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CRISPR/Cas9 screenings reveal the role of STX1A and CDK1 in Cathepsin G entering and killing colorectal cancer cells.

Neutrophils are the major populations of white blood cells and have been reported to facilitate cancer metastasis. Meanwhile, emerging evidence has recently suggested the anti-cancer role of neutrophils. Our previous study revealed that CB-839 and 5-FU-treated colorectal cancer (CRC) tumors recruited neutrophils and induced neutrophil extracellular traps (NETs). Cathepsin G (CTSG), which is released during NET formation, enters CRC cells through the receptor for advanced glycation end products (RAGE) and cleaves 14-3-3ε to promote apoptosis. However, the detailed mechanism underlying CTSG's anti-tumor function remains less studied. In this study, we report that CTSG enters CRC cells through RAGE-mediated endocytosis. Knocking out RAGE or inhibiting endocytosis blocks CTSG from entering CRC cells and attenuates CTSG-induced apoptosis. Furthermore, the clathrin coat assembly complex and SNARE proteins were enriched in an arrayed CRISPR/Cas9 screening targeting human membrane trafficking genes. Knocking out SNARE protein STX1A prevents the spread of CTSG in CRC cells and the induction of cleaved PARP. A pooled genome-wide CRISPR/Cas9 screening further identifies the role of CDK1 in the NET-induced killing of CRC cells. Inhibiting CDK1 protected CRC cells from killing by CTSG. Our study reveals novel mechanisms by which CTSG enters and kills CRC cells.

CDK1

Genome-Wide Association Analysis of Hippocampal Neuroplasticity as an Indicator of Stress Responsiveness in Laying Hens (Gallus gallus domesticus).

Environmental stressors in commercial poultry systems can negatively affect bird welfare, although individuals vary considerably in their responses. Neuroplasticity within the hippocampus, measured through the density of doublecortin-positive (DCX+) neurons, provides a potential biomarker of stress experience in laying hens. However, the genetic basis underlying variation in this biomarker remains poorly understood. A total of 42 H&N and Hy-Line Brown hens housed in a multitier free range and enriched cage system, respectively, were genotyped using Genotyping by Sequencing, yielding over 200 000 SNP markers after initial filtering. Hippocampal tissue sections were immunostained for DCX to quantify the density of highly plastic neurons. A genome-wide association analysis identified 19 genomic regions across eight chromosomes within the top 1% of windows explaining the greatest proportion of genetic variance in the neuroplasticity phenotype. Within ±100 kb of these regions, 39 annotated genes were identified, several of which are involved in cellular regulation and genetic information processing pathways. Notably, PIK3R6, VPS37D, STX1A, BAZ1B, HGH1, MAF1, MAPK15, and PIT54 emerged as positional candidate genes potentially contributing to variation in stress responsiveness. These findings provide preliminary insight into the genetic architecture of hippocampal neuroplasticity in laying hens and highlight candidate genes that may contribute to individual differences in stress response, with potential implications for breeding strategies aimed at improving poultry welfare.

Animals