Experimental and computational methods for studying the dynamics of RNA-RNA interactions in SARS-COV2 genomes.

Long-range ribonucleic acid (RNA)-RNA interactions (RRI) are prevalent in positive-strand RNA viruses, including Beta-coronaviruses, and these take part in regulatory roles, including the regulation of sub-genomic RNA production rates. Crosslinking of interacting RNAs and short read-based deep sequencing of resulting RNA-RNA hybrids have shown that these long-range structures exist in severe acute respiratory syndrome coronavirus (SARS-CoV)-2 on both genomic and sub-genomic levels and in dynamic topologies. Furthermore, co-evolution of coronaviruses with their hosts is navigated by genetic variations made possible by its large genome, high recombination frequency and a high mutation rate. SARS-CoV-2's mutations are known to occur spontaneously during replication, and thousands of aggregate mutations have been reported since the emergence of the virus. Although many long-range RRIs have been experimentally identified using high-throughput methods for the wild-type SARS-CoV-2 strain, evolutionary trajectory of these RRIs across variants, impact of mutations on RRIs and interaction of SARS-CoV-2 RNAs with the host have been largely open questions in the field. In this review, we summarize recent computational tools and experimental methods that have been enabling the mapping of RRIs in viral genomes, with a specific focus on SARS-CoV-2. We also present available informatics resources to navigate the RRI maps and shed light on the impact of mutations on the RRI space in viral genomes. Investigating the evolution of long-range RNA interactions and that of virus-host interactions can contribute to the understanding of new and emerging variants as well as aid in developing improved RNA therapeutics critical for combating future outbreaks.

A correlational study of uranium in groundwater with other physicochemical parameters using GIS mapping in Godda district of Jharkhand, India.

The present research concentrates on the cumulative use of GPS and GIS technologies, which are excellent resources for analyzing and monitoring divergent physicochemical parameters in groundwater, including pH, TDS, EC, ORP, Ca+2, Mg+2, NO3-, F-, SO4-2, Cl- and PO4-3 with explicit regard to uranium. Garmin GPS is used to record the locations of the sampling points in the Godda study area. The research aims to offer a thorough understanding of the relationship between soil and water, its impact on public health and the extent to which water can be used in various ways based on its quality. Utilizing the inverse distance weighted (IDW) technique, it is examined how these groundwater parameters and the Water Quality Index (WQI) can be estimated spatially. Additionally, a correlation analysis of the water quality parameters is computed to estimate the local population's cancer risk living in the study area. Except for calcium and magnesium, which are present in excess concentrations throughout the study area with the highest values of 325 and 406 mg/l, respectively at Amediha and Meherma, the results showed that the maximum concentration parameters are within limits with the standard. The main reason might be the area's predominance of Alfisol soil type. The radioactive element uranium is found to be in a limited  range. Chemo-toxicity and radiological risk assessment of the whole area lie far below the restricted cancer risk limit i.e., 30 ppb with the highest concentration of 14 ppb in the 'Sunderpahari' region, following the results obtained. The WQI for the area ranges from 'good' to 'very poor.' The results were favorable but a few sites such as 'Boarijor' and its surroundings, require additional attention to enhance groundwater quality. Given uranium's low availability in groundwater the region's cancer risk assessment is below average.

A Promising Approach of Dermal Targeting of Antipsoriatic Drugs via Engineered Nanocarriers Drug Delivery Systems for Tackling Psoriasis.

Psoriasis is a complex autoimmune skin condition with a significant genetic component. It causes skin inflammation and is characterized by flaky, silvery reddish spots that can worsen with age. This condition results from an impaired immunological response of T-cells and affects 2-5% of the global population. The severity of the illness determines the choice of treatment. Topical treatments are commonly used to treat psoriasis, but they can have several adverse effects. Biological therapy is another option for treating specific types of psoriasis. Recently, new nanoformulations have revolutionized psoriasis treatment. Various nanocarriers, such as liposomes, nanostructured lipid nanoparticles, niosomes, and nanoemulsions, have been developed and improved for drug delivery. The use of nanocarriers enhances patient compliance, precise drug delivery, and drug safety. This review aims to suggest new nanocarrier-based drug delivery systems for treating psoriasis. It discusses the importance of nanocarriers and compares them to traditional treatments. Anti-psoriatic drugs have also been investigated for cutaneous delivery using nanocarriers. The review also covers various factors that influence dermal targeting. By highlighting several relevant aspects of psoriasis treatment, the review emphasizes the current potential of nanotechnology. Using nanocarriers as a drug delivery technique may be a promising alternative treatment for psoriasis.

Acoel single-cell atlas reveals expression dynamics and heterogeneity of adult pluripotent stem cells.

Adult pluripotent stem cell (aPSC) populations underlie whole-body regeneration in many distantly-related animal lineages, but how the underlying cellular and molecular mechanisms compare across species is unknown. Here, we apply single-cell RNA sequencing to profile transcriptional cell states of the acoel worm Hofstenia miamia during postembryonic development and regeneration. We identify cell types shared across stages and their associated gene expression dynamics during regeneration. Functional studies confirm that the aPSCs, also known as neoblasts, are the source of differentiated cells and reveal transcription factors needed for differentiation. Subclustering of neoblasts recovers transcriptionally distinct subpopulations, the majority of which are likely specialized to differentiated lineages. One neoblast subset, showing enriched expression of the histone variant H3.3, appears to lack specialization. Altogether, the cell states identified in this study facilitate comparisons to other species and enable future studies of stem cell fate potentials.

A wound-induced differentiation trajectory for neurons.

Animals capable of whole-body regeneration can replace any missing cell type and regenerate fully-functional new organs, de novo . The regeneration of a new brain requires the formation of diverse neuronal cell types and their assembly into an organized structure and correctly-wired circuits. Recent work in various regenerative animals has revealed transcriptional programs required for the differentiation of distinct neuronal subpopulations, however how these transcriptional programs are initiated upon amputation remains unknown. Here, we focused on the highly regenerative acoel worm, Hofstenia miamia , to study wound-induced transcriptional regulatory events that lead to the production of neurons. Footprinting analysis using chromatin accessibility data on an improved genome assembly revealed that binding sites for the NFY transcription factor complex were significantly bound during regeneration, showing a dynamic increase in binding within one hour upon amputation specifically in tail fragments, which will regenerate a new brain. Strikingly, NFY targets were highly enriched for genes with neuronal functional. Single-cell transcriptome analysis combined with functional studies identified sox4 + stem cells as the likely progenitor population for multiple neuronal subtypes. Further, we found that wound-induced sox4 expression is likely under direct transcriptional control by NFY, uncovering a mechanism for how early wound-induced binding of a transcriptional regulator results in the initiation of a neuronal differentiation pathway. Highlights: A new chromosome-scale assembly for Hofstenia enables comprehensive analysis of transcription factor binding during regeneration NFY motifs become dynamically bound by 1hpa in regenerating tail fragments, particularly in the loci of neural genes A sox4 + neural-specialized stem cell is identified using scRNA-seq sox4 is wound-induced and required for differentiation of multiple neural cell types NFY regulates wound-induced expression of sox4 during regeneration.

Embryonic origins of adult pluripotent stem cells.

Although adult pluripotent stem cells (aPSCs) are found in many animal lineages, mechanisms for their formation during embryogenesis are unknown. Here, we leveraged Hofstenia miamia, a regenerative worm that possesses collectively pluripotent aPSCs called neoblasts and produces manipulable embryos. Lineage tracing and functional experiments revealed that one pair of blastomeres gives rise to cells that resemble neoblasts in distribution, behavior, and gene expression. In Hofstenia, aPSCs include transcriptionally distinct subpopulations that express markers associated with differentiated tissues; our data suggest that despite their heterogeneity, aPSCs are derived from one lineage, not from multiple tissue-specific lineages during development. Next, we combined single-cell transcriptome profiling across development with neoblast cell-lineage tracing and identified a molecular trajectory for neoblast formation that includes transcription factors Hes, FoxO, and Tbx. This identification of a cellular mechanism and molecular trajectory for aPSC formation opens the door for in vivo studies of aPSC regulation and evolution.

A NANOG-pERK reciprocal regulatory circuit regulates Nanog autoregulation and ERK signaling dynamics.

The self-renewal and differentiation potential of embryonic stem cells (ESCs) is maintained by the regulated expression of core pluripotency factors. Expression levels of the core pluripotency factor Nanog are tightly regulated by a negative feedback autorepression loop. However, it remains unclear how ESCs perceive NANOG levels and execute autorepression. Here, we show that a dose-dependent induction of Fgfbp1 and Fgfr2 by NANOG activates autocrine-mediated ERK signaling in Nanog-high cells to trigger autorepression. pERK recruits NONO to the Nanog locus to repress transcription by preventing POL2 loading. This Nanog autorepression process establishes a self-perpetuating reciprocal NANOG-pERK regulatory circuit. We further demonstrate that this reciprocal regulatory circuit induces pERK heterogeneity and ERK signaling dynamics in pluripotent stem cells. Collectively our data suggest that NANOG induces Fgfr2 and Fgfbp1 to activate ERK signaling in Nanog-high cells to establish a NANOG-pERK reciprocal regulatory circuit. This circuit regulates ERK signaling dynamics and Nanog autoregulation in pluripotent cells.

Assessing Chromatin Accessibility During WBR in Acoels.

Dynamic gene expression seen during whole-body regeneration is likely controlled by genomic regulatory elements that dictate the spatiotemporal activity of the regeneration transcriptome. Identifying and characterizing these non-coding regulatory sequences are key to understanding how genes are connected into networks to deploy the process of whole-body regeneration. Here, we describe the application of the Assay for Transposase Accessible Chromatin (ATAC-seq) in the acoel Hofstenia miamia to identify regions of open chromatin that represent putative regulatory elements. Notably, when paired with gene knockdown techniques such as RNAi, ATAC-seq can be implemented in a functional genomics approach to validate putative regulatory elements. ATAC-seq requires no species-specific reagents, is amenable to small input cell numbers, and can be completed in a single day, making it an ideal assay to identify dynamic chromatin at high resolution during whole-body regeneration in virtually any species with a quality genome assembly.

Studying development, regeneration, stem cells, and more in the acoel Hofstenia miamia.

Acoel worms represent an enigmatic lineage of animals (Acoelomorpha) that has danced around the tree of animal life. Morphology-based classification placed them as flatworms (Phylum Platyhelminthes), with much of their biology being interpreted as a variation on what is observed in better-studied members of that phylum. However, molecular phylogenies suggest that acoels belong to a clade (Xenacoelomorpha) that could be a sister group to other animals with bilateral symmetry (Bilateria) or could belong within deuterostomes, closely related to a group that includes sea stars (Ambulacraria). This change in phylogenetic position has led to renewed interest in the biology of acoels, which can now offer insights into the evolution of many bilaterian traits. The acoel Hofstenia miamia has emerged as a powerful new research organism that enables mechanistic studies of xenacoelomorph biology, especially of developmental and regenerative processes. This article explains the motivation for developing Hofstenia as a new model system, describes Hofstenia biology, highlights the tools and resources that make Hofstenia a good research organism, and considers the questions that Hofstenia is well-positioned to answer. Finally, looking to the future, this article serves as an invitation to new and established scientists to join the growing community of researchers studying this exciting model system.

CASowary: CRISPR-Cas13 guide RNA predictor for transcript depletion.

BACKGROUND: Recent discovery of the gene editing system - CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) associated proteins (Cas), has resulted in its widespread use for improved understanding of a variety of biological systems. Cas13, a lesser studied Cas protein, has been repurposed to allow for efficient and precise editing of RNA molecules. The Cas13 system utilizes base complementarity between a crRNA/sgRNA (crispr RNA or single guide RNA) and a target RNA transcript, to preferentially bind to only the target transcript. Unlike targeting the upstream regulatory regions of protein coding genes on the genome, the transcriptome is significantly more redundant, leading to many transcripts having wide stretches of identical nucleotide sequences. Transcripts also exhibit complex three-dimensional structures and interact with an array of RBPs (RNA Binding Proteins), both of which may impact the effectiveness of transcript depletion of target sequences. However, our understanding of the features and corresponding methods which can predict whether a specific sgRNA will effectively knockdown a transcript is very limited. RESULTS: Here we present a novel machine learning and computational tool, CASowary, to predict the efficacy of a sgRNA. We used publicly available RNA knockdown data from Cas13 characterization experiments for 555 sgRNAs targeting the transcriptome in HEK293 cells, in conjunction with transcriptome-wide protein occupancy information. Our model utilizes a Decision Tree architecture with a set of 112 sequence and target availability features, to classify sgRNA efficacy into one of four classes, based upon expected level of target transcript knockdown. After accounting for noise in the training data set, the noise-normalized accuracy exceeds 70%. Additionally, highly effective sgRNA predictions have been experimentally validated using an independent RNA targeting Cas system - CIRTS, confirming the robustness and reproducibility of our model's sgRNA predictions. Utilizing transcriptome wide protein occupancy map generated using POP-seq in HeLa cells against publicly available protein-RNA interaction map in Hek293 cells, we show that CASowary can predict high quality guides for numerous transcripts in a cell line specific manner. CONCLUSIONS: Application of CASowary to whole transcriptomes should enable rapid deployment of CRISPR/Cas13 systems, facilitating the development of therapeutic interventions linked with aberrations in RNA regulatory processes.

Transgenesis in the acoel worm Hofstenia miamia.

The acoel worm Hofstenia miamia, which can replace tissue lost to injury via differentiation of a population of stem cells, has emerged as a new research organism for studying regeneration. To enhance the depth of mechanistic studies in this system, we devised a protocol for microinjection into embryonic cells that resulted in stable transgene integration into the genome and generated animals with tissue-specific fluorescent transgene expression in epidermis, gut, and muscle. We demonstrate that transgenic Hofstenia are amenable to the isolation of specific cell types, investigations of regeneration, tracking of photoconverted molecules, and live imaging. Further, our stable transgenic lines revealed insights into the biology of Hofstenia, including a high-resolution three-dimensional view of cell morphology and the organization of muscle as a cellular scaffold for other tissues. Our work positions Hofstenia as a powerful system with multiple toolkits for mechanistic investigations of development, whole-body regeneration, and stem cell biology.

Mutational Landscape and Interaction of SARS-CoV-2 with Host Cellular Components.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its rapid evolution has led to a global health crisis. Increasing mutations across the SARS-CoV-2 genome have severely impacted the development of effective therapeutics and vaccines to combat the virus. However, the new SARS-CoV-2 variants and their evolutionary characteristics are not fully understood. Host cellular components such as the ACE2 receptor, RNA-binding proteins (RBPs), microRNAs, small nuclear RNA (snRNA), 18s rRNA, and the 7SL RNA component of the signal recognition particle (SRP) interact with various structural and non-structural proteins of the SARS-CoV-2. Several of these viral proteins are currently being examined for designing antiviral therapeutics. In this review, we discuss current advances in our understanding of various host cellular components targeted by the virus during SARS-CoV-2 infection. We also summarize the mutations across the SARS-CoV-2 genome that directs the evolution of new viral strains. Considering coronaviruses are rapidly evolving in humans, this enables them to escape therapeutic therapies and vaccine-induced immunity. In order to understand the virus's evolution, it is essential to study its mutational patterns and their impact on host cellular machinery. Finally, we present a comprehensive survey of currently available databases and tools to study viral-host interactions that stand as crucial resources for developing novel therapeutic strategies for combating SARS-CoV-2 infection.

Embryonic development in the acoel Hofstenia miamia.

Acoels are marine worms that belong to the phylum Xenacoelomorpha, a deep-diverging bilaterian lineage. This makes acoels an attractive system for studying the evolution of major bilaterian traits. Thus far, acoel development has not been described in detail at the morphological and transcriptomic levels in a species in which functional genetic studies are possible. We present a set of developmental landmarks for embryogenesis in the highly regenerative acoel Hofstenia miamia. We generated a developmental staging atlas from zygote to hatched worm based on gross morphology, with accompanying bulk transcriptome data. Hofstenia embryos undergo a stereotyped cleavage program known as duet cleavage, which results in two large vegetal pole 'macromeres' and numerous small animal pole 'micromeres'. These macromeres become internalized as micromere progeny proliferate and move vegetally. We also noted a second, previously undescribed, cell-internalization event at the animal pole, following which we detected major body axes and tissues corresponding to all three germ layers. Our work on Hofstenia embryos provides a resource for mechanistic investigations of acoel development, which will yield insights into the evolution of bilaterian development and regeneration.

Beyond Casual Resemblance: Rigorous Frameworks for Comparing Regeneration Across Species.

The majority of animal phyla have species that can regenerate. Comparing regeneration across animals can reconstruct the molecular and cellular evolutionary history of this process. Recent studies have revealed some similarity in regeneration mechanisms, but rigorous comparative methods are needed to assess whether these resemblances are ancestral pathways (homology) or are the result of convergent evolution (homoplasy). This review aims to provide a framework for comparing regeneration across animals, focusing on gene regulatory networks (GRNs), which are substrates for assessing process homology. The homology of the wound-induced activation of Wnt signaling and of adult stem cells provides examples of ongoing studies of regeneration that enable comparisons in a GRN framework. Expanding the study of regeneration GRNs in currently studied species and broadening taxonomic sampling for these approaches will identify processes that are unifying principles of regeneration biology across animals. These insights are important both for evolutionary studies of regeneration and for human regenerative medicine.

Transcriptome-wide high-throughput mapping of protein-RNA occupancy profiles using POP-seq.

Interaction between proteins and RNA is critical for post-transcriptional regulatory processes. Existing high throughput methods based on crosslinking of the protein-RNA complexes and poly-A pull down are reported to contribute to biases and are not readily amenable for identifying interaction sites on non poly-A RNAs. We present Protein Occupancy Profile-Sequencing (POP-seq), a phase separation based method in three versions, one of which does not require crosslinking, thus providing unbiased protein occupancy profiles on whole cell transcriptome without the requirement of poly-A pulldown. Our study demonstrates that ~ 68% of the total POP-seq peaks exhibited an overlap with publicly available protein-RNA interaction profiles of 97 RNA binding proteins (RBPs) in K562 cells. We show that POP-seq variants consistently capture protein-RNA interaction sites across a broad range of genes including on transcripts encoding for transcription factors (TFs), RNA-Binding Proteins (RBPs) and long non-coding RNAs (lncRNAs). POP-seq identified peaks exhibited a significant enrichment (p value < 2.2e-16) for GWAS SNPs, phenotypic, clinically relevant germline as well as somatic variants reported in cancer genomes, suggesting the prevalence of uncharacterized genomic variation in protein occupied sites on RNA. We demonstrate that the abundance of POP-seq peaks increases with an increase in expression of lncRNAs, suggesting that highly expressed lncRNA are likely to act as sponges for RBPs, contributing to the rewiring of protein-RNA interaction network in cancer cells. Overall, our data supports POP-seq as a robust and cost-effective method that could be applied to primary tissues for mapping global protein occupancies.

Transcription Factors in the Development and Pro-Allergic Function of Mast Cells.

Mast cells (MCs) are innate immune cells of hematopoietic origin localized in the mucosal tissues of the body and are broadly implicated in the pathogenesis of allergic inflammation. Transcription factors have a pivotal role in the development and differentiation of mast cells in response to various microenvironmental signals encountered in the resident tissues. Understanding the regulation of mast cells by transcription factors is therefore vital for mechanistic insights into allergic diseases. In this review we summarize advances in defining the transcription factors that impact the development of mast cells throughout the body and in specific tissues, and factors that are involved in responding to the extracellular milieu. We will further describe the complex networks of transcription factors that impact mast cell physiology and expansion during allergic inflammation and functions from degranulation to cytokine secretion. As our understanding of the heterogeneity of mast cells becomes more detailed, the contribution of specific transcription factors in mast cell-dependent functions will potentially offer new pathways for therapeutic targeting.

Role of SARS-CoV-2 in Altering the RNA-Binding Protein and miRNA-Directed Post-Transcriptional Regulatory Networks in Humans.

The outbreak of a novel coronavirus SARS-CoV-2 responsible for the COVID-19 pandemic has caused a worldwide public health emergency. Due to the constantly evolving nature of the coronaviruses, SARS-CoV-2-mediated alterations on post-transcriptional gene regulations across human tissues remain elusive. In this study, we analyzed publicly available genomic datasets to systematically dissect the crosstalk and dysregulation of the human post-transcriptional regulatory networks governed by RNA-binding proteins (RBPs) and micro-RNAs (miRs) due to SARS-CoV-2 infection. We uncovered that 13 out of 29 SARS-CoV-2-encoded proteins directly interacted with 51 human RBPs, of which the majority of them were abundantly expressed in gonadal tissues and immune cells. We further performed a functional analysis of differentially expressed genes in mock-treated versus SARS-CoV-2-infected lung cells that revealed enrichment for the immune response, cytokine-mediated signaling, and metabolism-associated genes. This study also characterized the alternative splicing events in SARS-CoV-2-infected cells compared to the control, demonstrating that skipped exons and mutually exclusive exons were the most abundant events that potentially contributed to differential outcomes in response to the viral infection. A motif enrichment analysis on the RNA genomic sequence of SARS-CoV-2 clearly revealed the enrichment for RBPs such as SRSFs, PCBPs, ELAVs, and HNRNPs, suggesting the sponging of RBPs by the SARS-CoV-2 genome. A similar analysis to study the interactions of miRs with SARS-CoV-2 revealed functionally important miRs that were highly expressed in immune cells, suggesting that these interactions may contribute to the progression of the viral infection and modulate the host immune response across other human tissues. Given the need to understand the interactions of SARS-CoV-2 with key post-transcriptional regulators in the human genome, this study provided a systematic computational analysis to dissect the role of dysregulated post-transcriptional regulatory networks controlled by RBPs and miRs across tissue types during a SARS-CoV-2 infection.

A Regulatory Program for Initiation of Wnt Signaling during Posterior Regeneration.

Whole-body regeneration relies on the re-establishment of body axes for patterning of new tissue. Wnt signaling is required to correctly regenerate tissues along the primary axis in many animals. However, the causal mechanisms that first launch Wnt signaling during regeneration are poorly characterized. We use the acoel worm Hofstenia miamia to identify processes that initiate Wnt signaling during posterior regeneration and find that the ligand wnt-3 is upregulated early in posterior-facing wounds. Functional studies reveal that wnt-3 is required to regenerate posterior tissues. wnt-3 is expressed in stem cells, it is needed for their proliferation, and its function is stem cell dependent. Chromatin accessibility data reveal that wnt-3 activation requires input from the general wound response. In addition, the expression of a different Wnt ligand, wnt-1, before amputation is required for wound-induced activation of wnt-3. Our study establishes a gene regulatory network for initiating Wnt signaling in posterior tissues in a bilaterian.