Miracle-dz/UniFAR-FTUs huggingface dataset:
Three-dimensional biofilm growth supports a mutualism involving matrix and nutrient sharing

Life in a three-dimensional structure such as a biofilm is typical for many bacteria, yet little is known about how strains with different genotypes interact in this context. Here, to systematically explore gene knockdowns across various three-dimensional contexts, we created arrayed libraries of essential-gene CRISPRi knockdowns in the model biofilm-forming bacterium Bacillus subtilis and measured competitive fitness during colony co-culture with a wild type-like parent on different media and at different knockdown levels. Partial knockdown led to a wide range of fitness phenotypes, with targeting of translation-related genes often leading to lower growth rates and rapid out-competition by the parent. Several knockdowns competed differentially in biofilms versus non-biofilm colonies, in some cases due to lack of a particular nutrient in one medium. Cells depleted for the alanine racemase AlrA died in monoculture, but co-cultures survived via nutrient sharing in a biofilm but not in liquid. This rescue was enhanced in biofilm co-culture with a parent unable to produce extracellular matrix, due to a mutualism involving nutrient and matrix sharing. Including alrA, we identified several examples of mutualism involving matrix sharing that occurred in a three-dimensional biofilm colony but not when growth was constrained to two dimensions. These findings demonstrate that growth in a three-dimensional biofilm can promote genetic diversity through sharing of secreted factors, and illustrate the role of matrix production in determining trajectories for biofilm evolution that may be relevant to pathogens and other environmental bacteria.
Genetic landscape of recessive diseases in the Vietnamese population from large-scale clinical exome sequencing

Purpose Accurate profiling of population-specific recessive diseases is essential for the design of cost-effective carrier screening programs. However, minority populations and ethnic groups, including Vietnamese, are still under-represented in existing genetic studies. Here we reported the first comprehensive study of recessive diseases in the Vietnamese population. Methods Clinical exome sequencing (CES) data of 4,503 disease-associated genes obtained from a cohort of 985 Vietnamese individuals was analyzed to identify pathogenic variants, associated diseases and their carrier frequencies in the population. Results Eighty-five recessive diseases were identified in the Vietnamese population, among which seventeen diseases had carrier frequencies of at least 1% (1 in 100 individuals). Three diseases were especially prevalent in the Vietnamese population with carrier frequencies of 2-12 times higher than in other East Asia or the world populations, including Beta-thalassemia (1 in 25), citrin deficiency (1 in 33) and phenylketonuria (1 in 40). Seven novel pathogenic and three likely pathogenic variants associated with nine recessive diseases were also discovered. Conclusions The comprehensive profile of recessive diseases identified in this study shall enable the design of cost-effective carrier screening programs specific to the Vietnamese population. The newly discovered pathogenic variants may also exist in other populations at extremely low frequencies, thus representing a valuable resource for future research. Our study has demonstrated the advantage of population-specific genetic studies to advance the knowledge and practice of medical genetics.
Extracellular vesicle-based vaccine platform displaying native viral envelope proteins elicits a robust anti-SARS-CoV-2 response in mice

Extracellular vesicles (EVs) emerge as essential mediators of intercellular communication. DNA vaccines encoding antigens presented on EVs efficiently induce T-cell responses and EV-based vaccines containing the Spike (S) proteins of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) are highly immunogenic in mice. Thus, EVs may serve as vaccine platforms against emerging diseases, going beyond traditional strategies, with the antigen displayed identically to the original protein embedded in the viral membrane and presented as such to the immune system. Compared to their viral and pseudotyped counterparts, EV-based vaccines overcome many safety issues including pre-existing immunity against these vectors. Here, we applied our technology in natural EV’s engineering, to express the S proteins of SARS-CoV-2 embedded in the EVs, which mimic the virus with its fully native spikes. Immunizations with a two component CoVEVax vaccine, comprising DNA vector (DNAS-EV) primes, allowing in situ production of Spike harbouring EVs, and a boost using S-EVs produced in mammalian cells, trigger potent neutralizing and cellular responses in mice, in the absence of any adjuvants. CoVEVax would be the prototype of vaccines, where the sole exchange of the envelope proteins on EVs leads to the generation of new vaccine candidates against emerging viruses.
ChronoRoot: High-throughput phenotyping by deep segmentation networks reveals novel temporal parameters of plant root system architecture

Deep learning methods have outperformed previous techniques in most computer vision tasks, including image-based plant phenotyping. However, massive data collection of root traits and the development of associated artificial intelligence approaches have been hampered by the inaccessibility of the rhizosphere. Here we present ChronoRoot, a system which combines 3D printed open-hardware with deep segmentation networks for high temporal resolution phenotyping of plant roots in agarized medium. We developed a novel deep learning based root extraction method which leverages the latest advances in convolutional neural networks for image segmentation, and incorporates temporal consistency into the root system architecture reconstruction process. Automatic extraction of phenotypic parameters from sequences of images allowed a comprehensive characterization of the root system growth dynamics. Furthermore, novel time-associated parameters emerged from the analysis of spectral features derived from temporal signals. Altogether, our work shows that the combination of machine intelligence methods and a 3D-printed device expands the possibilities of root high-throughput phenotyping for genetics and natural variation studies as well as the screening of clock-related mutants, revealing novel root traits.
The role of parvalbumin interneuron GIRK signaling in regulation of affect and cognition in male and female mice

Pathological impairments in the regulation of affect (i.e. emotion) and flexible decision-making are commonly observed across numerous neuropsychiatric disorders and are thought to reflect dysfunction of cortical and subcortical circuits that arise in part from imbalances in excitation and inhibition within these structures. Disruptions in GABA transmission, in particular that from parvalbumin-expressing interneurons (PVI), has been highlighted as a likely mechanism by which this imbalance arises, as they regulate excitation and synchronization of principle output neurons. G protein-gated inwardly rectifying potassium ion (GIRK/Kir3) channels are known to modulate excitability and output of pyramidal neurons in areas like the medial prefrontal cortex and hippocampus, however the role GIRK plays in PVI excitability and behavior is unknown. Male and female mice lacking GIRK1 in PVI (Girk1flox/flox:PVcre) and expressing td-tomato in PVI (Girk1flox/flox:PVCre:PVtdtom) exhibited increased open arm time in the elevated plus maze, while males showed an increase in immobile episodes during the forced swim test. Loss of GIRK1 did not alter motivated behavior for an appetitive reward or impair overall performance in an operant-based attention set-shifting model of cognitive flexibility, however it did alter types of errors committed during the visual cue test. Unexpectedly, baseline sex differences were also identified in these tasks, with females exhibiting overall poorer performance compared to males and distinct types of errors, highlighting potential differences in task-related problem solving. Interestingly, reductions in PVI GIRK signaling did not correspond to changes in membrane excitability but did increase action potential firing at higher current injections in PVI of males, but not females. This is the first investigation on the role that PVI GIRK-signaling has on membrane excitability, action potential firing, and their role on affect and cognition together increasing understanding of PVI cellular mechanisms and function.
Mechanisms of protein kinase C epsilon down-regulation by transforming growth factor-beta in lung cancer cells

Protein kinase C epsilon (PKCε), a diacylglycerol (DAG)/phorbol ester-regulated PKC isoform, has been widely linked to oncogenesis and metastasis. PKCε plays important roles in the regulation of motility and invasiveness in non-small cell lung cancer (NSCLC). We previously reported that this kinase becomes prominently down-regulated upon TGF-β-induced epithelial-to-mesenchymal transition (EMT), which leads to prominent phenotypic changes. While the phorbol ester PMA causes down-regulation of PKCα, δ and ε within hours, TGF-β requires at least 4 days to reduce the expression levels of PKCε without affecting the expression of other PKCs, an effect that parallels the acquisition of a mesenchymal phenotype. Despite the prominent transcriptional component involved in EMT, we found that PKCε down-regulation does not involve changes in PKCε mRNA levels and was entirely independent of transcriptional activation of the PRKCE gene. Further mechanistic analysis revealed that the reduction in PKCε expression is dependent on proteasomal and endolysosomal pathways, but independent of autophagy processing mechanisms. Site-directed mutagenesis of Lys312 and Lys321 in PKCε prevented its down-regulation in response to either TGF-β or the phorbol ester PMA. The shift in PKCε isozyme levels depending on cell plasticity underscores relevant functional consequences by modulating the expression of this oncogenic/metastatic kinase and highlights key roles of protein stability mechanisms in the control of PKCε phenotypic outcomes.
Counterfactual Hypothesis Testing of Tumor Microenvironment Scenarios Through Semantic Image Synthesis

Recent multiplexed protein imaging technologies make it possible to characterize cells, their spatial organization, and interactions within microenvironments at unprecedented resolution. Although observational data can reveal spatial associations, it does not allow users to infer biologically causative relationships and interactions between cells. To address this challenge, we develop a generative model that allows users to test hypotheses about the effect of cell-cell interactions on protein expression through in silico perturbation. Our Cell-Cell Interaction GAN (CCIGAN) model employs a generative adversarial network (GAN) architecture to generate biologically realistic multiplexed cell images from semantic cell segmentations. Our approach is unique in considering all imaging channels simultaneously, and we show that it successfully captures known tumor-immune cell interactions missed by other state-of-the-art GAN models, and yields biological insights without requiring in vivo manipulation. CCIGAN accepts data from multiple imaging technologies and can infer interactions from single images in any health or disease context.
RNA polymerase II is required for spatial chromatin reorganization following exit from mitosis

Mammalian chromosomes are three-dimensional entities shaped by converging and opposing forces. Mitotic cell division induces drastic chromosome condensation, but following reentry into the G1 cell cycle phase, condensed chromosomes unwind to reestablish interphase organization. Here, we use a cell line allowing auxin-mediated degradation of RNA polymerase II to test its role in this transition. In situ Hi-C showed that RNAPII is required for compartment and loop formation following mitosis. RNAPs often counteract loop extrusion and, in their absence, longer and more prominent loops arise. Evidence from chromatin fractionation, super-resolution imaging and in silico modeling attribute these effects to RNAPII-mediated cohesin loading at active promoters upon reentry into G1. Our findings reconcile the role of RNAPII in gene expression with that in chromatin architecture.
Modifying Adaptive Therapy to Enhance Competitive Suppression

Adaptive therapy is a promising new approach to cancer treatment. It is designed to leverage competition between drug-sensitive and drug-resistant cells in order to suppress resistance and maintain tumor control for longer. Prompted by encouraging results from a recent pilot clinical trial, we evaluate the design of this initial test of adaptive therapy and identify three simple modifications that should improve performance. These modifications are designed to increase competition and are easy to implement. Using the mathematical model that supported the recent adaptive therapy trial, we show that the suggested modifications further delay time to tumor progression and also increase the range of patients who can benefit from adaptive therapy.
A GO catalogue of human DNA-binding transcription factors

DNA-binding transcription factors recognise genomic addresses, specific sequence motifs in gene regulatory regions, to control gene transcription. A complete and reliable catalogue of all DNA-binding transcription factors is key to investigating the delicate balance of gene regulation in response to environmental and developmental stimuli. The need for such a catalogue of proteins is demonstrated by the many lists of DNA-binding transcription factors that have been produced over the past decade. The COST Action Gene Regulation Ensemble Effort for the Knowledge Commons (GREEKC) Consortium brought together experts in the field of transcription with the aim of providing high quality and interoperable gene regulatory data. The Gene Ontology (GO) Consortium provides strict definitions for gene product function, including factors that regulate transcription. The collaboration between the GREEKC and GO Consortia has enabled the application of those definitions to produce a new curated catalogue of human DNA-binding transcription factors, that can be accessed at https://www.ebi. ac.uk/QuickGO/targetset/dbTF. In addition, this curation effort has led to the GO annotation of almost sixty thousand DNA-binding transcription factors in over a hundred species. Thus, this work will aid researchers investigating the regulation of transcription in both biomedical and basic science.
Advanced systems for the rapid detection of anthelmintic drugs in food

Several surface plasmon resonance (SPR) biosensor assays were developed and validated for the detection of anthelmintic veterinary drugs in liver tissue and milk using a QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction procedure. The first screening assay was developed to detect 11 benzimidazole carbamates in milk and liver. In bovine milk the assay showed a limit of detection (LOD) of 2.7 μg kg-1 and a detection capability (CCβ) of 5 μg kg-1. Analyte recovery was in the range 81 to 116% and the assay was found to be fit for purpose when its performance was compared to UPLC-MS/MS analyses of milk from cows treated with benzimidazole products. In bovine liver the LOD (32 μg kg-1) and the CCβ (50 μg kg-1) were determined and the analyte recovery was in the range 77-132%. All non-compliant samples were identified when the assay performance was tested by analysing liver from animals treated with benzimidazole drugs and comparing the results with a UPLC-MS/MS confirmatory method. A screening assay was developed for four amino-benzimidazoles in liver. The LOD (41 μg kg-1) and the CCβ (75 μg kg-1) were determined and the analyte recovery was in the range 103-116%. A screening assay for thiabendazole and 5-hydroxy-thiabendazole in ovine liver tissue using a novel recombinant antibody fragment (Fab) was developed. The LOD (12.3 μg kg-1), the CCβ (20 μg kg-1) and analyte recovery (86-107%) satisfied the criteria required for thiabendazole screening in liver tissue. A biosensor to detect triclabendazole residues in liver tissue was developed through the immobilization of amino-triclabendazole via a glutaraldehyde homo-bifunctional crosslinker. Several experiments were required to reduce non-specific binding in this assay. An LOD of 105 μg kg-1 was determined which was close to the maximum residue limit (MRL) in liver matrix (100 μg kg-1). A biochip array was developed and validated to screen orange juice for fungicide and pesticide residues. The LOD for carbendazim (20 μg kg-1), 2-aminobenzimidazole (4.0 μg kg-1), thiabendazole (4.2 μg kg-1) and ivermectin (10.2 μg kg-1) residues were determined. The CCβ for carbendazim (50 μg kg-1), 2-aminobenzimidazole (10 μg kg- 1), thiabendazole (10 μg kg-1) and ivermectin (20 μg kg-1) residues were sufficient for the analysis of orange juice. When orange juice from retail outlets in the greater Dublin area (n = 15) Two samples contained thiabendazole residues above the CCβ (260 and 181 μg kg-1) however these concentrations were below the maximum residue limit.
Loss of KHSRP Increases Neuronal Growth and Synaptic Transmission and Alters Memory Consolidation Through RNA Stabilization

The KH-type splicing regulatory protein (KHSRP) is an RNA-binding protein linked to decay of AU-rich element containing mRNAs. We have previously shown that KHSRP destabilizes the mRNA encoding the growth-associated protein GAP-43 and decreases neurite growth in cultured embryonic neurons. In contrast, loss of KHSRP stabilizes Gap43 mRNA and increases neurite growth. Here, we have tested functions of neural KHSRP in vivo. We find upregulation of 1460 mRNAs in the neocortex of adult Khsrp−/− mice, of which 527 bind to KHSRP with high specificity. These KHSRP targets are involved in pathways for neuronal morphology, axon guidance, neurotransmission and long-term memory. Neocortical neurons show increased axon growth and dendritic spine density in Khsrp−/− mice. Analyses of neuronal cultures from embryonic Khsrp−/− mice point to a neuron-intrinsic alteration in axonal and dendritic growth and elevations in KHSRP-target mRNAs, including subcellularly localized mRNAs. Hippocampus and infralimbic cortex of Khsrp−/− mice show presynaptic elevations in neurotransmission. The Khsrp−/− mice have significant deficits in both trace conditioning and attention set-shifting tasks compared Khsrp+/+ mice, indicating impaired prefrontal- and hippocampal-dependent memory consolidation with loss of KHSRP. Overall, our results indicate that prenatal deletion of KHSRP impairs neuronal development resulting in alterations in neuronal morphology and function by changing post-transcriptional control of neuronal gene expression.
Differences in the gut microbiomes of distinct ethnicities within the same geographic area are linked to host metabolic health

Background The human gut microbiota exhibits marked variation around the world, which has been attributed to dietary intake and other environmental factors. However, the degree to which ethnicity-associated differences in gut microbial community structure and function are maintained following immigration or in the context of metabolic disease is poorly understood. Results We conducted a multi-omic study of 46 lean and obese East Asian (EA) and White (W) participants living in the San Francisco Bay Area. 16S rRNA gene sequencing revealed significant differences between ethnic groups in bacterial richness and community structure. W individuals were enriched for the mucin-degrading Akkermansia muciniphila. EA participants had increased levels of multiple Bacteroidetes species, fermentative pathways detected by metagenomics, and the short-chain fatty acid end products acetate, propionate, and isobutyrate. Differences in the gut microbiota between the EA and W groups could not be explained by reported dietary intake, were more pronounced for lean individuals, and were associated with current geographical location. Microbiome transplantations into germ-free mice confirmed that the differences in the gut microbiota of the EA and W individuals we analyzed are indeed independent of diet and that they differentially impact host body weight and adiposity in genetically identical mouse recipients. Conclusions The reported findings emphasize the utility of studying diverse ethnic groups within a defined geographical location and provide a starting point for dissecting the mechanisms contributing to the complex interactions between the gut microbiome and ethnicity-associated lifestyle, demographic, metabolic, and genetic factors.
Shannon entropy as a metric for conditional gene expression in Neurospora crassa

Neurospora crassa has been an important model organism for molecular biology and genetics for over 60 years. N. crassa has a complex life cycle, with over 28 distinct cell types and is capable of transcriptional responses to many environmental conditions including nutrient availability, temperature, and light. To quantify variation in N. crassa gene expression, we analyzed public expression data from 97 conditions and calculated the Shannon Entropy value for Neurospora’s approximately 11,000 genes. Entropy values can be used to estimate the variability in expression for a single gene over a range of conditions and be used to classify individual genes as constitutive or condition-specific. Shannon entropy has previously been used measure the degree of tissue specificity of multicellular plant or animal genes. We use this metric here to measure variable gene expression in a microbe and provide this information as a resource for the N. crassa research community. Finally, we demonstrate the utility of this approach by using entropy values to identify genes with constitutive expression across a wide range of conditions and to identify genes that are activated exclusively during sexual development.
Real-time linear prediction of simultaneous and independent movements of two finger groups using an intracortical brain-machine interface

Modern brain-machine interfaces can return function to people with paralysis, but current hand neural prostheses are unable to reproduce control of individuated finger movements. Here, for the first time, we present a real-time, high-speed, linear brain-machine interface in nonhuman primates that utilizes intracortical neural signals to bridge this gap. We created a novel task that systematically individuates two finger groups, the index finger and the middle-ring-small fingers combined, presenting separate targets for each group. During online brain control, the ReFIT Kalman filter demonstrated the capability of individuating movements of each finger group with high performance, enabling a nonhuman primate to acquire two targets simultaneously at 1.95 targets per second, resulting in an average information throughput of 2.1 bits per second. To understand this result, we performed single unit tuning analyses. Cortical neurons were active for movements of an individual finger group, combined movements of both finger groups, or both. Linear combinations of neural activity representing individual finger group movements predicted the neural activity during combined finger group movements with high accuracy, and vice versa. Hence, a linear model was able to explain how cortical neurons encode information about multiple dimensions of movement simultaneously. Additionally, training ridge regressing decoders with independent component movements was sufficient to predict untrained higher-complexity movements. Our results suggest that linear decoders for brain-machine interfaces may be sufficient to execute high-dimensional tasks with the performance levels required for naturalistic neural prostheses.