputs in multiple stages. Compared to traditional ML, DL models have provided significant improvements across many domains and applications. In this work, we examine DL applications in genomics, transcriptomics, spatial transcriptomics, and multi-omics integration, and address whether DL techniques will prove to be advantageous or if the single-cell omics domain poses unique challenges. Through a systematic literature review, we find that DL has not yet revolutionized or addressed the most pressing challenges of the single-cell omics field. However, using DL models for single-cell omics has shown promising results (in many cases outperforming the previous state-of-the-art models) in data preprocessing and downstream analysis, but many DL models still lack the needed biological interpretability. Although developments of DL algorithms for single-cell omics have generally been gradual, recent advances reveal that DL can offer valuable resources in fast-tracking and advancing research in single-cell.

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title: NODeJ: an ImageJ plugin for 3D segmentation of nuclear objects

abstract:
BackgroundThe three-dimensional nuclear arrangement of chromatin impacts many cellular processes operating at the DNA level in animal and plant systems. Chromatin organization is a dynamic process that can be affected by biotic and abiotic stresses. Three-dimensional imaging technology allows to follow these dynamic changes, but only a few semi-automated processing methods currently exist for quantitative analysis of the 3D chromatin organization.

ResultsWe present an automated method, Nuclear Object DetectionJ (NODeJ), developed as an imageJ plugin. This program segments and analyzes high intensity domains in nuclei from 3D images. NODeJ performs a Laplacian convolution on the mask of a nucleus to enhance the contrast of intra-nuclear objects and allows their detection. We reanalyzed public datasets and determined that NODeJ is able to accurately identify heterochromatin domains from a diverse set of Arabidopsis thaliana nuclei stained with DAPI or Hoechst. NODeJ is also able to detect signals in nuclei from DNA FISH experiments, allowing for the analysis of specific targets of interest.

Conclusion and availabilityNODeJ allows for efficient automated analysis of subnuclear structures by avoiding the semi-automated steps, resulting in reduced processing time and analytical bias. NODeJ is written in Java and provided as an ImageJ plugin with a command line option to perform more high-throughput analyses. NODeJ can be downloaded from https://gitlab.com/axpoulet/image2danalysis/-/releases with source code, documentation and further information avaliable at https://gitlab.com/axpoulet/image2danalysis. The images used in this study are publicly available at https://www.brookes.ac.uk/indepth/images/ and https://doi.org/10.15454/1HSOIE.

category: bioinformatics


title: ENNGene: an Easy Neural Network model building tool for Genomics

abstract:
BackgroundThe recent big data revolution in Genomics, coupled with the emergence of Deep Learning as a set of powerful machine learning methods, has shifted the standard practices of machine learning for Genomics. Even though Deep Learning methods such as Convolutional Neural Networks (CNNs) and Recurrent Neural Networks (RNNs) are becoming widespread in Genomics, developing and training such models is outside the ability of most researchers in the field.

ResultsHere we present ENNGene - Easy Neural Network model building tool for Genomics. This tool simplifies training of custom CNN or hybrid CNN-RNN models on genomic data via an easy-to-use Graphical User Interface. ENNGene allows multiple input branches, including sequence, evolutionary conservation, and secondary structure, and performs all the necessary preprocessing steps, allowing simple input such as genomic coordinates. The network architecture is selected and fully customized by the user, from the number and types of the layers to each layer's precise set-up. ENNGene then deals with all steps of training and evaluation of the model, exporting valuable metrics such as multi-class ROC and precision-recall curve plots or TensorBoard log files. To facilitate interpretation of the predicted results, we deploy Integrated Gradients, providing the user with a graphical representation of an attribution level of each input position. To showcase the usage of ENNGene, we train multiple models on the RBP24 dataset, quickly reaching the state of the art while improving the performance on more than half of the proteins by including the evolutionary conservation score and tuning the network per protein.

ConclusionsAs the role of DL in big data analysis in the near future is indisputable, it is important to make it available for a broader range of researchers. We believe that an easy-to-use tool such as ENNGene can allow Genomics researchers without a background in Computational Sciences to harness the power of DL to gain better insights into and extract important information from the large amounts of data available in the field.

Contactpanagiotis.alexiou@ceitec.muni.cz


title: Identifying Microbial Interaction Networks Based on Irregularly Spaced Longitudinal 16S rRNA sequence data

abstract: The microbial interactions within the human microbiome are complex and temporally dynamic, but few methods are available to model this system within a longitudinal network framework. Based on general longitudinal 16S rRNA sequence data, we propose a stationary Gaussian graphical model (SGGM) for microbial interaction networks (MIN) which can accommodate the possible correlations between the high-dimensional observations. For SGGM, an EM-type algorithm is devised to compute the L1-penalized maximum likelihood estimate of MIN which employs the classic graphical LASSO algorithm as the building block and can therefore be implemented easily. Simulation studies show that the proposed algorithms can significantly outperform the conventional algorithms when the correlations between measurements grow large. The algorithms are then applied to a real 16S rRNA gene sequence data set for gut microbiome. With the estimated MIN in hand, module-preserving permutation test is proposed to test the independence of the MIN and the corresponding phylogenetic tree. The results demonstrate strong evidence of an association between the MIN and the phylogenetic tree which indicates that the genetically related taxa tend to have more/stronger interactions. The proposed algorithms are implemented in R package lglasso at https://CRAN.R-project.org/package=lglasso.


title: HiCArch: A Deep Learning-based Hi-C Data Predictor

abstract: AO_SCPLOWBSTRACTC_SCPLOWHi-C sequencing analysis is one of the most popular methods to study three-dimensional (3D) genome structures, which affect the gene expression and other cellular activities by allowing distal regulations in spatial proximity. Hi-C sequencing analysis enhances understanding of chromatin functionality. However, due to the high cost of Hi-C sequencing, the publicly available Hi-C data of high resolutions (such as 10kb) are limited in only a few cell types. In this paper we present HiCArch, a light-weight deep neural network that predicts Hi-C contact matrices from 11 common 1D epigenomic features. HiCArch identifies topological associated domains (TADs) of 10kb resolution within the distance of 10Mb. HiCArch obtains train Pearson correlation score at 0.9123 and test Pearson correlation score at 0.9195 when trained on K562 cell line. which are significantly higher than previous approaches, such as HiC-Reg[1], Akita[2], DeepC[3], and Epiphany[4].


title: Multi-state Modeling of G-protein Coupled Receptors at Experimental Accuracy

abstract: The family of G-protein coupled receptors (GPCRs) is one of the largest protein families in the human genome. GPCRs transduct chemical signals from extracellular to intracellular regions via a conformational switch between active and inactive states upon ligand binding. While experimental structures of GPCRs remain limited, high-accuracy computational predictions are now possible with AlphaFold2. However, AlphaFold2 only predicts one state and is biased towards either the active or inactive conformation depending on the GPCR class. Here, a multi-state prediction protocol is introduced that extends AlphaFold2 to predict either active or inactive states at very high accuracy using state-annotated templated GPCR databases. The predicted models accurately capture the main structural changes upon activation of the GPCR at the atomic level. For most of the benchmarked GPCRs (10 out of 15), models in the active and inactive states were closer to their corresponding activation state structures. Median RMSDs of the transmembrane regions were 1.12 [A] and 1.41 [A] for the active and inactive state models, respectively. The models were more suitable for protein-ligand docking than the original AlphaFold2 models and template-based models. Finally, our prediction protocol predicted accurate GPCR structures and GPCR-peptide complex structures in GPCR Dock 2021, a blind GPCR-ligand complex modeling competition. We expect that high accuracy GPCR models in both activation states will promote understanding in GPCR activation mechanisms and drug discovery for GPCRs. At the time, the new protocol paves the way towards capturing the dynamics of proteins at high-accuracy via machine-learning methods.

category: biophysics


title: easier: interpretable predictions of antitumor immune response from bulk RNA-seq data

abstract: Immunotherapy with immune checkpoint blockers (ICB) is associated with striking clinical success, but only in a small fraction of patients. Thus, we need computational biomarker-based methods that can anticipate which patients will respond to treatment. Current established biomarkers are imperfect due to their incomplete view of the tumor and its microenvironment. We have recently presented a novel approach that integrates transcriptomics data with biological knowledge to study tumors at a more holistic level. Validated in four different solid cancers, our approach outperformed the state-of-the-art methods to predict response to ICB. Here, we introduce estimate systems immune response (easier), an R/Bioconductor package that applies our approach to quantify biomarkers and assess patients likelihood to respond to immunotherapy, providing just the patients baseline bulk-tumor RNA-sequencing (RNA-seq) data as input.

category: systems biology


title: Divergence and convergence of cortical encoding during word reading in bilinguals

abstract:
Word reading includes a series of cognitive processes that convert low-level visual characteristics to neural representations. However, the consistency of the neural mechanisms for processing these cognitive components across different writing systems in bilinguals remains inconclusive. Here, we explored this question by employing representational similarity analysis with a semantic access task involving Chinese words, English words and Chinese pinyin. Distinct anatomical distribution patterns were detected for each type of brain representation across ideographic and alphabetic languages, resulting in 100% classification accuracy. Meanwhile, convergent cognitive components processing was found in the core language-related regions in left hemisphere, including the inferior frontal gyrus, temporal pole, superior and middle temporal gyrus, precentral gyrus and supplementary motor areas. Broadly, our findings indicated that the neural basis for word recognition of different writing systems in bilinguals was divergent in anatomical locations of neural representations for specialized processing but convergent in cognitive domains, which supported and enriched the assimilation-accommodation hypothesis.

TeaserCortical encoding linguistic processing across languages was support by parallel interdigitated distributed networks but convergent in cognitive domains.

category: neuroscience


title: Cortical encoding during word reading in bilinguals: parallel interdigitated distributed networks support convergent linguistic functioning


title: Identification of transcription factors involved in the specification of photoreceptor subtypes

abstract: During development, retinal progenitors navigate a complex landscape of fate decisions that culminates with an array of unique cell types that are required for proper vision. Here, we aim to identify factors that are required for fate decisions in photoreceptors. These factors help create a diversity of photoreceptor subtypes that sustain vision in day and night, enable the detection of colors, of prey and predators, and other aspects of vision. To identify these factors, we generate a high-quality and deep transcriptomic profile of each photoreceptor subtype in zebrafish. From these profiles, we focus on transcription factors--key players in cell-fate decisions. We apply CRISPR-FO screening as a versatile platform to explore the involvement of transcription factors in photoreceptor subtype-specification. We find that three differentially-expressed transcription factors (Foxq2, Tbx2a and Tbx2b) play unique roles in controling the identity of photoreceptor subtypes within the retina. Our results provide novel insights into the function of these factors and how photoreceptors acquire their final identities. Furthermore, we have made our transcriptomic dataset openly available and easy to explore. This dataset and the screening method will be valuable to the scientific community and will enable the exploration of genes involved in many essential aspects of photoreceptor biology.


title: STAMP: Simultaneous Training and Model Pruning for Low Data Regimes in Medical Image Segmentation

abstract: Acquisition of high quality manual annotations is vital for the development of segmentation algorithms. However, to create them we require a substantial amount of expert time and knowledge. Large numbers of labels are required to train convolutional neural networks due to the vast number of parameters that must be learned in the optimisation process. Here, we develop the STAMP algorithm to allow the simultaneous training and pruning of a UNet architecture for medical image segmentation with targeted channelwise dropout to make the network robust to the pruning. We demonstrate the technique across segmentation tasks and imaging modalities. It is then shown that, through online pruning, we are able to train networks to have much higher performance than the equivalent standard UNet models while reducing their size by more than 85% in terms of parameters. This has the potential to allow networks to be directly trained on datasets where very low numbers of labels are available.


title: OViTAD: Optimized Vision Transformer to Predict Various Stages of Alzheimer's Disease Using Resting-State fMRI and Structural MRI Data

abstract: Advances in applied machine learning techniques for neuroimaging have encouraged scientists to implement models to diagnose brain disorders such as Alzheimers disease at early stages. Predicting the exact stage of Alzheimers disease is challenging; however, complex deep learning techniques can manage this with precision. While successful, these complex architectures are difficult to interrogate and computationally expensive. Therefore, using novel, simpler architectures with more efficient pattern extraction capabilities, such as transformers, is of interest to neuroscientists. This study introduced an optimized vision transformer architecture to predict the group membership by separating healthy adults, mild cognitive impairment, and Alzheimers brains within the same age group (>75 years) using resting-state functional (rs-fMRI) and structural magnetic resonance imaging (sMRI) data. Our optimized architecture known as OViTAD is currently the sole vision transformer-based end-to-end pipeline and outperformed the existing transformer models and most state-of-the-art solutions. Our model achieved F1-scores of 97%{+/-}0.0 and 99.55%{+/-}0.39 from the testing sets for the rs-fMRI and sMRI modalities in the triple-class prediction experiments. Furthermore, our model reached these performances using 30% fewer parameters than a vanilla transformer. The model was robust and repeatable, producing similar estimates across three runs (we reported the averaged evaluation metrics). Finally, to challenge the model, we observed how it handled increasing noise levels by inserting varying numbers of healthy brains into the two dementia groups. Our findings suggest that optimized vision transformers are a promising and exciting new approach for neuroimaging applications, especially for Alzheimers disease prediction.


title: RNA Profiling of Neuropathic Pain-Associated Human DRGs Reveal Sex-differences in Neuro-immune Interactions Promoting Pain

abstract: Neuropathic pain is a leading cause of high impact pain, is often disabling and is poorly managed by current therapeutics. Here we focused on a unique group of neuropathic pain patients undergoing thoracic vertebrectomy where the DRG is removed as part of the surgery allowing for molecular characterization and identification of mechanistic drivers of neuropathic pain independently of preclinical models. Our goal was to quantify whole transcriptome RNA abundances using RNA-seq in pain-associated human DRGs from these patients, allowing comprehensive identification of molecular changes in these samples by contrasting them with non-pain associated DRGs. We sequenced 70 human DRGs, including over 50 having mRNA libraries with neuronal mRNA. Our expression analysis revealed profound sex differences in differentially expressed genes including increase of IL1B, TNF, CXCL14, and OSM in male and including CCL1, CCL21, PENK and TLR3 in female DRGs associated with neuropathic pain. Co-expression modules revealed enrichment in members of JUN-FOS signaling in males, and centromere protein coding genes in females. Neuro-immune signaling pathways revealed distinct cytokine signaling pathways associated with neuropathic pain in males (OSM, LIF, SOCS1) and females (CCL1, CCL19, CCL21). We validated cellular expression profiles of a subset of these findings using RNAscope in situ hybridization. Our findings give direct support for sex differences in underlying mechanisms of neuropathic pain in patient populations.


title: Deeper than you think: partisan-dependent brain response

abstract: Recent political polarization has highlighted the extent to which individuals with opposing views experience ongoing events in markedly different ways. In this study, we explored the neural mechanisms underpinning this phenomenon. We conducted functional magnetic resonance image (fMRI) scanning right- and left-wing participants watching political videos just before the 2019 elections in Israel. Behavioral results demonstrated significant differences between left- and right-wing participants in their interpretation of the videos content. Neuroimaging results revealed partisanship-dependent differences in both high-order regions and early-motor and somato-sensory regions, although no such differences were found with regard to neutral content. Moreover, we found that most of the political content was more potent in synchronizing participants with right-wing views, and that this synchronization was observed already in early visual and auditory cortices. These results suggest that political polarization is not limited to higher-order processes as previously thought, but rather emerges already in motor and sensory regions.


title: Identification of bacteria by poly-aromatic hydrocarbons biosensors

abstract: Human health is consistently threatened by different species of pathogenic bacteria. To fight the spread of diseases, it is important to develop rapid methods for bacterial identification. Over the years, different kinds of biosensors were developed for this cause. Another environmental risk are poly-aromatic hydrocarbons (PAHs) that may be emitted from industrial facilities and pollute environmental water and soil. One of the methods for their purification is conducted by the addition of bacteria that can degrade the PAHs, while the bacteria itself can be filtrated at the end of the process. Although many studies reported monitoring of the PAHs degradation by fluorescence, not much attention was dedicated to studying the influence of the PAHs on the intrinsic fluorescence of the degrading bacteria. In this work, we apply synchronous fluorescence (SF) measurements to study the ability of the 5 PAHs: 9-Antracene carboxylic acid (9ACA), Pyrene, Perylene, Pentacene, and Chrysene to interact with bacteria and change its fluorescence spectra. We show that upon incubation of each PAH with the bacterium E.coli only the 2 PAHs 9ACA and Perylene cause an intensity decrease in the emission at {lambda} = 300 - 375 nm, which derives from the emission of Tyrosine and Tryptophane (TT). Also, we show that upon incubation of 9ACA and Perylene with 5 different pathogenic bacteria, the intensity increase or decrease in the TT emission is unique to each bacterial species. Based on this observation, we suggest that the PAHs 9ACA and Perylene can be utilized as biosensors for bacterial identification.

category: microbiology


title: A new replication medium enables a rapid identification of {varphi}x-174 virus by synchronous fluorescence of Tryptophan.

abstract: Development of rapid methods for detection of virus particles based on their intrinsic fluorescence is challenging. Pure viruses may be detected in filtered solutions, based on the strong fluorescence of the amino acid Tryptophan (Trp) in their proteins. Nevertheless, Trp also exists in high quantities in the hosts and host cultivation media. In this work, we show that a separation of the bacteriophage{varphi} x-174 from its E. coli host (grown on the standard cultivation medium "Nutrient Agar") by simple extraction and filtration is not sufficient for its detection based on the intrinsic fluorescence since ~70 % of the Trp fluorescence is derived from impurities. We formulate a new cultivation medium with very low Trp concentration. We apply synchronous fluorescence measurements to show that no Trp fluorescence is detected in the extract solution upon incubation of this medium substrate with ammonium acetate extraction buffer. Finally, we detect{varphi} x-174 based on the spectral fingerprint of its intrinsic Trp content by synchronous fluorescence measurements. The concept of coupling intrinsic fluorescence-based methods to impurities reduction in the source, may pave the way towards future development of simple, cheap, and rapid methods for detection of viral pathogens.


title: Salmonella enterica serovar Typhimurium from Wild Birds in the United States Represent Distinct Lineages Defined by Bird Type

abstract:
Salmonella enterica serovar Typhimurium is typically considered a host generalist, however certain strains are associated with specific hosts and show genetic features of host adaptation. Here, we sequenced 131 S. Typhimurium strains from wild birds collected in 30 U.S. states during 1978-2019. We found that isolates from broad taxonomic host groups including passerine birds, water birds (Aequornithes), and larids (gulls and terns) represented three distinct lineages and certain S. Typhimurium CRISPR types presented in individual lineages. We also showed that lineages formed by wild bird isolates differed from most strains originating from domestic animal sources, and genomes from these lineages substantially improved source attribution of Typhimurium genomes to wild birds by a machine learning classifier. Furthermore, virulence gene signatures that differentiated S. Typhimurium from passerines, water birds, and larids were detected. Passerine isolates tended to lack S. Typhimurium-specific virulence plasmids. Isolates from the passerine, water bird, and larid lineages had close genetic relatedness with human clinical isolates, including those from a 2021 U.S. outbreak linked to passerine birds. These observations indicate that S. Typhimurium from wild birds in the U.S. are likely host-adapted, and the representative genomic dataset examined in this study can improve source prediction and facilitate outbreak investigation.

IMPORTANCEWithin-host evolution of S. Typhimurium may lead to pathovars adapted to specific hosts. Here, we report the emergence of disparate avian S. Typhimurium lineages with distinct virulence gene signatures. The findings highlight the importance of wild birds as a reservoir for S. Typhimurium and contribute to our understanding of the genetic diversity of S. Typhimurium from wild birds. Our study indicates that S. Typhimurium may have undergone adaptive evolution within wild birds in the U.S. The representative S. Typhimurium genomes from wild birds, together with the virulence gene signatures identified in these bird isolates, are valuable for S. Typhimurium source attribution and epidemiological surveillance.


title: Physiological roles of short-chain and long-chain menaquinones (vitamin K2) in Lactococcus cremoris

abstract: Lactococcus cremoris and L. lactis are well-known for their occurrence and applications in dairy fermentations, but their niche extends to a range of natural and food production environments. L. cremoris and L. lactis produce MKs (vitamin K2), mainly as the long-chain forms represented by MK-9 and MK-8, and a detectable amount of short-chain forms represented by MK-3. The physiological significance of the different MK forms in the lifestyle of these bacterial species has not been investigated extensively. In this study, we used L. cremoris MG1363 to construct mutants producing different MK profiles by deletion of genes encoding (i) a menaquinone-specific isochorismate synthase, (ii) a geranyltranstransferase and (iii) a prenyl diphosphate synthase. These gene deletions resulted in (i) a non-MK producer ({Delta}menF), (ii) a presumed MK-1 producer ({Delta}ispA) and (iii) a MK-3 producer ({Delta}llmg_0196), respectively. By examining the phenotypes of the MG1363 wildtype strain and respective mutants, including biomass accumulation, stationary phase survival, oxygen consumption, primary metabolites, azo dye/copper reduction, and proteomes, under aerobic, anaerobic and respiration-permissive conditions, we could infer that short-chain MKs like MK-1 and MK-3 are preferred to mediate extracellular electron transfer and reaction with extracellular oxygen, while the long-chain MKs like MK-9 and MK-8 are more efficient in aerobic respiratory electron transport chain. The different electron transfer routes mediated by short-chain and long-chain MKs likely support growth and survival of L. cremoris in a range of (transiently) anaerobic and aerobic niches including food fermentations, highlighting the physiological significance of diverse MKs in L. cremoris.


title: Isolation of a Bacillus safensis from mine tailings in Peru, genomic characterization and characterization of its cyanide- degrading enzyme CynD

abstract: Cyanide is widely used in industry as a potent lixiviant due to its capacity to tightly bind metals. This property imparts cyanide enormous toxicity to all known organisms. Thus, industries that utilize this compound must reduce its concentration in recycled or waste waters. Physical, chemical, and biological treatments have been used for cyanide remediation; however, none of them meet all the desired characteristics: efficiency, low cost and low environmental impact. A better understanding of metabolic pathways and biochemistry of enzymes involved in cyanide degradation is a necessary step to improve cyanide bioremediation efficacy to satisfy the industry requirements. Here, we used several approaches to explore this topic. We have isolated three cyanide-degrading Bacillus strains from water in contact with mine tailings from Lima, Peru, and classified them as Bacillus safensis PER-URP-08, Bacillus licheniformis PER-URP-12, and Bacillus subtilis PER-URP-17 based on 16S rRNA gene sequencing and core genome analyses. Additionally, core genome analyses of 132 publicly available genomes of Bacillus pumilus group including B. safensis and B. altitudinis allowed us to reclassify some strains and identify two strains that did not match with any known species of the Bacillus pumilus group. We searched for possible routes of cyanide-degradation in the genomes of these three strains and identified putative B. licheniformis PER-URP-12 and B. subtilis PER-URP-17 rhodaneses and B. safensis PER-URP-08 cyanide dihydratase (CynD) sequences possibly involved cyanide degradation. We identified characteristic C-terminal residues that differentiate CynD from B. pumilus and B. safensis, and showed that, differently from CynD from B. pumilus C1, recombinant CynD from the Bacillus safensis PER-URP-08 strain remains active up to pH 9 and presents a distinct oligomerization pattern at pH 8 and 9. Moreover, transcripts of B. safensis PER-URP-08 CynD (CynDPER-URP-08) are strongly induced in the presence of cyanide. Our results warrant further investigation of B. safensis PER-URP-08 and CynDPER-URP-08 as potential tools for cyanide-bioremediation.


title: Nanopore-based genome-wide DNA methylation sequencing data of Nosema ceranae-inoculated and un-inoculated eastern honeybee workers' midguts

abstract:
Apis cerana cerana is an excellent subspecies of Apis cerana, playing a vital role in pollination for wild flowers and crops as well as ecological balance. Nosema ceranae, an emergent fungal parasite infecting various bee species, originates from eastern honeybee. In this article, midguts of N. ceranae-inoculated A. c. cerana workers at 7 days post inoculation (dpi) and 10 dpi (AcT1 and AcT2) and un-inoculated workers midguts (AcCK1, AcCK2) were subjected to Nanopore-based genome-wide DNA methylation sequencing. Totally, 1773258, 2151476, 1927874 and 2109961 clean reads were generated from AcCK1, AcCK2, AcT1, and AcT2 groups, with the N50 lengths of 7548, 7936, 7678, and 7291 and the average quality value of 8.97, 8.95, 9.24, and 8.98, respectively. Among these, 93.85%, 94.49%, 88.69%, and 81.27% clean reads could be mapped to the reference genome of A. c. cerana. In the aforementioned four groups, 2149685, 2614513, 1637018 and 2726985 CHG sites were identified; the numbers of CHH sites were 9581990, 11801082, 7178559, and 12342423, whereas those of CpG sites were 14325356, 15703508, 14856284 and 13956849, respectively. Additionally, there were 36114, 118867, 30249, and 82984 6mA methylation sites respectively discovered. These data can be used for identifying differential 5mC methylation and 6mA methylation engaged in response of eastern honeybee workers to N. ceranae infestation, and for investigating the 5mC or 6mA methylation-mediated mechanism underlying host response.

Value of the dataO_LIThe data provide enrichment for information about 5mC and 6mA methylation in eastern honeybees.
C_LIO_LIOur data contributes to clarification of the epigenetic mechanism underlying eastern honeybee workers response to microsporidian infestation.
C_LIO_LIThis presented data offer novel insights into interaction between Apis cerana and Nosema ceranae.
C_LI

category: molecular biology


title: A spatial multi-omics atlas of the human lung reveals a novel immune cell survival niche

abstract: Multiple distinct cell types of the human lung and airways have been defined by single cell RNA sequencing (scRNAseq). Here we present a multi-omics spatial lung atlas to define novel cell types which we map back into the macro- and micro-anatomical tissue context to define functional tissue microenvironments. Firstly, we have generated single cell and nuclei RNA sequencing, VDJ-sequencing and Visium Spatial Transcriptomics data sets from 5 different locations of the human lung and airways. Secondly, we define additional cell types/states, as well as spatially map novel and known human airway cell types, such as adult lung chondrocytes, submucosal gland (SMG) duct cells, distinct pericyte and smooth muscle subtypes, immune-recruiting fibroblasts, peribronchial and perichondrial fibroblasts, peripheral nerve associated fibroblasts and Schwann cells. Finally, we define a survival niche for IgA-secreting plasma cells at the SMG, comprising the newly defined epithelial SMG-Duct cells, and B and T lineage immune cells. Using our transcriptomic data for cell-cell interaction analysis, we propose a signalling circuit that establishes and supports this niche. Overall, we provide a transcriptional and spatial lung atlas with multiple novel cell types that allows for the study of specific tissue microenvironments such as the newly defined gland-associated lymphoid niche (GALN).

category: cell biology


title: Life Strategies in Placozoa

abstract: Placozoans are essential reference species for understanding the origins and evolution of the animal organization. However, little is known about their life strategies in natural habitats. Here, by establishing long-term culturing for four species of Trichoplax and Hoilungia, we extend our knowledge about feeding and reproductive adaptations relevant to their ecology and immune mechanisms. Three modes of population growth depended upon feeding sources, including induction of social behaviors and different reproductive strategies. In addition to fission, representatives of all haplotypes produced  swarmers, which could be formed from the lower epithelium (with greater cell- type diversity) as a separate asexual reproduction stage. In aging culture, we reported the formation of specialized structures ( spheres) from the upper cell layer as a part of the innate immune defense response with the involvement of fiber cells. Finally, we showed that regeneration could be a part of the adaptive reproductive strategies in placozoans and a unique model for regenerative biology in general.

category: developmental biology


title: Dispersal of southern elephant seals (Mirounga leonina) from Davis Base, Antarctica: Combining population genetics and tracking data

abstract: Marine animals such as the southern elephant seal (Mirounga leonina) rely on a productive marine environment and are vulnerable to oceanic changes that can affect their reproduction and survival rates. Davis Base, Antarctica, acts as a moulting site for southern elephant seals that forage in Prydz Bay, but the genetic diversity and natal source populations of these seals has not been characterized. Determining the genetic diversity of moulting populations like this one provides essential information on seal dispersal, inter-population mixing, and foraging behaviours. In this study, we combined genetic and animal tracking data on these moulting seals to identify levels of genetic diversity, natal source population, and movement behaviours during foraging and haul-out periods. Using mitochondrial sequence data, we identified two major breeding lineages of seals at Davis Base. We found that the majority of the seals originated from breeding stocks within the South Atlantic Ocean and South Indian Ocean. One seal was grouped with the Macquarie Island breeding stock (South Pacific Ocean). The Macquarie Island population, unlike the other two stocks, is decreasing in size. Tracking data revealed long-distance foraging activity of the Macquarie Island seal around Crozet Islands. We speculate that changes to the Antarctic marine environment have resulted in a shift in foraging and dispersal strategies, which subsequently affects seal population growth rates. These findings have implications for conservation management plans aimed at improving the population status of the southern elephant seal.

category: ecology


title: Avian recolonization of unrestored and restored bogs in Eastern Canada

abstract: Over the last several decades, peat has been extracted from bogs of temperate, populated regions of Eastern Canada, leaving large areas devoid of vegetation if unrestored. For the last 25 years, projects have been conducted in these regions to re-establish vegetation and facilitate recolonization by wildlife. We tested whether vegetation structure and bird species assemblages 10 to 20 years post extraction differ among natural, unrestored and restored bogs at the scales of individual sites and entire bogs. We conducted bird counts and vegetation surveys between 1993 and 2019, using both point counts (309 sites) and Autonomous Recording Units (80 sites). According to our vegetation surveys, restoration of sites that were previously used for peat harvesting accelerated the establishment of Sphagnum and herbaceous strata, but ericaceous and tree strata were unaffected over a 17-year period. None of the bird species with large home ranges were associated specifically to natural, unrestored, or restored areas at the bog level. Bird species diversity was similar in restored and natural sites, but lower in unrestored sites. Alder Flycatcher and American Goldfinch occupied restored and unrestored sites more frequently than natural sites, independent of the number of years post extraction. Occupancy of restored sites by Palm and Yellow-rumped Warblers increased over the years, reaching levels similar to those in natural sites 20 years after restoration was implemented. Occupancy of restored sites by Song and Savannah sparrows increased from 1993-2019 and diverged from their declining occupancy of natural sites. Species assemblages of restored and unrestored sites differed significantly from those of natural sites soon after peat extraction ceased or post restoration. But assemblages from restored and unrestored sites became progressively similar to those of natural sites during the first 20 years, especially in restored sites. We conclude that bird species assemblages of restored bog sites are converging toward those of natural sites, and that restoration provides novel habitats for regionally declining species, e.g., Savannah Sparrows.


title: On selection-regulated population dynamics in birds and mammals

abstract:
I use the North American Breeding Bird Survey (Sauer et al. 2017) to construct 462 population trajectories with about 50 yearly abundance estimates each. Applying AIC model-selection, I find that selection-regulated population dynamics is 25, 000 (95%:0.42-1.7e17) times more probable than density-regulated growth. Selection is essential in 94% of the best models explaining 82% of the population dynamics variance across the North American continent. Similar results are obtained for 111, 215, and 420 populations of British birds (BTO 2020), Danish birds (DOF 2020), and birds and mammals in the Global Population Dynamic Database (GPDD 2010).

The traditional paradigm--that the population dynamic growth rate is a function of the environment, with maximal per-capita growth at low population densities, and sub-optimal reproduction from famine at carrying capacities with strong competition for limited resources--is not supported. Selection regulation generates a new paradigm where the world is green and individuals are selected to survive and reproduce at optimal levels at population dynamic equilibria with sufficient resources. It is only the acceleration of the population dynamic growth rate, and not the growth rate itself, that is determined by the density-dependent environment, with maximal growth occurring at the densities of the population dynamic equilibrium.


title: Intralocus sexual conflict can maintain alternative reproductive tactics

abstract: Alternative reproductive tactics (ARTs) are ubiquitous throughout the animal kingdom and widely regarded as an outcome of high variance in reproductive success. Proximate mechanisms underlying ARTs include genetically based polymorphisms, environmentally induced polymorphisms, and those mediated by a combination of genetic and environmental factors. However, few ultimate mechanisms have been proposed to explain the maintenance of ARTs over time, the most important of which have been disruptive and negative frequency-dependent selection. Here we explore the role that intralocus sexual conflict may play in the maintenance of sex-specific ARTs. We use a genetically explicit individual-based model in which body size influences both female fecundity and male tactic through a shared genetic architecture. By modeling ART maintenance under varying selection regimes and levels of sex-specific gene expression, we explore the conditions under which intralocus sexual conflict can maintain a hypothetical ART defined by larger (alpha) and smaller (beta) tactics. Our models consistently revealed that sexual conflict can result in the persistence of a sex-specific polymorphism over hundreds of generations, even in the absence of negative frequency-dependent selection. ARTs were maintained through correlated selection when one male ART has lower fitness but produces daughters with higher fitness. These results highlight the importance of understanding selection on both sexes when attempting to explain the maintenance of ARTs. Our results are consistent with a growing literature documenting genetic correlations between male ARTs and female fitness, suggesting that the maintenance of sex-specific ARTs through intralocus sexual conflict may be common and widespread in nature.

category: evolutionary biology


title: Genome assembly of Danaus chrysippus and comparison with the Monarch Danaus plexippus

abstract: Milkweed butterflies in the genus Danaus are studied in a diverse range of research fields including the neurobiology of migration, biochemistry of plant detoxification, host-parasite interactions, evolution of sex chromosomes, and speciation. We have assembled a nearly chromosomal genome for Danaus chrysippus (known as the African Monarch, African Queen, and Plain Tiger) using long read sequencing data. This species is of particular interest for the study of genome structural change and its consequences for evolution. Comparison with the genome of the North American Monarch Danaus plexippus reveals generally strong synteny, but highlights three inversion differences. The three chromosomes involved were previously found to carry peaks of intra-specific differentiation in D. chrysippus in Africa, suggesting that these inversions may be polymorphic and associated with local adaptation. The D. chrysippus genome is over 40% larger than that of D. plexippus, and nearly all of the additional [~]100 Megabases of DNA comprises repeats. Future comparative genomic studies within this genus will shed light on the evolution of genome architecture.

category: genomics


title: Diverse digital and fuzzy composite transcriptional elements are prevalent features of mammalian cis-regulomes

abstract: Mammalian transcriptional regulatory sequences are comprised of complex combinations of simple transcription factor (TF) motifs. Stereospecific juxta-positioning of simple TF motifs generates composite elements (CEs), that increase combinatorial and regulatory specificity of TF-DNA interactions. Although a small number of CEs and their cooperative or anti-cooperative modes of TF binding have been thoroughly characterized, a systematic analysis of CE diversity, prevalence and properties in cis-regulomes has not been undertaken. We developed a computational pipeline termed CEseek to discover >20,000 CEs in open chromatin regions of diverse immune cells and validated many using CAP-SELEX, ChIP-Seq and STARR-seq datasets. Strikingly, the CEs manifested a bimodal distribution of configurations, termed digital and fuzzy, based on their stringent or relaxed stereospecific constraints, respectively. Digital CEs mediate cooperative as well as anti-cooperative binding of structurally diverse TFs that likely reflect AND/OR genomic logic gates. In contrast, fuzzy CEs encompass a less diverse set of TF motif pairs that are selectively enriched in p300 associated, multi-genic enhancers. The annotated CEs greatly expand the regulatory DNA motif lexicon and the universe of TF-TF interactions that underlie combinatorial logic of gene regulation.


title: The base-editing enzyme APOBEC3A catalyzes cytosine deamination in RNA with low proficiency and high selectivity

abstract: Human APOBEC3A (A3A) is a nucleic acid-modifying enzyme that belongs to the cytidine deaminase family. Canonically, A3A catalyzes the deamination of cytosine into uracil in single-stranded DNA, an activity that makes A3A both a critical antiviral defense factor and a useful tool for targeted genome editing. However, off-target mutagenesis by A3A has been readily detected in both cellular DNA and RNA, which has been shown to promote oncogenesis. Given the importance of substrate discrimination for the physiological, pathological, and biotechnological activities of A3A, here we explore the mechanistic basis for its preferential targeting of DNA over RNA. Using a chimeric substrate containing a target ribocytidine within an otherwise DNA backbone, we demonstrate that a single hydroxyl at the sugar of the target base acts as a major selectivity determinant for deamination. To assess the contribution of bases neighboring the target cytosine, we show that overall RNA deamination is greatly reduced relative to that of DNA, but can be observed when ideal features are present, such as preferred sequence context and secondary structure. A strong dependence on idealized substrate features can also be observed with a mutant of A3A (eA3A, N57G) which has been employed for genome editing due to altered selectivity for DNA over RNA. Altogether, our work reveals a relationship between the overall decreased reactivity of A3A and increased substrate selectivity, and our results hold implications both for characterizing off-target mutagenesis and for engineering optimized DNA deaminases for base-editing technologies.

category: biochemistry


title: α-Helix stabilization by co-operative side chain charge-reinforced interactions to phosphoserine in a basic kinase-substrate motif

abstract: How cellular functions are regulated through protein phosphorylation events that promote or inhibit protein-protein interactions (PPIs) is key to understanding regulatory molecular mechanisms. Whilst phosphorylation can orthosterically or allosterically influence protein recognition, phospho-driven changes in the conformation of recognition motifs are less well explored. We recently discovered that clathrin heavy chain recognises phosphorylated TACC3 through a helical motif that, in the unphosphorylated protein, is disordered. However, it was unclear whether and how phosphorylation could stabilize a helix in a broader context. In the current manuscript, we address this challenge using poly-Ala based model peptides and a suite of circular dichroism and nuclear magnetic resonance spectroscopies. We show that phosphorylation of a Ser residue stabilizes the -helix in the context of an Arg(i - 3)pSeri Lys(i + 4) triad through charge-reinforced side chain interactions with positive co-operativity, whilst phosphorylation of Thr induces an opposing response. This is significant as it may represent a general method for control of PPIs by phosphorylation; basic kinase-substrate motifs are common with 55 human protein kinases recognising an Arg at a position -3 from the phosphorylated Ser, whilst the Arg(i - 3)pSeri Lys(i + 4) is a motif found in over 2000 human proteins.


title: Phenotype-Centric Modeling for Rational Metabolic Engineering

abstract: Phenotype-centric modeling enables a paradigm shift in the analysis of kinetic models. It brings the focus to a networks biochemical phenotypes and their relationship with measurable traits (e.g., product yields, system dynamics, signal amplification factors, etc.) and away from computationally intensive parameter sampling and numerical simulation. Here, we explore applications of this new modeling strategy in the field of Rational Metabolic Engineering using the amorphadiene biosynthetic network as a case study. Our phenotype-centric approach not only identifies known beneficial intervention strategies for this network, but it also provides an understanding of mechanistic context for the validity of these predictions. Additionally, we propose a set of hypothetical strains with the potential to outperform reported production strains and enhance the mechanistic understanding of the amorphadiene biosynthetic network. We believe that phenotype-centric modeling can advance the field of Rational Metabolic Engineering by enabling the development of next generation kinetics-based algorithms and methods that do not rely on a priori knowledge of kinetic parameters but allow a structured, global analysis of the design space of parameter values.

category: bioengineering


title: DLL4 and VCAM1 enhance the emergence of T cell-competent hematopoietic progenitors from human pluripotent stem cells

abstract: T cells are key mediators of the adaptive immune response and show tremendous efficacy as cellular therapeutics. However, obtaining primary T cells from human donors is expensive and variable. Pluripotent stem cells (PSCs) have the potential to serve as a consistent and renewable source of T cells, but differentiating PSCs into hematopoietic progenitors with T cell potential remains a significant challenge. Here, we developed an efficient serum- and feeder-free protocol for differentiating human PSCs into hematopoietic progenitors and T cells. This defined method allowed us to study the impact of individual recombinant proteins on blood emergence and lineage potential. We demonstrate that the presence of DLL4 and VCAM1 during the endothelial-to-hematopoietic transition (EHT) enhances downstream progenitor T cell output by >80-fold. Using single cell transcriptomics, we showed that these two proteins synergise to drive strong notch signalling in nascent hematopoietic stem and progenitor cells and that VCAM1 additionally drives a pro-inflammatory transcriptional program. Finally, we applied this differentiation method to study the impact of cytokine concentration dynamics on T cell maturation. We established optimised media formulations that enabled efficient and chemically defined differentiation of CD8{beta}+, CD4-, CD3+, TCR{beta}+ T cells from PSCs.


title: AlphaFill: enriching the AlphaFold models with ligands and co-factors

abstract: Artificial intelligence (AI) methods for constructing structural models of proteins on the basis of their sequence are having a transformative effect in biomolecular sciences. The AlphaFold protein structure database makes available hundreds of thousands of protein structures. However, all these structures lack cofactors essential for their structural integrity and molecular function (e.g. hemoglobin lacks a bound heme), key ions essential for structural integrity (e.g. zinc-finger motifs) or catalysis (e.g. Ca2+ or Zn2+ in metalloproteases), and ligands that are important for biological function (e.g. kinase structures lack ADP or ATP). Here, we present AlphaFill, an algorithm based on sequence and structure similarity, to "transplant" such "missing" small molecules and ions from experimentally determined structures to predicted protein models. These publicly available structural annotations are mapped to predicted protein models, to help scientists interpret biological function and design experiments.


title: Type II muscle fibre properties are not associated with balance recovery following large perturbations during walking in young and older adults

abstract: Falls among older adults are often attributed to declining muscle strength with ageing. Associations between muscle strength and balance control have been reported, but the evidence for, and key mechanisms of resistance exercise in fall prevention are unclear. No studies have directly examined the relationship between muscle fibre characteristics and reactive balance control. Here, we address whether or not Type II muscle fibre characteristics associate with reactive balance during walking in young and older adults with varying muscle fibre type composition. We analyse muscle biopsy-derived fibre characteristics and stability during a treadmill-based walking perturbation (trip-like) task of healthy young adults, healthy, normally active older adults, trained older adults and physically impaired older adults. We find no significant associations between Type II muscle fibre properties and reactive balance during walking, indicating that practitioners and researchers should consider more than just the muscle tissue properties when assessing and intervening on fall risk.

category: physiology


title: Ionotropic glutamate receptors in the retina - a bioinformatical meta-analysis

abstract: Glutamate is an essential neurotransmitter for signal processing in the vertical pathway of the mammalian retina, where it is involved in the distribution of visual information into several parallel channels. The excitatory effects of glutamate are mediated by AMPA-, kainate-, and NMDA-type ionotropic glutamate receptors (iGluRs). The expression patterns of these receptors in the vertebrate retina have been investigated so far with mainly immunocytochemical, in-situ hybridization, and electrophysiological/pharmacological techniques. Here, we have used scRNA sequencing data from chicken, mouse, macaque, and human retina to describe and compare the profile of iGluR expression in major retinal cell types across species. Our results suggest that major retinal cell types each express a unique set of iGluRs with substantial differences between non-mammalian and mammalian retinae. Expression of iGluRs has been investigated in more detail for amacrine and bipolar cell types of the human retina, each showing minor variations of a common pattern. The differential expression of iGluRs is likely to convey unique signal processing properties to individual elements of the retinal circuitry.


title: GnRH pulse generator frequency is modulated by kisspeptin and GABA-glutamate interactions in the posterodorsal medial amygdala in female mice

abstract: Kisspeptin neurons in the arcuate nucleus of the hypothalamus generate GnRH pulses, and act as critical initiators of functional gonadotrophin secretion, and reproductive competency. However, kisspeptin in other brain regions, most notably the posterodorsal subnucleus of the medial amygdala (MePD), plays a significant modulatory role over the hypothalamic kisspeptin population; our recent studies using optogenetics have shown that low frequency light stimulation of MePD kisspeptin results in increased LH pulse frequency. Nonetheless, the neurochemical pathways that underpin this regulatory function remain unknown. To study this, we have utilised an optofluid technology, precisely combining optogenetic stimulation with pharmacological receptor antagonism, to investigate the neurotransmission involved in this circuitry. We have shown that functional neurotransmission of both GABAA and glutamate is a requirement for effective modulation of the GnRH pulse generator by amygdala kisspeptin neurons.


title: Synaptic Activity-Dependent Changes in the Hippocampal Palmitoylome

abstract:
Dynamic protein S-palmitoylation is critical for neuronal function, development, and synaptic plasticity. Activity-dependent changes in palmitoylation have been observed for several neuronal substrates, however a full characterization of the activity-regulated palmitoylome is lacking. Here, we use an unbiased approach to identify differentially palmitoylated proteins in the mouse hippocampus following context-dependent fear conditioning. Of the 121 differentially palmitoylated proteins identified 63 were synaptic proteins, while others were associated with metabolic functions, cytoskeletal organization, and signal transduction. The vast majority of synaptic proteins exhibited increased palmitoylation following fear conditioning, whereas proteins that exhibited decreased palmitoylation were predominantly associated with metabolic processes. We show a link between dynamic palmitoylation and synapse plasticity by demonstrating that the palmitoylation of one of our identified proteins, PRG-1/LPPR4, is essential for activity-induced insertion of AMPA receptors into the postsynaptic membrane. Together, this study identifies networks of synaptic proteins whose dynamic palmitoylation may play a central role in learning and memory.

SUMMARYThis study identifies networks of proteins that undergo dynamic post-translational palmitoylation in response to fear conditioning and demonstrates that palmitoylation of one of these proteins is essential for synapse plasticity. Together, this illustrates the importance of palmitoylation in learning/memory and synapse plasticity.


title: White Matter Hyperintensity Distribution Differences in Aging and Neurodegenerative Disease Cohorts

abstract:
IntroductionWhite matter hyperintensities (WMHs) are common magnetic resonance imaging (MRI) findings in the aging population in general, as well as in patients with neurodegenerative diseases. They are known to exacerbate the cognitive deficits and worsen the clinical outcomes in the patients. However, it is not well-understood whether there are disease-specific differences in prevalence and distribution of WMHs in different neurodegenerative disorders.

MethodsData included 976 participants with cross-sectional T1-weighted and fluid attenuated inversion recovery (FLAIR) MRIs from the Comprehensive Assessment of Neurodegeneration and Dementia (COMPASS-ND) cohort of the Canadian Consortium on Neurodegeneration in Aging (CCNA) with eleven distinct diagnostic groups: cognitively intact elderly (CIE), subjective cognitive impairment (SCI), mild cognitive impairment (MCI), vascular MCI (V-MCI), Alzheimers dementia (AD), vascular AD (V-AD), frontotemporal dementia (FTD), Lewy body dementia (LBD), cognitively intact elderly with Parkinsons disease (PD-CIE), cognitively impaired Parkinsons disease (PD-CI), and mixed dementias. WMHs were segmented using a previously validated automated technique. WMH volumes in each lobe and hemisphere were compared against matched CIE individuals, as well as each other, and between men and women.

ResultsAll cognitively impaired diagnostic groups had significantly greater overall WMH volumes than the CIE group. Vascular groups (i.e. V-MCI, V-AD, and mixed dementia) had significantly greater WMH volumes than all other groups, except for FTD, which also had significantly greater WMH volumes than all non-vascular groups. Women tended to have lower WMH burden than men in most groups and regions, controlling for age. The left frontal lobe tended to have a lower WMH burden than the right in all groups. In contrast, the right occipital lobe tended to have greater WMH loads than the left.

ConclusionsThere were distinct differences in WMH prevalence and distribution across diagnostic groups, sexes, and in terms of asymmetry. WMH burden was significantly greater in all neurodegenerative dementia groups, likely encompassing areas exclusively impacted by neurodegeneration as well as areas related to cerebrovascular disease pathology.


title: Remote Deep Brain Stimulation by Transgene-free Magnetomechanical Approach

abstract: Various physical stimulation methods are developed to minimize the invasiveness of deep brain stimulation (DBS)1-3. Among them, only magnetic field can penetrate into the biological tissues without scattering or absorption4, which makes it ideal for untethered DBS. Recently developed magnetogenetics have shown the potential of developing treatments for neurological disorders5. However, magnetogenetic approaches have potential side effects from overexpression of exogenous ion channels and gene delivery with viral vectors6,7. Here, we demonstrated that the iron oxide magnetic nanodiscs (~270 nm) can be used as transducers to trigger calcium responses in the wild-type cultured neurons during the application of slow varying weak magnetic fields (50 mT at 10 Hz). Moreover, we identified that the intrinsic mechanosensitive ion channel transient receptor potential canonical (TRPC), which were widely expressed in the brain8, plays the main roles in this magnetomechanical stimulation. Finally, when we applied magnetic fields to the awake mice with magnetic nanodiscs injecting into subthalamic nucleus, the magnetomechanical stimulation triggered neuronal activities in the targeted region and the downstream region. Overall, this research demonstrated a magnetomechanical approach that can be used for wireless neuronal stimulation in vitro and untethered DBS in awake mice in vivo without implants or genetic manipulation.


title: Timing is everything: event-related transcranial direct current stimulation improves motor adaptation

abstract:
BackgroundThere is a current discord between the foundational theories underpinning motor learning and how we currently apply transcranial direct current stimulation (TDCS): the former is dependent on tight coupling of events while the latter is conducted with very low temporal resolution.

ObjectiveHere we aimed to investigate the temporal specificity of stimulation by applying TDCS in short epochs, and coincidentally with movement, during a motor adaptation task.

MethodsParticipants simultaneously adapted a reaching movement to two opposing velocity-dependent force-fields (clockwise and counter-clockwise), distinguished by a contextual leftward or rightward shift in the task display and cursor location respectively. Brief bouts (< 3 second) of event-related TDCS (er-TDCS) were applied over M1 or the cerebellum during movements for only one of these learning contexts.

ResultsWe show that when short duration stimulation is applied to the cerebellum and yoked to movement, only those reaching movements performed simultaneously with stimulation are selectively enhanced, whilst similar and interleaved movements are left unaffected. We found no evidence of improved adaptation following M1 er-TDCS, as participants displayed equivalent levels of error during both stimulated and unstimulated movements. Similarly, participants in the sham stimulation group adapted comparably during left and right-shift trials.

ConclusionsIt is proposed that the coupling of cerebellar stimulation and movement influences timing-dependent (i.e., Hebbian-like) mechanisms of plasticity to facilitate enhanced learning in the stimulated context.


title: A chemical-genetic map of the pathways controlling drug potency in Mycobacterium tuberculosis

abstract: Mycobacterium tuberculosis (Mtb) infection is notoriously difficult to treat. Treatment efficacy is limited by Mtbs intrinsic drug resistance, as well as its ability to evolve acquired resistance to all antituberculars in clinical use. A deeper understanding of the bacterial pathways that govern drug efficacy could facilitate the development of more effective therapies to overcome resistance, identify new mechanisms of acquired resistance, and reveal overlooked therapeutic opportunities. To define these pathways, we developed a CRISPR interference chemical-genetics platform to titrate the expression of Mtb genes and quantify bacterial fitness in the presence of different drugs. Mining this dataset, we discovered diverse and novel mechanisms of intrinsic drug resistance, unveiling hundreds of potential targets for synergistic drug combinations. Combining chemical-genetics with comparative genomics of Mtb clinical isolates, we further identified numerous new potential mechanisms of acquired drug resistance, one of which is associated with the emergence of a multidrug-resistant tuberculosis (TB) outbreak in South America. Lastly, we make the unexpected discovery of an "acquired drug sensitivity." We found that the intrinsic resistance factor whiB7 was inactivated in an entire Mtb sublineage endemic to Southeast Asia, presenting an opportunity to potentially repurpose the macrolide antibiotic clarithromycin to treat TB. This chemical-genetic map provides a rich resource to understand drug efficacy in Mtb and guide future TB drug development and treatment.


title: Life histories of 9,488 bird and 4,865 mammal species

abstract: I use 56,214 life history data to estimate equilibrium life history models for birds and mammals with body mass estimates. Missing parameters were estimated by allometric correlations at the lowest taxonomic level (genus, family, order, class) with data. The estimation is optimised to predict the existing data, with precision estimated separately for the different taxonomic levels of the estimator. This provides complete life history models for 9,488 species of birds, and 4,865 species of mammals. Each model includes estimates of metabolism, net assimilated energy, individual growth, mortality, fecundity, age of reproductive maturity, generation time, life span, home range, population density, biomass, population consumption, and a relative measure of intra-specific interactive competition, providing 387,531 parameter estimates in total.


title: Similarity in evoked responses does not imply similarity in macroscopic network states across tasks

abstract:
There is an ongoing debate as to whether cognitive processes arise from a group of functionally specialized brain modules (modularism) or as the result of a distributed nonlinear process (dynamical systems theory). The former predicts that tasks that recruit similar brain areas should have an equivalent degree of similarity in their connectivity. The latter allows for differential connectivity, even when the areas recruited are largely the same. Here we evaluated both views by comparing activation and connectivity patterns from a large sample of healthy subjects (N=242) that performed two executive control tasks, color-word Stroop task and Multi-Source Interference Task (MSIT), known to recruit similar brain areas. Using a measure of instantaneous connectivity based on edge time series as outcome variables, we estimated task-related network profiles as connectivity changes between incongruent and congruent information conditions. The degree of similarity of such profiles at the group level between both tasks was substantially smaller than their overlapping activation responses. A similar finding was observed at the subject level and when employing a different method for defining task-related connectivity. Our results are consistent with the perspective of the brain as a dynamical system, suggesting that task representations should be understood at both node and edge (connectivity) levels.

Significance StatementThere exist two contrasting views of the brain that yield different predictions of how cognition is represented. In the modular view, similar cognitive processes should have similar connectivity profiles. In contrast, a dynamical systems view allows for multiple network configurations in these situations. Here we tested both views using two tasks, color-word Stroop and Multi-Source Interference Task (MSIT), that evoke a similar brain response. Using a novel approach of instantaneous connectivity we first estimated the connectivity changes during both tasks and subsequently found that their similarity was substantially smaller than the patterns of brain activation. These findings reinforce the view of brain as a dynamical system and shed light onto how cognitive processes may be quantitatively modeled.


title: Dopamine release in nucleus accumbens is under tonic inhibition by adenosine A1 receptors regulated by astrocytic ENT1 and dysregulated by ethanol

abstract:
Striatal adenosine A1 receptor (A1R) activation can inhibit dopamine release. A1Rs on other striatal neurons are activated by an adenosine tone that is limited by equilibrative nucleoside transporter 1 (ENT1) that is enriched on astrocytes and is ethanol-sensitive. We explored whether dopamine release in nucleus accumbens core is under tonic inhibition by A1Rs, and is regulated by astrocytic ENT1 and ethanol. In ex vivo striatal slices from male and female mice, A1R agonists inhibited dopamine release evoked electrically or optogenetically and detected using fast-scan cyclic voltammetry, most strongly for lower stimulation frequencies and pulse numbers, thereby enhancing the activity-dependent contrast of dopamine release. Conversely, A1R antagonists reduced activity-dependent contrast but enhanced evoked dopamine release levels, even for single optogenetic pulses indicating an underlying tonic inhibition. The ENT1 inhibitor NBTI reduced dopamine release and promoted A1R-mediated inhibition, and conversely, virally-mediated astrocytic overexpression of ENT1 enhanced dopamine release and relieved A1R-mediated inhibition. By imaging the genetically encoded fluorescent adenosine sensor GRAB-Ado, we identified a striatal extracellular adenosine tone that was elevated by the ENT1 inhibitor and sensitive to gliotoxin fluorocitrate. Finally, we identified that ethanol (50 mM) promoted A1R-mediated inhibition of dopamine release, through diminishing adenosine uptake via ENT1. Together, these data reveal that dopamine output dynamics are gated by a striatal adenosine tone, limiting amplitude but promoting contrast, regulated by ENT1, and promoted by ethanol. These data add to the diverse mechanisms through which ethanol modulates striatal dopamine, and to emerging datasets supporting astrocytic transporters as important regulators of striatal function.

SIGNIFICANCE STATEMENTDopamine axons in the mammalian striatum are emerging as strategic sites where neuromodulators can powerfully influence dopamine output in health and disease. We found that ambient levels of the neuromodulator adenosine tonically inhibit dopamine release in nucleus accumbens core via adenosine A1 receptors (A1Rs), to a variable level that promotes the contrast in dopamine signals released by different frequencies of activity. We reveal that the equilibrative nucleoside transporter 1 (ENT1) on astrocytes limits this tonic inhibition, and that ethanol promotes it by diminishing adenosine uptake via ENT1. These findings support the hypotheses that A1Rs on dopamine axons inhibit DA release and, furthermore, that astrocytes perform important roles in setting the level of striatal dopamine output, in health and disease.


title: More than meets the Kappa for Antibody Superantigen Protein L (PpL)

abstract: Immunoglobulin superantigens play an important role in the affinity purification of antibodies and underlie the microbiota-immune axis at mucosal areas Focussing on the Staphylococcal Protein A (SpA), Streptococcal Protein G (SpG), and the Finegoldia Protein L (PpL) that were previously thought to bind to only specific regions of human antibodies, a systematic and holistic analysis of the antibody regions using 63 antibody permutations involving six V{kappa} and seven VH region IgG1 revealed showed novel PpL-antibody interactions. While SpA and SpG showed relatively consistent interactions with the antibodies, our findings showed PpL binding to certain VH-V{kappa}2, 5 and 6 interactions had contribution by other antibody regions. The findings of this have implications on PpL-based affinity antibody purifications and antibody design as well as provides novel insights to PpL-based microbiota-immune axis effects.

category: immunology


title: Mural cell SRF controls pericyte migration, vessel patterning and blood flow

abstract:
RationalePericytes (PCs) and vascular smooth muscle cells (vSMCs), collectively known as mural cells (MCs), are recruited through PDGFB-PDGFRB signaling. MCs are essential for vascular integrity, and their loss has been associated with numerous diseases. Most of this knowledge is based on studies in which MCs are insufficiently recruited or fully absent upon inducible ablation. In contrast, little is known about the physiological consequences that result from impairment of specific MC functions.

ObjectiveHere, we characterize the role of the transcription factor serum response factor (SRF) in MCs and study its function in developmental and pathological contexts.

Methods and ResultsWe generated a mouse model of MC-specific inducible Srf gene deletion and studied its consequences during retinal angiogenesis. By postnatal day (P)6, PCs lacking SRF were morphologically abnormal and failed to properly co-migrate with angiogenic sprouts. As a consequence, PC-deficient vessels at the retinal sprouting front became dilated and leaky. By P12, also the vSMCs had lost SRF, which coincided with the formation of pathological arteriovenous (AV) shunts. Mechanistically, we show that PDGFB-dependent SRF activation is mediated via MRTF co-factors. We further show that MRTF-SRF signaling promotes pathological PC activation during ischemic retinopathy. RNA-sequencing, immunohistology, in vivo live imaging and in vitro experiments demonstrated that SRF regulates expression of contractile SMC proteins essential to maintain the vascular tone.

ConclusionsSRF is crucial for distinct functions in PCs and vSMCs. SRF directs PC migration downstream of PDGFRB signaling and mediates pathological PC activation during ischemic retinopathy. In vSMCs, SRF is essential for expression of the contractile machinery, and its deletion triggers formation of AV shunts. These essential roles in physiological and pathological contexts provide a rational for novel therapeutic approaches through targeting SRF activity in MCs.


title: Time course of EEG power during creative problem-solving with insight or remote thinking

abstract: Problem-solving often requires creativity and is critical in everyday life. However, the neurocognitive mechanisms underlying creative problem-solving remain poorly understood. Two mechanisms have been highlighted: forming new connections from and between the problem elements and insight solving (with a sudden realization of a solution). We examined EEG activity during an adapted version of a classical insight problem task, the Remote Associates Test, that requires finding a word connecting three words. It allowed us to explore remoteness in semantic connections (by varying the remoteness of the solution word across trials) and insight solving (identified as a "Eureka" moment reported by the participants). Semantic remoteness was associated with a power increase in alpha band (8-12Hz) in a left parieto-temporal cluster, beta band (13-30Hz) in a right fronto-temporal cluster in the early phase of the task, and theta band (3-7Hz) in frontal cluster before the participants responded. Insight solving was associated with power increase preceding the response in alpha and gamma band (31-60Hz) in left temporal clusters and theta band in a frontal cluster. Source reconstructions show the brain regions associated with these clusters. Overall, our findings shed new light on the dynamic of some of the mechanisms involved in creative problem-solving.


title: Obesity impairs therapeutic efficacy of mesenchymal stem cells by inhibiting cardiolipin-dependent mitophagy and intercellular mitochondrial transfer in mouse models of airway allergic inflammation

abstract: Mesenchymal stem cell (MSC) transplantation alleviates metabolic defects in diseased recipient cells by intercellular mitochondrial transport (IMT). However, the effect of host metabolic conditions on MSCs in general, and IMT in particular, has largely remained unexplored. This study has identified a molecular pathway that primarily governs the metabolic function and IMT of MSCs. We found underlying mitochondrial dysfunction, impaired mitophagy, and reduced IMT in MSCs derived from high-fat diet (HFD)-induced obese mice (MSC-Ob). Mechanistically, MSC-Ob failed to sequester their damaged mitochondria into LC3-dependent autophagosomes due to decrease in mitochondrial cardiolipin content, which we propose as a putative mitophagy receptor for LC3 in MSCs. Functionally, MSC-Ob exhibited diminished potential to rescue metabolic deficits and cell death in stress-induced epithelial cells. In a small molecule screen, we found pyrroloquinoline quinone (PQQ) as a regulator of mitophagy and IMT. Long-term culture of MSC-Ob with PQQ (MSC-ObPQQ) restored cardiolipin content and sequestration of mitochondria to autophagosomes with concomitant activation of mitophagy. Upon co-culture, MSC-ObPQQ rescued cell death in stress-induced epithelial cells by enhancing IMT. The beneficial effect of PQQ was also evident in MSCs derived from human subjects in an in vitro model. In two independent mice models, the transplantation of MSC-ObPQQ restored IMT to airway epithelial cells, improved their mitochondrial metabolism and attenuated features of allergic airway inflammation (AAI). However, unmodulated MSC-Ob failed to do so. In summary, we uncover the molecular mechanism leading to the therapeutic decline of obese-derived MSCs and highlight the importance of pharmacological modulation of these cells for therapeutic intervention.


title: Single-cell generalized trend model (scGTM): a flexible and interpretable model of gene expression trend along cell pseudotime

abstract:
MotivationModeling single-cell gene expression trends along cell pseudotime is a crucial analysis for exploring biological processes. Most existing methods rely on nonparametric regression models for their flexibility; however, nonparametric models often provide trends too complex to interpret. Other existing methods use interpretable but restrictive models. Since model interpretability and flexibility are both indispensable for understanding biological processes, the single-cell field needs a model that improves the interpretability and largely maintains the flexibility of nonparametric regression models.

ResultsHere we propose the single-cell generalized trend model (scGTM) for capturing a genes expression trend, which may be monotone, hill-shaped, or valley-shaped, along cell pseudotime. The scGTM has three advantages: (1) it can capture non-monotonic trends that are still easy to interpret, (2) its parameters are biologically interpretable and trend informative, and (3) it can flexibly accommodate common distributions for modeling gene expression counts. To tackle the complex optimization problems, we use the particle swarm optimization algorithm to find the constrained maximum likelihood estimates for the scGTM parameters. As an application, we analyze several single-cell gene expression data sets using the scGTM and show that it can capture interpretable gene expression trends along cell pseudotime and reveal molecular insights underlying the biological processes.

Availability and implementationThe Python package scGTM is open-access and available at https://github.com/ElvisCuiHan/scGTM.

Contactjli@stat.ucla.edu


title: Two Liberibacter Effectors Combine to Suppress Critical Innate Immune Defenses and Facilitate Huanglongbing Pathogenesis in Citrus

abstract: Genome sequence analyses predicted the presence of effectors in the gram-negative Candidatus Liberibacter asiaticus (CLas) even without the presence of a classical type III secretion system. Since CLas is not culturable, it is not possible to perform traditional gene knockout experiments to determine the role of various effectors in Huanglongbing (HLB) pathogenesis. Therefore, we followed an alternative functional genomics approach to examine the role of the CLas effectors in HLB pathogenesis in general and more specifically in suppressing citrus innate immune response. Here, we focused on the CLas effectors, P235 and Effector 3, to perform the following studies. First, proteomic studies by LC-MS/MS were conducted to screen the putative interacting citrus protein partners of P235 and Effector 3 from the healthy and CLas-infected Hamlin extracts and the most probable candidates were identified based upon their high protein scores from LC-MS/MS. Second, a transgenic tobacco split GFP system was designed for in planta detection of the most probable citrus interacting protein partners of P235 and Effector 3. Third, in vitro and in planta studies were performed to show that each of two effectors interacts with and inhibits the functions of multiple citrus proteins belonging to the innate immune pathways. These inhibitory interactions led to a high level of reactive oxygen species (ROS), blocking of bactericidal lipid binding protein (LTP), and induction of premature programmed cell death (PCD), thereby supporting CLas infection and HLB pathogenesis. Finally, an LTP mimic was designed to sequester and block the CLas effector and to rescue the bactericidal activity of LTP.

category: plant biology


title: Archaic introgression and variation in pharmacogenes and implications for local adaptation

abstract: Modern humans carry Neanderthal and Denisovan (archaic) genome elements which may have been adaptive. These effects may be particularly evident in pharmacogenes - genes responsible for the processing of exogenous substances such as food, pollutants, and medications. However, the health implications and contribution of archaic ancestry in pharmacogenes of modern humans remains understudied. We characterize eleven key cytochrome P450 (CYP450) genes involved in drug metabolizing reactions in three Neanderthal and one Denisovan individuals and examine archaic introgression in modern human populations. We infer the metabolizing efficiency of these eleven genes in archaic individuals and show important genetic differences relative to modern human variants. We identify archaic-specific SNVs in each CYP450 gene, including some that are potentially damaging, which may result in altered metabolism in modern human people carrying these variants. We highlight four genes which display interesting patterns of archaic variation: CYP2B6 - we find a large number of unique variants in the Vindija Neanderthal, some of which are shared with a small subset of African modern humans; CYP2C9 - containing multiple variants that are shared between Europeans and Neanderthals; CYP2A6*12 - a variant defined by a hybridization event that was found in humans and Neanderthals, suggesting the recombination event predates both species; and CYP2J2 - in which we hypothesize a Neanderthal variant was re-introduced in non-African populations by archaic admixture. The genetic variation identified in archaic individuals suggests that selective pressures from the environment may be involved in the evolution of CYP450 genes.


title: Deletion of two genes improves heterologous secretion of fungal lignin-degrading heme peroxidases in S. cerevisiae

abstract: Efficient, large-scale heterologous production of enzymes is a crucial component of the biomass valorization industry. Whereas cellulose utilization has been successful in applications such as bioethanol, its counterpart lignin remains significantly underutilized despite being an abundant potential source of aromatic commodity chemicals. Fungal lignin-degrading heme peroxidases are thought to be the major agents responsible for lignin depolymerization in nature, but their large-scale production remains inaccessible due to the genetic intractability of basidiomycete fungi and the challenges in the heterologous production of these enzymes. In this study, we employ a strain engineering approach based on functional genomics to identify mutants of the model yeast Saccharomyces cerevisiae with enhanced heterologous production of lignin-degrading heme peroxidases. We show that our screening method coupling an activity-based readout with fluorescence-assisted cell sorting enables identification of two single null mutants of S. cerevisiae, pmt2 and cyt2, with up to 11-fold improved secretion of a versatile peroxidase from the lignin-degrading fungus Pleurotus eryngii. We demonstrate that the double deletion strain pmt2cyt2 displays positive epistasis, improving and even enabling production of members from all three classes of lignin-degrading fungal peroxidases. We anticipate that these mutant strains will be broadly applicable for improved heterologous production of this biotechnologically important class of enzymes.


title: Enemy or ally: a genomic approach to elucidate the lifestyle of Phyllosticta citrichinaensis

abstract: Members of the fungal genus Phyllosticta can colonize a variety of plant hosts, including several Citrus species such as Citrus sinensis (orange), Citrus limon (lemon), and Citrus maxima (pomelo). Some Phyllosticta species have the capacity to cause disease, such as Citrus Black Spot, while others have only been observed as endophytes. Thus far, genomic differences underlying lifestyle adaptations of Phyllosticta species have not yet been studied. Furthermore, the lifestyle of Phyllosticta citrichinaensis is ambiguous, as it has been described as a weak pathogen but Kochs postulates may not have been established and the presence of this species was never reported to cause any crop or economic losses. Here, we examined the genomic differences between pathogenic and endophytic Phyllosticta spp. colonizing Citrus and specifically aimed to elucidate the lifestyle of Phyllosticta citrichinaensis. We found several genomic differences between species of different lifestyles, including groups of genes that were only present in pathogens or endophytes. We also observed that species, based on their carbohydrate active enzymes, group independent of their phylogenetic association, and this clustering correlated with trophy prediction. Phyllosticta citrichinaensis shows an intermediate lifestyle, sharing genomic and phenotypic attributes of both pathogens and endophytes. We thus present the first genomic comparison of multiple citrus-colonizing pathogens and endophytes of the genus Phyllosticta, and therefore provide the basis for further comparative studies into the lifestyle adaptations within this genus.


title: Population Dynamics of Hybrid State during Adaptive Therapy in Cancer

abstract: Drug resistance emerges due to drug-induced phenotypic switching of drug-sensitive to drug-resistant subpopulations in cancer during therapy. Existing models indicate the competitive advantage of sensitive over resistant population to regulate tumor and reducing the treatment cost with increased time to progression of tumor ultimately benefiting the patient in a clinical setting. Here, we present a Lotka Volterra (LV) based population dynamics (PD) model of the drug-sensitive, drug-resistant, and transient drug-hybrid state along with phenotypic switching during adaptive therapy based on a simple cancer biomarker (CB) to decide the adaptive therapy dosage to regulate cancer. We identified that the strength of intra competition along with phenotypic switching parameters is crucial to mediate the effectiveness of adaptive therapy and also investigated the significance of the initial fraction of subpopulations on AT. We hypothesize and predict the dynamics of drug-induced transient hybrid state playing a key role in the cancer cells undergoing metastasis.

category: cancer biology


title: Improved reference genome annotation of Brassica rapa by PacBio RNA sequencing

abstract: The species Brassica rapa includes several important vegetable crops. The draft reference genome of B. rapa ssp. pekinensis was completed in 2011, and it has since been updated twice. The pangenome with structural variations of 18 B. rapa accessions was published in 2021. Although extensive genomic analysis has been conducted on B. rapa, a comprehensive genome annotation including gene structure, alternative splicing events, and non-coding genes is still lacking. Therefore, we used the Pacific Biosciences (PacBio) single-molecular long-read technology to improve gene models and produced the annotated genome version 3.5. In total, we obtained 753,041 full-length non-chimeric (FLNC) reads and collapsed these into 92,810 non-redundant consensus isoforms, capturing 48% of the genes annotated in the B. rapa reference genome annotation v3.1. Based on the isoform data, we identified 830 novel protein-coding genes that were missed in previous genome annotations, defined the UTR regions of 20,340 annotated genes and corrected 886 wrongly-spliced genes. We also identified 28,564 alternative splicing (AS) events and 1,480 long non-coding RNAs (lncRNAs). We produced a relatively complete and high-quality reference transcriptome for B. rapa that can facilitate further functional genomic research.


title: Parameter estimation in the age of degeneracy and unidentifiability

abstract: Parameter estimation from observable or experimental data is a crucial stage in any modeling study. Identifiability refers to ones ability to uniquely estimate the model parameters from the available data. Structural unidentifiability in dynamic models, the opposite of identifiability, is associated with the notion of degeneracy where multiple parameter sets produce the same pattern. Therefore, the inverse function of determining the model parameters from the data is not well defined. Degeneracy is not only a mathematical property of models, but it has also been reported in biological experiments. Classical studies on structural unidentifiability focused on the notion that one can at most identify combinations of unidentifiable model parameters. We have identified a different type of structural degeneracy/unidentifiability present in a family of models, which we refer to as the Lambda-Omega ({Lambda}-{Omega}) models. These are an extension of the classical lambda-omega ({lambda}-{omega}) models that have been used to model biological systems, and display a richer dynamic behavior and waveforms that range from sinusoidal to square-wave to spike-like. We show that the {Lambda}-{Omega} models feature infinitely many parameter sets that produce identical stable oscillations, except possible for a phase-shift (reflecting the initial phase). These degenerate parameters are not identifiable combinations of unidentifiable parameters as is the case in structural degeneracy. In fact, reducing the number of model parameters in the {Lambda}-{Omega} models is minimal in the sense that each one controls a different aspect of the model dynamics and the dynamic complexity of the system would be reduced by reducing the number of parameters. We argue that the family of {Lambda}-{Omega} models serves as a framework for the systematic investigation of degeneracy and identifiability in dynamic models and for the investigation of the interplay between structural and other forms of unidentifiability resulting on the lack of information from the experimental/observational data.


title: The bacterial hitchhiker's guide to COI: Universal primer-based COI capture probes fail to exclude bacterial DNA, but 16S capture leaves metazoa behind

abstract: Environmental DNA (eDNA) metabarcoding from water samples has, in recent years, shown great promise for biodiversity monitoring. However, universal primers targeting the cytochrome oxidase I (COI) marker gene popular in metazoan studies have displayed high levels of nontarget amplification. To date, enrichment methods bypassing amplification have not been able to match the detection levels of conventional metabarcoding. This study evaluated the use of universal metabarcoding primers as capture probes to either isolate target DNA or to remove nontarget DNA, prior to amplification, by using biotinylated versions of universal metazoan and bacterial barcoding primers, namely metazoan COI (mlCOIintF) and bacterial 16S (515F). Additionally, each step of the protocol was assessed by amplifying for both metazoan COI (mlCOIintF/jgHCO2198) and bacterial 16S (515F/806R) to investigate the effect on the metazoan and bacterial communities. Bacterial read abundance increased significantly in response to the captures (COI library), while the quality of the captured DNA was also improved. The metazoan-based probe captured bacterial DNA in a range that was also amplifiable with the 16S primers, demonstrating the ability of universal capture probes to isolate larger fragments of DNA from eDNA. Although the use of the tested COI probe cannot be recommended for metazoan enrichment, based on the experimental results, the concept of capturing longer fragments could be applied to metazoan metabarcoding. By using a truly conserved site without a high-level taxonomic resolution as a target for capture, it may be possible to isolate DNA fragments large enough to span over a nearby barcoding region (e.g., COI), which can then be processed through a conventional metabarcoding-by-amplification protocol.


title: High throughput screening of a new fluorescent G-quadruplex ligand having telomerase inhibitory activity in human A549 cells

abstract:
Genome-wide analysis showed that putative G-quadruplex DNA structures are prevalent in the human genome. The presence of G-quadruplex structure in the telomere and promoter region of certain oncogenes inspired people to use G-quadruplex ligand as anti-cancer agents. G-quadruplex structures, stabilized by ligand at telomere are resolved by telomerase making the cancer cells resistant to G-quadruplex ligand. So, identification of a new G-quadruplex ligand having anti-telomerase activity would be a promising strategy for cancer therapy as about 85% of human cancers are telomerase positive. A set of the drug-like compounds were screened from the ZINC database randomly and 2284 ligands were chosen following Lipinskis rule of five that were docked with five different G-quadruplex DNA sequences in idock. We screened 43 potential G-quadruplex binders using Z-score as a normalization scoring function. The compound (ZINC ID-05220992) gave the best score (average idock = -10.17 kcal/mol, average normalized idock = -3.42). We performed G4 FID assay, CD analysis to understand its binding with three different G-quadruplex DNA sequences, and checked its anti-telomerase activity in A549 cells using TRAP assay. We observed that this compound had an intrinsic fluorescence, capability to stain live cells with a blue fluorescence, and a specific affinity to only 22AG out of three different G-quadruplex DNA sequences under study. It showed cytotoxicity, good permeability to live cells, and a significant reduction of telomerase activity in human A549 cells at a very low dose. So, this compound has strong potential to be an anti-cancer drug.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/470216v1_ufig1.gif" ALT="Figure 1">
View larger version (41K):
org.highwire.dtl.DTLVardef@116cce1org.highwire.dtl.DTLVardef@1e500c1org.highwire.dtl.DTLVardef@194a0b3org.highwire.dtl.DTLVardef@c258fe_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIA set of compounds were screened randomly by a High throughput method and Lipinskis rule of five from ZINC database to identify potential G4-binders.
C_LIO_LIThe compound (ZINC ID-05220992) was screened after docking with five G-quadruplex DNA
C_LIO_LIIt binds G-quadruplex DNA 22AG as detected by TO displacement and CD spectroscopy.
C_LIO_LIIt inhibits telomerase activity in A549 cells and also cytotoxic to this cell.
C_LIO_LIIt penetrates live A549 cell in culture and stains it with blue fluorescence
C_LI


title: The effects of weak selection on neutral diversity at linked sites

abstract: The effects of selection on variability at linked sites have an important influence on levels and patterns of within-population variation across the genome. Most theoretical models of these effects have assumed that selection is sufficiently strong that allele frequency changes at the loci concerned are largely deterministic. These models have led to the conclusion that directional selection for selectively favorable mutations, or against recurrent deleterious mutations, reduces nucleotide site diversity at linked neutral sites. Recent work has shown, however, that fixations of weakly selected mutations, accompanied by significant stochastic changes in allele frequencies, can sometimes cause higher diversity at linked sites when compared with the effects of fixations of neutral mutations. The present paper extends this work by deriving approximate expressions for the mean conditional times to fixation and loss of mutations subject to selection, and analysing the conditions under which selection increases rather than reduces these times. Simulations are used to examine the relations between diversity at a neutral site and the fixation and loss times of mutations at a linked site that is subject to selection. It is shown that the long-term level of neutral diversity can be increased over the purely neutral value by recurrent fixations and losses of linked, weakly selected dominant or partially dominant favorable mutations, or linked recessive or partially recessive deleterious mutations. The results are used to examine the conditions under which associative overdominance, as opposed to background selection, is likely to operate.