Biology subjects

Tesfaigzi, Y.

Publications and source records attributed to Tesfaigzi, Y..

7 recordsLinked to original sources

bioRxiv2026

A framework for designing splice-junction experiments in deep 3' single-cell RNA sequencing

Alternative splicing is cell-type-specific and disease-relevant, but single-cell RNA sequencing is optimized for gene-level quantification, and how sequencing depth governs splice-junction recovery in the dominant 3'-biased chemistries has not been quantified, so experiments cannot be designed to a depth target and datasets cannot be interpreted against expected recovery. We generated deep single-cell RNA sequencing of human airway epithelial cells (12 samples, 102 to 803 million uniquely mapped reads per sample) and benchmarked junction recovery in five annotated samples against matched bulk RNA sequencing from a 190-donor cohort. Junction detection did not saturate with depth, and validity was governed by read support rather than annotation status. Recovering 90% of the observed full-depth junction complement required approximately 52,250 reads per cell, several-fold more than gene-level analysis. Quantifying splicing was more depth-limited than detecting it, and rare cell types were limited by cell number rather than depth. Against matched bulk, single-cell recall plateaued at a practical ceiling set by sample size and sparse sampling, and was independent of distance from the 3' end. These results establish a framework for designing splicing experiments in deep 3'-biased single-cell RNA sequencing, and provide an airway-epithelial dataset with matched bulk for benchmarking splicing methods.

bioinformatics↗

bioRxiv2026

Improving splice site usage prediction with SPLAIRE

BackgroundAlternative splicing, the mechanism by which intronic sequences are excised from pre-mRNAs to produce mature mRNA, affects >95% of human protein-coding genes and is a major driver of human disease states. The spliceosome, a protein-RNA complex responsible for splicing pre-mRNA, identifies candidate splice sites partly through the recognition of characteristic sequence motifs at exon-intron junctions. Deep learning models that predict the presence of splice sites from pre-mRNA sequence have achieved breakthrough performance relative to previous machine-learning techniques, and these models have improved our ability to identify pathogenic genetic variants that alter splicing. ResultsWe show that, while overall performance measures from these models suggest near-perfect performance, substantial gaps in prediction remain, including the identification of splice sites with low usage rates and tissue-specific splice sites. We leverage one of the largest paired RNA and genotyping datasets used to date to train a novel splicing model optimized for a specific cell type, human airway epithelial cells. We trained a dilated convolutional neural network on data from cultured airway epithelial cells from 100 donors, and showed that this model outperforms current state-of-the-art models on splice site identification and splice site usage quantification, including on multiple tissues not included in the model training data. ConclusionsWe present the most comprehensive evaluation of state-of-the-art splicing models published to date, revealing reasonable performance across models for genetic variant effect prediction along with important performance gaps and insights into directions for future model development.

genomics↗

bioRxiv2025

High-throughput Mucus Microrheology for Phenotyping and Disease Modeling

Mucus mechanics regulate barrier, clearance, and transport functions across epithelial tissues, yet quantitative rheology remains difficult to scale because available assays require large volumes, specialized equipment, or extensive manual analysis. Here, a high-throughput microrheology workflow based on Differential Dynamic Microscopy is established for frequency-dependent measurements from 3-10 uL mucus samples and intact mucosal surfaces using standard epifluorescence microscopy. The workflow combines preloaded tracer chambers, controlled sample handling, short video acquisition, and automated analysis to reduce operator intervention while preserving sensitivity to mucus relaxation on 1--20 s time scales. Validation against shear rheometry and particle tracking in reconstituted mucin gels shows robust recovery of viscoelastic trends across mucus-like material states. Applications in human airway epithelial air--liquid interface cultures resolve culture-medium, age, donor, COPD, cigarette-smoke, cystic-fibrosis treatment, and IL-13-associated differences, including spatial heterogeneity in situ. Measurements of clinical cervical mucus further demonstrate parallel testing of scarce specimens and sensitivity to osmotic swelling. This platform provides a scalable route to quantitative mucus phenotyping for disease modeling, therapeutic testing, and future precision-medicine studies.

bioengineering↗

bioRxiv2023

A Drug-Free Pathogen Capture and Neutralizing Nasal Spray to Prevent Emerging Respiratory Infections

Respiratory infections pose a global health crisis. Vaccines are pathogen specific, and new vaccines are needed for mutants and emerging pathogens. Here, we report a "drug free" prophylactic platform - a "Pathogen Capture and Neutralizing Spray" (PCANS) that acts via a multi-pronged approach to prevent a broad spectrum of respiratory infections. PCANS forms a protective coating in the nasal cavity that enhances the capture of large respiratory droplets. The coating acts as a physical barrier against a broad spectrum of viruses and bacteria, and rapidly neutralizes them, reducing the pathogen load by >99.99%. In mice, PCANS showed nasal retention for at least 8 h and was safe for daily administration. A single prophylactic dose of PCANS protected mice against supra-lethal dosages of a mouse-adapted H1N1 Influenza virus (PR8), reduced lung viral titer by >99.99%, improved survival, and suppressed pathological manifestations. Together, our data suggest PCANS as a promising daily-use prophylactic approach against current and emerging respiratory infections.

bioengineering↗

bioRxiv2022

Single-cell transcriptomics reveal hyperacute cytokine and immune checkpoint axis in patients with poor neurological outcomes after cardiac arrest

Neurological injury is a major driver of mortality among patients hospitalized after cardiac arrest (CA). The early systemic inflammatory response after CA is associated with neurological injury and mortality but remains poorly defined. We determine the innate immune network induced by clinical CA at single-cell resolution. Immune cell states diverge as early as 6h post-CA between patients with good or poor neurological outcomes at hospital discharge. Nectin-2+ monocyte and Tim-3+ natural killer (NK) cell subpopulations associate with poor outcomes, and interactome analysis highlights their crosstalk via cytokines and immune checkpoints. Ex vivo studies on peripheral blood cells from CA patients demonstrate that immune checkpoints are a compensatory mechanism against inflammation after CA. IFN{gamma}/IL-10 induce Nectin-2 on monocytes; in a negative feedback loop, Nectin-2 suppresses IFN{gamma} production by NK cells. The initial hours after CA may represent a window for therapeutic intervention in the resolution of inflammation via immune checkpoints.

immunology↗

bioRxiv2020

Paradoxical effects of cigarette smoke and COPD on SARS-CoV2 infection and disease

IntroductionHow cigarette smoke (CS) and chronic obstructive pulmonary disease (COPD) affect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and severity is controversial. We investigated the protein and mRNA expression of SARS-CoV-2 entry receptor ACE2 and proteinase TMPRSS2 in lungs from COPD patients and controls, and lung tissue from mice exposed acutely and chronically to CS. Also, we investigated the effects of CS exposure on SARS-CoV-2 infection in human bronchial epithelial cells. MethodsIn Cohort 1, ACE2-positive cells were quantified by immunostaining in FFPE sections from both central and peripheral airways. In Cohort 2, we quantified pulmonary ACE2 protein levels by immunostaining and ELISA, and both ACE2 and TMPRSS2 mRNA levels by RT-qPCR. In C57BL/6 WT mice exposed to air or CS for up to 6 months, pulmonary ACE2 protein levels were quantified by triple immunofluorescence staining and ELISA. The effects of CS exposure on SARS-CoV-2 infection were evaluated after 72hr in vitro infection of Calu-3 cells. After SARS-CoV-2 infection, the cells were fixed for IF staining with dsRNA-specific J2 monoclonal Ab, and cell lysates were harvested for WB of viral nucleocapsid (N) protein. Supernatants (SN) and cytoplasmic lysates were obtained to measure ACE2 levels by ELISA. ResultsIn both human cohorts, ACE2 protein and mRNA levels were decreased in peripheral airways from COPD patients versus both smoker and NS controls, but similar in central airways. TMPRSS2 levels were similar across groups. Mice exposed to CS had decreased ACE2 protein levels in their bronchial and alveolar epithelia versus air-exposed mice exposed to 3 and 6 months of CS. In Calu3 cells in vitro, CS-treatment abrogated infection to levels below the limit of detection. Similar results were seen with WB for viral N protein, showing peak viral protein synthesis at 72hr. ConclusionsACE2 levels were decreased in both bronchial and alveolar epithelial cells from uninfected COPD patients versus controls, and from CS-exposed versus air-exposed mice. CS-pre-treatment did not affect ACE2 levels but potently inhibited SARS-CoV-2 replication in this in vitro model. These findings urge to further investigate the controversial effects of CS and COPD on SARS-CoV2 infection.

immunology↗

bioRxiv2020

Bmf Facilitates Protein Degradation and Reduces Beclin1 Ubiquitination to Inhibit Autophagy Independent of mTOR

Previous observations suggested that Bcl-2 modifying factor (Bmf) affects autophagy but the underlying mechanisms were unknown. The present studies show that Bmf inhibited the initiation and flux of autophagy in a manner that is independent of the mTOR pathway and inhibition of mTOR increased Bmf expression to temper the autophagic cell death. In mice, emphysema was observed in Bmf-deficient mice, suggesting that Bmf suppresses autophagic cell death of alveolar type II cells. Bmf deficiency increased ubiquitination of Beclin1 with K63 chains and released Beclin1 from Bcl-2. However, Bmf deficiency also increased the levels of polyubiquitinated proteins in general. In mice, Bmf-deficiency robustly increased p62 levels in all tissues analyzed, but LC3-II levels were reduced only in the hearts of old mice. Also, Bmf-deficiency caused persistent mucous cell metaplasia in mice exposed to allergen and increased levels of polyubiquitinated Muc5ac in differentiated airway epithelial cells. The reduction of ubiquitinated proteins was mediated by the BH3- and dynein binding-domains of Bmf. Together, these findings show that the primary role of Bmf is to reduce protein levels and affects K63- and K48-ubiqutination.

cell biology↗