NCK-associated protein 1 like (nckap1l) minor splice variant regulates intrahepatic biliary network morphogenesis.

Impaired formation of the intrahepatic biliary network leads to cholestatic liver diseases, which are frequently associated with autoimmune disorders. Using a chemical mutagenesis strategy in zebrafish combined with computational network analysis, we screened for novel genes involved in intrahepatic biliary network formation. We positionally cloned a mutation in the nckap1l gene, which encodes a cytoplasmic adaptor protein for the WAVE regulatory complex. The mutation is located in the last exon after the stop codon of the primary splice isoform, only disrupting a previously unannotated minor splice isoform, which indicates that the minor splice isoform is responsible for the intrahepatic biliary network phenotype. CRISPR/Cas9-mediated nckap1l deletion, which disrupts both the primary and minor isoforms, showed the same defects. In the liver of nckap1l mutant larvae, WAVE regulatory complex component proteins are degraded specifically in biliary epithelial cells, which line the intrahepatic biliary network, thus disrupting the actin organization of these cells. We further show that nckap1l genetically interacts with the Cdk5 pathway in biliary epithelial cells. These data together indicate that although nckap1l was previously considered to be a hematopoietic cell lineage-specific protein, its minor splice isoform acts in biliary epithelial cells to regulate intrahepatic biliary network formation.

Enhanced mitochondrial fission suppresses signaling and metastasis in triple-negative breast cancer.

BACKGROUND: Mitochondrial dynamics underlies malignant transformation, cancer progression, and response to treatment. Current research presents conflicting evidence for functions of mitochondrial fission and fusion in tumor progression. Here, we investigated how mitochondrial fission and fusion states regulate underlying processes of cancer progression and metastasis in triple-negative breast cancer (TNBC). METHODS: We enforced mitochondrial fission and fusion states through chemical or genetic approaches and measured migration and invasion of TNBC cells in 2D and 3D in vitro models. We also utilized kinase translocation reporters (KTRs) to identify single cell effects of mitochondrial state on signaling cascades, PI3K/Akt/mTOR and Ras/Raf/MEK/ERK, commonly activated in TNBC. Furthermore, we determined effects of fission and fusion states on metastasis, bone destruction, and signaling in mouse models of breast cancer. RESULTS: Enforcing mitochondrial fission through chemical or genetic approaches inhibited migration, invasion, and metastasis in TNBC. Breast cancer cells with predominantly fissioned mitochondria exhibited reduced activation of Akt and ERK both in vitro and in mouse models of breast cancer. Treatment with leflunomide, a potent activator of mitochondrial fusion proteins, overcame inhibitory effects of fission on migration, signaling, and metastasis. Mining existing datasets for breast cancer revealed that increased expression of genes associated with mitochondrial fission correlated with improved survival in human breast cancer. CONCLUSIONS: In TNBC, mitochondrial fission inhibits cellular processes and signaling pathways associated with cancer progression and metastasis. These data suggest that therapies driving mitochondrial fission may benefit patients with breast cancer.

Single-cell RNA-sequencing of migratory breast cancer cells: discovering genes associated with cancer metastasis.

Considerable evidence suggests breast cancer metastasis arises from cells undergoing epithelial-to-mesenchymal-transition (EMT) and cancer stem-like cells (CSCs). Using a microfluidic device that enriches migratory breast cancer cells with enhanced capacity for tumor formation and metastasis, we identified genes differentially expressed in migratory cells by high-throughput single-cell RNA-sequencing. Migratory cells exhibited overall signatures of EMT and CSCs with variable expression of marker genes, and they retained expression profiles of EMT over time. With single-cell resolution, we discovered intermediate EMT states and distinct epithelial and mesenchymal sub-populations of migratory cells, indicating breast cancer cells can migrate rapidly while retaining an epithelial state. Migratory cells showed differential profiles for regulators of oxidative stress, mitochondrial morphology, and the proteasome, revealing potential vulnerabilities and unexpected consequences of drugs. We also identified novel genes correlated with cell migration and outcomes in breast cancer as potential prognostic biomarkers and therapeutic targets to block migratory cells in metastasis.

Impact of anemia on acute ischemic stroke outcomes: A systematic review of the literature.

INTRODUCTION: Anemia has been reported in nearly 40% of acute ischemic stroke (AIS) patients and is linked to significant morbidity and disability. The presence of anemia is associated with worse outcomes in AIS, specifically in the presence of large vessel occlusion (LVO). An optimal hemoglobin (Hb) target specific to this pathology has not yet been established. The goal of this review is to systematically review literature that observes the association that exists between AIS outcomes and hemoglobin (Hb) levels. METHODS: A systematic review was performed in accordance with guidelines for the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) to identify studies from 2008-2022. The following inclusion and exclusion criteria were used: studies of adult patients with AIS; must describe outcomes with regard to Hb levels in AIS (not limited to LVO); must be written in English. The clinical variables extracted included Length of Stay (LOS), modified rankin score (mRS), Hb levels, and mortality. RESULTS: A total of 1,154 studies were gathered, with 116 undergoing full text review. 31 studies were included in this review. The age of patients ranged from 61.4 to 77.8. The presence of anemia in AIS increased LOS by 1.7 days on average and these patients also have a 15.2% higher rate of mortality at one year, on average. DISCUSSION: This data suggests that the contemporary thresholds for treating anemia in AIS patients may be inadequate because anemia is strongly associated with poor outcomes (e.g., mRS>2 or mortality) and increased LOS in AIS patients. The current generalized Hb threshold for transfusion (7 g/dL) is also used in AIS patients, however, a more aggressive transfusion parameter should be further explored based on these findings. Further studies are required to confirm these findings and to determine if a more liberal RBCT threshold will result in clinical benefits.

Hydro-Seq enables contamination-free high-throughput single-cell RNA-sequencing for circulating tumor cells.

Molecular analysis of circulating tumor cells (CTCs) at single-cell resolution offers great promise for cancer diagnostics and therapeutics from simple liquid biopsy. Recent development of massively parallel single-cell RNA-sequencing (scRNA-seq) provides a powerful method to resolve the cellular heterogeneity from gene expression and pathway regulation analysis. However, the scarcity of CTCs and the massive contamination of blood cells limit the utility of currently available technologies. Here, we present Hydro-Seq, a scalable hydrodynamic scRNA-seq barcoding technique, for high-throughput CTC analysis. High cell-capture efficiency and contamination removal capability of Hydro-Seq enables successful scRNA-seq of 666 CTCs from 21 breast cancer patient samples at high throughput. We identify breast cancer drug targets for hormone and targeted therapies and tracked individual cells that express markers of cancer stem cells (CSCs) as well as of epithelial/mesenchymal cell state transitions. Transcriptome analysis of these cells provides insights into monitoring target therapeutics and processes underlying tumor metastasis.