Establishment of fishing cat cell biobanking for sustainable conservation.

The fishing cat (Prionailurus viverrinus) is a vulnerable wild felid that is currently under threat from habitat destruction and other human activities. The zoo provides insurance to ensure the survival of the fishing cat population. Creating a biobank of fishing cats is a critical component of recent zoo strategies for securely stocking cell samples for long-term survival. Here, our goal was to compare cell biobanking techniques (tissue collection, primary culture, and reprogramming) and tissue sources (ear skin, abdominal skin, testis) from captive (n = 6)/natural (n = 6) vs. living (n = 8)/postmortem (n = 4) fishing cats. First, we show that dermal fibroblasts from the medial border of the helix of the ear pinna and abdominal tissues of living fishing cats can be obtained, whereas postmortem animals provided far fewer fibroblasts from the ears than from the testes. Furthermore, we can extract putative adult spermatogonial stem cells from the postmortem fishing cat's testes. The main barrier to expanding adult fibroblasts was early senescence, which can be overcome by overexpressing reprogramming factors through felid-specific transfection programs, though we demonstrated that reaching iPSC state from adult fibroblasts of fishing cats was ineffective with current virus-free mammal-based induction approaches. Taken together, the success of isolating and expanding primary cells is dependent on a number of factors, including tissue sources, tissue handling, and nature of limited replicative lifespan of the adult fibroblasts. This study provides recommendations for tissue collection and culture procedures for zoological research to facilitate the preservation of cells from both postmortem and living felids.

Phosphoproteomic Analysis Defines BABAM1 as mTORC2 Downstream Effector Promoting DNA Damage Response in Glioblastoma Cells.

Glioblastoma (GBM) is a devastating primary brain cancer with a poor prognosis. GBM is associated with an abnormal mechanistic target of rapamycin (mTOR) signaling pathway, consisting of two distinct kinase complexes: mTORC1 and mTORC2. The complexes play critical roles in cell proliferation, survival, migration, metabolism, and DNA damage response. This study investigated the aberrant mTORC2 signaling pathway in GBM cells by performing quantitative phosphoproteomic analysis of U87MG cells under different drug treatment conditions. Interestingly, a functional analysis of phosphoproteome revealed that mTORC2 inhibition might be involved in double-strand break (DSB) repair. We further characterized the relationship between mTORC2 and BRISC and BRCA1-A complex member 1 (BABAM1). We demonstrated that pBABAM1 at Ser29 is regulated by mTORC2 to initiate DNA damage response, contributing to DNA repair and cancer cell survival. Accordingly, the inactivation of mTORC2 significantly ablated pBABAM1 (Ser29), reduced DNA repair activities in the nucleus, and promoted apoptosis of the cancer cells. Furthermore, we also recognized that histone H2AX phosphorylation at Ser139 (γH2AX) could be controlled by mTORC2 to repair the DNA. These results provided a better understanding of the mTORC2 function in oncogenic DNA damage response and might lead to specific mTORC2 treatments for brain cancer patients in the future.

COVID-19 active case findings based on self-collected saliva samples with CRISPR-Cas12a detection.

COVID-19 is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus affecting the world population. Early detection has become one of the most successful strategies to alleviate the epidemic and pandemic of this contagious coronavirus. Surveillance testing programs have been initiated in many countries worldwide to prevent the outbreak of COVID-19. In this study, we demonstrated that our previously established clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based assay could detect variants of concern during 2021 in Thailand, including Alpha, Beta, and Delta strains as well as Omicron strain in early 2022. In combination with the newly designed saliva collection funnel, we established a safe, simple, economical, and efficient self-collection protocol for the COVID-19 screening process. We successfully utilized the assay in an active case finding with a total number of 578 asymptomatic participants to detect the SARS-CoV-2 in saliva samples. We finally demonstrated that the validation and evaluation in a large-scale setting could provide valuable information and elaborate the practicality of the test in real-world settings. Our optimized protocol yielded effective results with high sensitivity, specificity, and diagnostic accuracy (96.86%). In addition, this study demonstrates COVID-19 active case findings in low-resource settings, which would be feasible and attractive for surveillance and outbreak prevention in the future.