STAT5B SH2 variants disrupt mammary enhancers and the stability of genetic programs during pregnancy.

During pregnancy, mammary tissue undergoes expansion and differentiation, leading to lactation, a process regulated by the hormone prolactin through the JAK2-STAT5 pathway. STAT5 activation is key to successful lactation making the mammary gland an ideal experimental system to investigate the impact of human missense mutations on mammary tissue homeostasis. Here, we investigated the effects of two human variants in the STAT5B SH2 domain, which convert tyrosine 665 to either phenylalanine (Y665F) or histidine (Y665H), both shown to activate STAT5B in cell culture. We ported these mutations into the mouse genome and found distinct and divergent functions. Homozygous Stat5bY665H mice failed to form functional mammary tissue, leading to lactation failure, with impaired alveolar development and greatly reduced expression of key differentiation genes. STAT5BY665H failed to recognize mammary enhancers and impeded STAT5A binding. In contrast, mice carrying the Stat5bY665F mutation exhibited abnormal precocious development, accompanied by an early activation of the mammary transcription program and the induction of otherwise silent genetic programs. Physiological adaptation was observed in Stat5bY665H mice as continued exposure to pregnancy hormones led to lactation. In summary, our findings highlight that human STAT5B variants can modulate their response to cytokines and thereby impact mammary homeostasis and lactation.

SARS-CoV-2 Spike Protein 1 Causes Aggregation of α-Synuclein via Microglia-Induced Inflammation and Production of Mitochondrial ROS: Potential Therapeutic Applications of Metformin.

Abnormal aggregation of α-synuclein is the hallmark of neurodegenerative diseases, classified as α-synucleinopathies, primarily occurring sporadically. Their onset is associated with an interaction between genetic susceptibility and environmental factors such as neurotoxins, oxidative stress, inflammation, and viral infections. Recently, evidence has suggested an association between neurological complications in long COVID (sometimes referred to as 'post-acute sequelae of COVID-19') and α-synucleinopathies, but its underlying mechanisms are not completely understood. In this study, we first showed that SARS-CoV-2 Spike protein 1 (S1) induces α-synuclein aggregation associated with activation of microglial cells in the rodent model. In vitro, we demonstrated that S1 increases aggregation of α-synuclein in BE(2)M-17 dopaminergic neurons via BV-2 microglia-mediated inflammatory responses. We also identified that S1 directly affects aggregation of α-synuclein in dopaminergic neurons through increasing mitochondrial ROS, though only under conditions of sufficient α-Syn accumulation. In addition, we observed a synergistic effect between S1 and the neurotoxin MPP+ S1 treatment. Combined with a low dose of MPP+, it boosted α-synuclein aggregation and mitochondrial ROS production compared to S1 or the MPP+ treatment group. Furthermore, we evaluated the therapeutic effects of metformin. The treatment of metformin suppressed the S1-induced inflammatory response and α-synucleinopathy. Our findings demonstrate that S1 promotes α-synucleinopathy via both microglia-mediated inflammation and mitochondrial ROS, and they provide pathological insights, as well as a foundation for the clinical management of α-synucleinopathies and the onset of neurological symptoms after the COVID-19 outbreak.

Sexually dimorphic renal expression of Klotho is directed by a kidney-specific distal enhancer responsive to HNF1b.

Transcription enhancers are genomic sequences regulating common and tissue-specific genes and their disruption can contribute to human disease development and progression. Klotho, a sexually dimorphic gene specifically expressed in kidney, is well-linked to kidney dysfunction and its deletion from the mouse genome leads to premature aging and death. However, the sexually dimorphic regulation of Klotho is not understood. Here, we characterize two candidate Klotho enhancers using H3K27ac epigenetic marks and transcription factor binding and investigate their functions, individually and combined, through CRISPR-Cas9 genome engineering. We discovered that only the distal (E1), but not the proximal (E2) candidate region constitutes a functional enhancer, with the double deletion not causing Klotho expression to further decrease. E1 activity is dependent on HNF1b transcription factor binding site within the enhancer. Further, E1 controls the sexual dimorphism of Klotho as evidenced by qPCR and RNA-seq. Despite the sharp reduction of Klotho mRNA, unlike germline Klotho knockouts, mutant mice presented normal phenotype, including weight, lifespan, and serum biochemistry. Lastly, only males lacking E1 display more prominent acute, but not chronic kidney injury responses, indicating a remarkable range of potential adaptation to isolated Klotho loss, especially in female E1 knockouts, retaining renoprotection despite over 80% Klotho reduction.

Machine Learning to Identify Critical Biomarker Profiles in New SARS-CoV-2 Variants.

The global dissemination of SARS-CoV-2 resulted in the emergence of several variants, including Alpha, Alpha + E484K, Beta, and Omicron. Our research integrated the study of eukaryotic translation factors and fundamental components in general protein synthesis with the analysis of SARS-CoV-2 variants and vaccination status. Utilizing statistical methods, we successfully differentiated between variants in infected individuals and, to a lesser extent, between vaccinated and non-vaccinated infected individuals, relying on the expression profiles of translation factors. Additionally, our investigation identified common causal relationships among the translation factors, shedding light on the interplay between SARS-CoV-2 variants and the host's translation machinery.

Generation of an induced pluripotent stem cell line (KNIHi001-A) by reprogramming peripheral blood mononuclear cells isolated from a patient with Parkinson's disease.

Parkinson's disease is a degenerative brain disorder characterized by dopamine neuronal degeneration and dopamine transporter loss. In this study, we generated an induced pluripotent stem cell (iPSC) line, KNIHi001-A, from the peripheral blood mononuclear cells (PBMCs) of a 76-year-old man with Parkinson's disease. The non-integrating Sendai virus was used to reprogram iPSCs. iPSCs exhibit pluripotent markers, a normal karyotype, viral clearance, and the ability to differentiate into the three germ layers.

Sexually dimorphic renal expression of Klotho is directed by a kidney-specific distal enhancer responsive to HNF1b.

Transcription enhancers are genomic sequences regulating common and tissue-specific genes and their disruption can contribute to human disease development and progression. Klotho, a sexually dimorphic gene specifically expressed in kidney, is well-linked to kidney dysfunction and its deletion from the mouse genome leads to premature aging and death. However, the sexually dimorphic regulation of Klotho is not understood. Here, we characterize two candidate Klotho enhancers using H3K27ac epigenetic marks and transcription factor binding and investigate their functions, individually and combined, through CRISPR-Cas9 genome engineering. We discovered that only the distal (E1), but not the proximal (E2) candidate region constitutes a functional enhancer, with the double deletion not causing Klotho expression to further decrease. E1 activity is dependent on HNF1b transcription factor binding site within the enhancer. Further, E1 controls the sexual dimorphism of Klotho as evidenced by qPCR and RNA-seq. Despite the sharp reduction of Klotho mRNA, unlike germline Klotho knockouts, mutant mice presented normal phenotype, including weight, lifespan, and serum biochemistry. Lastly, only males lacking E1 display more prominent acute, but not chronic kidney injury responses, indicating a remarkable range of potential adaptation to isolated Klotho loss, especially in female E1 knockouts, retaining renoprotection despite over 80% Klotho reduction.

Blood transcriptomics analysis offers insights into variant-specific immune response to SARS-CoV-2.

Bulk RNA sequencing (RNA-seq) of blood is typically used for gene expression analysis in biomedical research but is still rarely used in clinical practice. In this study, we propose that RNA-seq should be considered a diagnostic tool, as it offers not only insights into aberrant gene expression and splicing but also delivers additional readouts on immune cell type composition as well as B-cell and T-cell receptor (BCR/TCR) repertoires. We demonstrate that RNA-seq offers insights into a patient's immune status via integrative analysis of RNA-seq data from patients infected with various SARS-CoV-2 variants (in total 196 samples with up to 200 million reads sequencing depth). We compare the results of computational cell-type deconvolution methods (e.g., MCP-counter, xCell, EPIC, quanTIseq) to complete blood count data, the current gold standard in clinical practice. We observe varying levels of lymphocyte depletion and significant differences in neutrophil levels between SARS-CoV-2 variants. Additionally, we identify B and T cell receptor (BCR/TCR) sequences using the tools MiXCR and TRUST4 to show that-combined with sequence alignments and BLASTp-they could be used to classify a patient's disease. Finally, we investigated the sequencing depth required for such analyses and concluded that 10 million reads per sample is sufficient. In conclusion, our study reveals that computational cell-type deconvolution and BCR/TCR methods using bulk RNA-seq analyses can supplement missing CBC data and offer insights into immune responses, disease severity, and pathogen-specific immunity, all achievable with a sequencing depth of 10 million reads per sample.

Variant- and vaccination-specific alternative splicing profiles in SARS-CoV-2 infections.

The COVID-19 pandemic, driven by the SARS-CoV-2 virus and its variants, highlights the important role of understanding host-viral molecular interactions influencing infection outcomes. Alternative splicing post-infection can impact both host responses and viral replication. We analyzed RNA splicing patterns in immune cells across various SARS-CoV-2 variants, considering immunization status. Using a dataset of 190 RNA-seq samples from our prior studies, we observed a substantial deactivation of alternative splicing and RNA splicing-related genes in COVID-19 patients. The alterations varied significantly depending on the infecting variant and immunization history. Notably, Alpha or Beta-infected patients differed from controls, while Omicron-infected patients displayed a splicing profile closer to controls. Particularly, vaccinated Omicron-infected individuals showed a distinct dynamic in alternative splicing patterns not widely shared among other groups. Our findings underscore the intricate interplay between SARS-CoV-2 variants, vaccination-induced immunity, and alternative splicing, emphasizing the need for further investigations to deepen understanding and guide therapeutic development.

Direct and robust citramalate production from brown macroalgae using fast-growing Vibrio sp. dhg.

Brown macroalgae is a promising feedstock for biorefinery owing to its high biomass productivity and contents of carbohydrates such as alginate and mannitol. However, the limited availability of microbial platforms efficiently catabolizing the brown macroalgae sugars has restricted its utilization. In this study, the direct production of citramalate, an important industrial compound, was demonstrated from brown macroalgae by utilizing Vibrio sp. dhg, which has a remarkably efficient catabolism of alginate and mannitol. Specifically, citramalate synthase from Methanocaldococcus jannaschii was synthetically expressed, and competing pathways were removed to maximally redirect the carbon flux toward citramalate production. Notably, a resulting strain, VXHC, produced citramalate up to 9.8 g/L from a 20 g/L mixture of alginate and mannitol regardless of their ratios. Citramalate was robustly produced even when diverse brown macroalgae were provided directly. Collectively, this study showcased the high potential of brown macroalgae biorefinery using Vibrio sp. dhg.

Variant- and Vaccination-Specific Alternative Splicing Profiles in SARS-CoV-2 Infections.

The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, and its subsequent variants has underscored the importance of understanding the host-viral molecular interactions to devise effective therapeutic strategies. A significant aspect of these interactions is the role of alternative splicing in modulating host responses and viral replication mechanisms. Our study sought to delineate the patterns of alternative splicing of RNAs from immune cells across different SARS-CoV-2 variants and vaccination statuses, utilizing a robust dataset of 190 RNA-seq samples from our previous studies, encompassing an average of 212 million reads per sample. We identified a dynamic alteration in alternative splicing and genes related to RNA splicing were highly deactivated in COVID-19 patients and showed variant- and vaccination-specific expression profiles. Overall, Omicron-infected patients exhibited a gene expression profile akin to healthy controls, unlike the Alpha or Beta variants. However, significantly, we found identified a subset of infected individuals, most pronounced in vaccinated patients infected with Omicron variant, that exhibited a specific dynamic in their alternative splicing patterns that was not widely shared amongst the other groups. Our findings underscore the complex interplay between SARS-CoV-2 variants, vaccination-induced immune responses, and alternative splicing, emphasizing the necessity for further investigations into these molecular cross-talks to foster deeper understanding and guide strategic therapeutic development.

Blood transcriptomics analysis offers insights into variant-specific immune response to SARS-CoV-2.

Bulk RNA sequencing (RNA-seq) of blood is typically used for gene expression analysis in biomedical research but is still rarely used in clinical practice. In this study, we argue that RNA-seq should be considered a routine diagnostic tool, as it offers not only insights into aberrant gene expression and splicing but also delivers additional readouts on immune cell type composition as well as B-cell and T-cell receptor (BCR/TCR) repertoires. We demonstrate that RNA-seq offers vital insights into a patient's immune status via integrative analysis of RNA-seq data from patients infected with various SARS-CoV-2 variants (in total 240 samples with up to 200 million reads sequencing depth). We compare the results of computational cell-type deconvolution methods (e.g., MCP-counter, xCell, EPIC, quanTIseq) to complete blood count data, the current gold standard in clinical practice. We observe varying levels of lymphocyte depletion and significant differences in neutrophil levels between SARS-CoV-2 variants. Additionally, we identify B and T cell receptor (BCR/TCR) sequences using the tools MiXCR and TRUST4 to show that - combined with sequence alignments and pBLAST - they could be used to classify a patient's disease. Finally, we investigated the sequencing depth required for such analyses and concluded that 10 million reads per sample is sufficient. In conclusion, our study reveals that computational cell-type deconvolution and BCR/TCR methods using bulk RNA-seq analyses can supplement missing CBC data and offer insights into immune responses, disease severity, and pathogen-specific immunity, all achievable with a sequencing depth of 10 million reads per sample.

Network medicine-based epistasis detection in complex diseases: ready for quantum computing.

Most heritable diseases are polygenic. To comprehend the underlying genetic architecture, it is crucial to discover the clinically relevant epistatic interactions (EIs) between genomic single nucleotide polymorphisms (SNPs)1-3. Existing statistical computational methods for EI detection are mostly limited to pairs of SNPs due to the combinatorial explosion of higher-order EIs. With NeEDL (network-based epistasis detection via local search), we leverage network medicine to inform the selection of EIs that are an order of magnitude more statistically significant compared to existing tools and consist, on average, of five SNPs. We further show that this computationally demanding task can be substantially accelerated once quantum computing hardware becomes available. We apply NeEDL to eight different diseases and discover genes (affected by EIs of SNPs) that are partly known to affect the disease, additionally, these results are reproducible across independent cohorts. EIs for these eight diseases can be interactively explored in the Epistasis Disease Atlas (https://epistasis-disease-atlas.com). In summary, NeEDL is the first application that demonstrates the potential of seamlessly integrated quantum computing techniques to accelerate biomedical research. Our network medicine approach detects higher-order EIs with unprecedented statistical and biological evidence, yielding unique insights into polygenic diseases and providing a basis for the development of improved risk scores and combination therapies.

Cell-specific and shared regulatory elements control a multigene locus active in mammary and salivary glands.

Regulation of high-density loci harboring genes with different cell-specificities remains a puzzle. Here we investigate a locus that evolved through gene duplication and contains eight genes and 20 candidate regulatory elements, including one super-enhancer. Casein genes (Csn1s1, Csn2, Csn1s2a, Csn1s2b, Csn3) are expressed in mammary glands, induced 10,000-fold during pregnancy and account for 50% of mRNAs during lactation, Prr27 and Fdcsp are salivary-specific and Odam has dual specificity. We probed the function of 12 candidate regulatory elements, individually and in combination, in the mouse genome. The super-enhancer is essential for the expression of Csn3, Csn1s2b, Odam and Fdcsp but largely dispensable for Csn1s1, Csn2 and Csn1s2a. Csn3 activation also requires its own local enhancer. Synergism between local enhancers and cytokine-responsive promoter elements facilitates activation of Csn2 during pregnancy. Our work identifies the regulatory complexity of a multigene locus with an ancestral super-enhancer active in mammary and salivary tissue and local enhancers and promoter elements unique to mammary tissue.

circRNA-sponging: a pipeline for extensive analysis of circRNA expression and their role in miRNA sponging.

Motivation: Circular RNAs (circRNAs) are long noncoding RNAs (lncRNAs) often associated with diseases and considered potential biomarkers for diagnosis and treatment. Among other functions, circRNAs have been shown to act as microRNA (miRNA) sponges, preventing the role of miRNAs that repress their targets. However, there is no pipeline to systematically assess the sponging potential of circRNAs. Results: We developed circRNA-sponging, a nextflow pipeline that (i) identifies circRNAs via backsplicing junctions detected in RNA-seq data, (ii) quantifies their expression values in relation to their linear counterparts spliced from the same gene, (iii) performs differential expression analysis, (iv) identifies and quantifies miRNA expression from miRNA-sequencing (miRNA-seq) data, (v) predicts miRNA binding sites on circRNAs, (vi) systematically investigates potential circRNA-miRNA sponging events, (vii) creates a network of competing endogenous RNAs and (viii) identifies potential circRNA biomarkers. We showed the functionality of the circRNA-sponging pipeline using RNA sequencing data from brain tissues, where we identified two distinct types of circRNAs characterized by a specific ratio of the number of the binding site to the length of the transcript. The circRNA-sponging pipeline is the first end-to-end pipeline to identify circRNAs and their sponging systematically with raw total RNA-seq and miRNA-seq files, allowing us to better indicate the functional impact of circRNAs as a routine aspect in transcriptomic research. Availability and implementation: https://github.com/biomedbigdata/circRNA-sponging. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Electrochemical and fluorescent dual-mode sensor of acetylcholinesterase activity and inhibition based on MnO2@PD-coated surface.

We developed an electrochemical and fluorescent dual-mode sensor for assessing acetylcholinesterase (AChE) activity and inhibition by taking advantage of the high redox sensitivity of surface-coated mesoporous MnO2@polymer dot (MnO2@PD) towards AChE. The following phenomena constitute the basis of the detection mechanism: fluorescence resonance energy transfer (FRET) effect between MnO2 and PD; catalytic hydrolysis of acetylthiocholine (ATCh) to thiocholine (TCh) by AChE expressed by PC-12 cells, inducing fluorescence restoration and change in the conductivity of the system due to MnO2 decomposition; the presence of the inhibitor neostigmine preventing the conversion of ATCh to TCh. The surface-coated biosensor presents both fluorescence-based and electrochemical approaches for effectively monitoring AChE activity and inhibition. The fluorescence approach is based on the fluorescent "on/off" property of the system caused by MnO2 breakdown after interaction with TCh and the subsequent release of PDs. The conductivity of the coated electrode decreased dramatically as AChE concentration increased, resulting in electrochemical sensing of AChE activity and inhibition screening. Real-time wireless sensing can be conducted using a smartphone to monitor the resistance change, investigating the potential use of MnO2@PD nanocomposites in biological studies, and offering a real-time redox-fluorescent test for AChE activity monitoring and inhibitor screening.

Cell-specific and shared enhancers control a high-density multi-gene locus active in mammary and salivary glands.

Regulation of high-density loci harboring genes with different cell-specificities remains a puzzle. Here we investigate a locus that evolved through gene duplication 1 and contains eight genes and 20 candidate regulatory elements, including a super-enhancer. Five genes are expressed in mammary glands and account for 50% of all mRNAs during lactation, two are salivary-specific and one has dual specificity. We probed the function of eight candidate enhancers through experimental mouse genetics. Deletion of the super-enhancer led to a 98% reduced expression of Csn3 and Fdcsp in mammary and salivary glands, respectively, and Odam expression was abolished in both tissues. The other three casein genes were only marginally affected. Notably, super-enhancer activity requires the additional presence of a distal Csn3 -specific enhancer. Our work identifies an evolutionary playground on which regulatory duality of a multigene locus was attained through an ancestral super-enhancer active in mammary and salivary tissue and gene-specific mammary enhancers.

A cytokine-responsive promoter is required for distal enhancer function mediating the hundreds-fold increase in milk protein gene expression during lactation.

During lactation, specialized cells in the mammary gland produce milk to nourish the young. Milk protein genes are controlled by distal enhancers activating expression several hundred-fold during lactation. However, the role of promoter elements is not understood. We addressed this issue using the Csn2 gene, which accounts for 10% of mRNA in mammary tissue. We identified STAT5 and other mammary transcription factors binding to three distal candidate enhancers and a cytokine-response promoter element. While deletion of the enhancers or the introduction of an inactivating mutation in a single promoter element had a marginable effect, their combined loss led to a 99.99% reduction of Csn2 expression. Our findings reveal the essential role of a promoter element in the exceptional activation of a milk protein gene and highlight the importance of analyzing regulatory elements in their native genomic context to fully understand the multifaceted functions of enhancer clusters and promoters.

Cell-specific and shared enhancers control a high-density multi-gene locus active in mammary and salivary glands.

Regulation of high-density loci harboring genes with different cell-specificities remains a puzzle. Here we investigate a locus that evolved through gene duplication 1 and contains eight genes and 20 candidate regulatory elements, including a super-enhancer. Five genes are expressed in mammary glands and account for 50% of all mRNAs during lactation, two are salivary-specific and one has dual specificity. We probed the function of eight candidate enhancers through experimental mouse genetics. Deletion of the super-enhancer led to a 98% reduced expression of Csn3 and Fdcsp in mammary and salivary glands, respectively, and Odam expression was abolished in both tissues. The other three casein genes were only marginally affected. Notably, super-enhancer activity requires the additional presence of a distal Csn3 -specific enhancer. Our work identifies an evolutionary playground on which regulatory duality of a multigene locus was attained through an ancestral super-enhancer active in mammary and salivary tissue and gene-specific mammary enhancers.

circRNA-sponging: a pipeline for extensive analysis of circRNA expression and their role in miRNA sponging.

MOTIVATION: Circular RNAs (circRNAs) are long non-coding RNAs (lncRNAs) often associated with diseases and considered potential biomarkers for diagnosis and treatment. Among other functions, circRNAs have been shown to act as microRNA (miRNA) sponges, preventing the role of miRNAs that repress their targets. However, there is no pipeline to systematically assess the sponging potential of circRNAs. RESULTS: We developed circRNA-sponging, a nextflow pipeline that (1) identifies circRNAs via backsplicing junctions detected in RNA-seq data, (2) quantifies their expression values in relation to their linear counterparts spliced from the same gene, (3) performs differential expression analysis, (4) identifies and quantifies miRNA expression from miRNA-sequencing (miRNA-seq) data, (5) predicts miRNA binding sites on circRNAs, (6) systematically investigates potential circRNA-miRNA sponging events, (7) creates a network of competing endogenous RNAs, and (8) identifies potential circRNA biomarkers. We showed the functionality of the circRNA-sponging pipeline using RNA sequencing data from brain tissues, where we identified two distinct types of circRNAs characterized by a specific ratio of the number of the binding site to the length of the transcript. The circRNA-sponging pipeline is the first end-to-end pipeline to identify circRNAs and their sponging systematically with raw total RNA-seq and miRNA-seq files, allowing us to better indicate the functional impact of circRNAs as a routine aspect in transcriptomic research. AVAILABILITY: https://github.com/biomedbigdata/circRNA-sponging Contact: markus.daniel.hoffmann@tum.de; markus.list@tum.de Supplementary Material: Supplementary data are available at Bioinformatic Advances online.