A multivariate cell-based liquid biopsy for lung nodule risk stratification: Analytical validation and early clinical evaluation.

Background: The Indicator Cell Assay Platform (iCAP) is a novel tool for blood-based diagnostics that uses living cells as biosensors to integrate and amplify weak, multivalent disease signals present in patient serum. In the platform, standardized cells are exposed to small volumes of patient serum, and the resulting transcriptomic response is analyzed using machine learning tools to develop disease classifiers.

Methods: We developed a lung cancer-specific iCAP (LC-iCAP) as a rule-out test for the management of indeterminate pulmonary nodules detected by low-dose CT screening.

Early and opposing neutrophil and CD4 T cell responses shape pulmonary tuberculosis pathology.

Pulmonary Mycobacterium tuberculosis (Mtb) infection results in a variety of heterogeneous lesion structures, from necrotic granulomas to alveolitis, but the mechanisms regulating their development remain unclear. Using a mouse model of concomitant immunity and subsequent aerosol infection, we demonstrate that counter regulation between neutrophils and CD4 T cells occurs very early during infection and governs these distinct pathologies. In primary Mtb infection, a dysregulated feed-forward circuit of neutrophil recruitment occurs, in which neutrophils hinder CD4 T cell interactions with infected macrophages, cause granuloma necrosis, and establish a replicative niche that drives a two-log increase in lung bacterial burden.

APOE protects against severe infection with Mycobacterium tuberculosis by restraining production of neutrophil extracellular traps.

Mice lacking apolipoprotein E (APOE, Apoe-/- mice) on a high cholesterol (HC) diet are highly susceptible to infection with Mycobacterium tuberculosis (Mtb) but the underlying immune dysregulation has been unclear. While neutrophils are often the predominant cell type in the lungs of humans with severe tuberculosis (TB), they are relatively scarce in the lungs of some strains of mice that are used to study the disease. The neutrophil levels in the lungs of Mtb-infected Apoe-/- HC mice are very high, and thus studies in this model offer the opportunity to examine the role of specific neutrophil functions in the pathology of severe TB.

Exploiting host kinases to combat dengue virus infection and disease.

The burden of dengue on human health has dramatically increased in recent years, underscoring the urgent need for effective therapeutic interventions. Despite decades of research since the discovery of the dengue virus, no specific antiviral treatments are available and strategies to reliably prevent severe disease remain limited. Direct-acting antivirals against dengue are under active investigation but have shown limited efficacy to date.

A multivariate cell-based assay for blood-based diagnostics enhances lung cancer risk stratification.

Unlabelled: The indicator cell assay platform (iCAP) is a tool for blood-based diagnostics that addresses the low signal-to-noise ratio of blood biomarkers by using cells as biosensors. The assay exposes small volumes of patient serum to standardized cells in culture and classifies disease by machine learning analysis of the gene expression readout from the cells. We developed the lung cancer iCAP (LC-iCAP) as a rule-out test for nodule management in computed tomography (CT)-based lung-cancer screening.

A Conditional Cas9 System for Stage-Specific Gene Editing in .

Unlabelled: The malaria parasite has a complex lifecycle involving various host cell environments in both human and mosquito hosts. The parasite must tightly regulate gene expression at each stage in order to adapt to its current environment while continuing development. However, it is challenging to study gene function and regulation of essential genes across the parasite's multi-host lifecycle.

Preclinical development of a replication-competent vesicular stomatitis virus-based Lassa virus vaccine candidate advanced into human clinical trials.

Background: Lassa fever (LF) is a zoonotic haemorrhagic disease caused by Lassa virus (LASV), which is endemic in West African countries. The multimammate rat is the main animal reservoir and its geographic range is expected to expand due to influences like climate change and land usage, and this will place larger parts of Africa at risk. We conducted preclinical development on a promising experimental vaccine that allowed its advancement into human trials.

TOR and heat shock response pathways regulate peroxisome biogenesis during proteotoxic stress.

Peroxisomes are versatile organelles mediating energy homeostasis and redox balance. While peroxisome dysfunction is linked to numerous diseases, the molecular mechanisms and signaling pathways regulating peroxisomes during cellular stress remain elusive. Using yeast, we show that perturbations disrupting protein homeostasis including loss of ER or cytosolic chaperone function, impairments in ER protein translocation, blocking ER N-glycosylation, or reductive stress, cause peroxisome proliferation.

DeleteomeTools: utilizing a compendium of yeast deletion strain transcriptomes to identify co-functional genes.

We introduce DeleteomeTools, an R package that leverages the Deleteome compendium of yeast single-gene deletion transcriptomes to predict gene function. Primarily, the package provides functions for identifying similarities between the transcriptomic signatures of deletion strains, thereby associating genes of interest with others that may be functionally related. We describe how our software predicted a novel relationship between the yeast nucleoporin Nup170 and the Ctf18-RFC complex, which was confirmed experimentally, revealing a previously unknown link between nuclear pore complexes and the DNA replication machinery.

APOE Protects Against Severe Infection with by Restraining Production of Neutrophil Extracellular Traps.

While neutrophils are the predominant cell type in the lungs of humans with active tuberculosis (TB), they are relatively scarce in the lungs of most strains of mice that are used to study the disease. However, similar to humans, neutrophils account for approximately 45% of CD45+ cells in the lungs of mice on a high-cholesterol (HC) diet following infection with (Mtb). We hypothesized that the susceptibility of HC mice might arise from an unrestrained feed-forward loop in which production of neutrophil extracellular traps (NETs) stimulates production of type I interferons by pDCs which in turn leads to the recruitment and activation of more neutrophils, and demonstrated that depleting neutrophils, depleting plasmacytoid dendritic cells (pDCs), or blocking type I interferon signaling, improved the outcome of infection.

Systems-level reconstruction of kinase phosphosignaling networks regulating endothelial barrier integrity using temporal data.

Phosphosignaling networks control cellular processes. We built kinase-mediated regulatory networks elicited by thrombin stimulation of brain endothelial cells using two computational strategies: Temporal Pathway Synthesizer (TPS), which uses phosphoproteomics data as input, and Temporally REsolved KInase Network Generation (TREKING), which uses kinase inhibitor screens. TPS and TREKING predicted overlapping barrier-regulatory kinases connected with unique network topology.

Systems-level reconstruction of kinase phosphosignaling networks regulating endothelial barrier integrity using temporal data.

Phosphosignaling networks control cellular processes. We built kinase-mediated regulatory networks elicited by thrombin stimulation of brain endothelial cells using two computational strategies: Temporal Pathway Synthesizer (TPS), which uses phosphoproteomics data as input, and Temporally REsolved KInase Network Generation (TREKING), which uses kinase inhibitor screens. TPS and TREKING predicted overlapping barrier-regulatory kinases connected with unique network topology.

Automated, image-based quantification of peroxisome characteristics with perox-per-cell.

Summary: perox-per-cell automates cumbersome, image-based data collection tasks often encountered in peroxisome research. The software processes microscopy images to quantify peroxisome features in yeast cells. It uses off-the-shelf image processing tools to automatically segment cells and peroxisomes and then outputs quantitative metrics including peroxisome counts per cell and spatial areas.

Predicting host-based, synthetic lethal antiviral targets from omics data.

Traditional antiviral therapies often have limited effectiveness due to toxicity and the emergence of drug resistance. Host-based antivirals are an alternative, but can cause nonspecific effects. Recent evidence shows that virus-infected cells can be selectively eliminated by targeting synthetic lethal (SL) partners of proteins disrupted by viral infection.

Nanobody repertoire generated against the spike protein of ancestral SARS-CoV-2 remains efficacious against the rapidly evolving virus.

To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single-domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies.

CD4-mediated immunity shapes neutrophil-driven tuberculous pathology.

Pulmonary (Mtb) infection results in highly heterogeneous lesions ranging from granulomas with central necrosis to those primarily comprised of alveolitis. While alveolitis has been associated with prior immunity in human post-mortem studies, the drivers of these distinct pathologic outcomes are poorly understood. Here, we show that these divergent lesion structures can be modeled in C3HeB/FeJ mice and are regulated by prior immunity.

Automated, image-based quantification of peroxisome characteristics with .

automates cumbersome, image-based data collection tasks often encountered in peroxisome research. The software processes microscopy images to quantify peroxisome features in yeast cells. It uses off-the-shelf image processing tools to automatically segment cells and peroxisomes and then outputs quantitative metrics including peroxisome counts per cell and spatial areas.

Multiple receptor tyrosine kinases regulate dengue infection of hepatocytes.

Introduction: Dengue is an arboviral disease causing severe illness in over 500,000 people each year. Currently, there is no way to constrain dengue in the clinic. Host kinase regulators of dengue virus (DENV) infection have the potential to be disrupted by existing therapeutics to prevent infection and/or disease progression.

SARS-CoV-2 Orf6 is positioned in the nuclear pore complex by Rae1 to inhibit nucleocytoplasmic transport.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) accessory protein Orf6 works as an interferon antagonist, in part, by inhibiting the nuclear import activated p-STAT1, an activator of interferon-stimulated genes, and the export of the poly(A) RNA. Insight into the transport regulatory function of Orf6 has come from the observation that Orf6 binds to the nuclear pore complex (NPC) components: Rae1 and Nup98. To gain further insight into the mechanism of Orf6-mediated transport inhibition, we examined the role of Rae1 and Nup98.

DeleteomeTools: Utilizing a compendium of yeast deletion strain transcriptomes to identify co-functional genes.

We introduce DeleteomeTools, an R package that leverages the Deleteome compendium of yeast single-gene deletion transcriptomes to predict gene function. Primarily, the package provides functions for identifying similarities between the transcriptomic signatures of deletion strains, thereby associating genes of interest with others that may be functionally related. We describe how our software predicted a novel relationship between the yeast nucleoporin Nup170 and the Ctf18-RFC complex, which was confirmed experimentally, revealing a previously unknown link between nuclear pore complexes and the DNA replication machinery.

Predicting host-based, synthetic lethal antiviral targets from omics data.

Traditional antiviral therapies often have limited effectiveness due to toxicity and development of drug resistance. Host-based antivirals, while an alternative, may lead to non-specific effects. Recent evidence shows that virus-infected cells can be selectively eliminated by targeting synthetic lethal (SL) partners of proteins disrupted by viral infection.

Nanobody repertoire generated against the spike protein of ancestral SARS-CoV-2 remains efficacious against the rapidly evolving virus.

To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies.

SUMOylation at the inner nuclear membrane facilitates nuclear envelope biogenesis during mitosis.

As eukaryotic cells progress through cell division, the nuclear envelope (NE) membrane must expand to accommodate the formation of progeny nuclei. In Saccharomyces cerevisiae, closed mitosis allows visualization of NE biogenesis during mitosis. During this period, the SUMO E3 ligase Siz2 binds the inner nuclear membrane (INM) and initiates a wave of INM protein SUMOylation.

Nuclear pore complexes mediate subtelomeric gene silencing by regulating PCNA levels on chromatin.

The nuclear pore complex (NPC) physically interacts with chromatin and regulates gene expression. The Saccharomyces cerevisiae inner ring nucleoporin Nup170 has been implicated in chromatin organization and the maintenance of gene silencing in subtelomeric regions. To gain insight into how Nup170 regulates this process, we used protein-protein interactions, genetic interactions, and transcriptome correlation analyses to identify the Ctf18-RFC complex, an alternative proliferating cell nuclear antigen (PCNA) loader, as a facilitator of the gene regulatory functions of Nup170.