In vivo CRISPR screening directly targeting testicular cells.

CRISPR-Cas9 short guide RNA (sgRNA) library screening is a powerful approach to understand the molecular mechanisms of biological phenomena. However, its in vivo application is currently limited. Here, we developed our previously established in vitro revival screening method into an in vivo one to identify factors involved in spermatogenesis integrity by utilizing sperm capacitation as an indicator. By introducing an sgRNA library into testicular cells, we successfully pinpointed the retinal degeneration 3 (Rd3) gene as a significant factor in spermatogenesis. Single-cell RNA sequencing (scRNA-seq) analysis highlighted the high expression of Rd3 in round spermatids, and proteomics analysis indicated that Rd3 interacts with mitochondria. To search for cell-type-specific signaling pathways based on scRNA-seq and proteomics analyses, we developed a computational tool, Hub-Explorer. Through this, we discovered that Rd3 modulates oxidative stress by regulating mitochondrial distribution upon ciliogenesis induction. Collectively, our screening system provides a valuable in vivo approach to decipher molecular mechanisms in biological processes.

Fatal Community-Acquired Clostridioides difficile Infection as a Cause of Listeria Meningitis: A Case Report.

A 77-year-old woman with a history of total gastrectomy was transferred to our hospital with complaints of fever and consciousness disturbance for five days. She had fever and consciousness disturbance with positive meningeal signs. Laboratory findings indicated an elevated inflammatory response and hypoalbuminemia, and computed tomography (CT) of the body indicated intestinal gas retention and mild ascites. Cerebrospinal fluid analysis revealed pleocytosis with elevated protein levels and a diagnosis of Listeria meningitis was made. Treatment with ampicillin/sulbactam was started, and her fever and consciousness disturbance resolved on day 2. However, on day 3, her fever and conscious disturbance deteriorated, and she went into shock subsequently. Laboratory findings revealed deteriorated inflammatory response and hypoalbuminemia. Body CT showed an obvious distended bowel loop and intestinal edema. A stool culture revealed positive Clostridioides difficile toxin B, and we diagnosed her with Clostridioides difficile infection (CDI). Although intravenous metronidazole was initiated, she died due to prolonged hypovolemic shock. We considered she had community-acquired CDI because her CDI emerged immediately after the initiation of antibiotics, symptom deterioration within 48 hours of admission, and abnormal abdominal CT findings at admission. Listeria meningitis can develop based on community-acquired CDI. Because CDI can have a very rapid and fatal course and is sometimes complicated by other infectious diseases, clinicians should pay attention to this complication.

An Infection Model for SARS-CoV-2 Using Rat Transplanted with hiPSC-Airway Epithelial Cells.

Investigating the infection mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the airway epithelium and developing effective defense strategies against infection are important. To achieve this, establishing appropriate infection models is crucial. Therefore, various in vitro models, such as cell lines and primary cultures, and in vivo models involving animals that exhibit SARS-CoV-2 infection and genetically humanized animals have been used as animal models. However, no animal model has been established that allows infection experiments with human cells under the physiological environment of airway epithelia. Therefore, we aimed to establish a novel animal model that enables infection experiments using human cells. Human induced pluripotent stem cell-derived airway epithelial cell-transplanted nude rats (hiPSC-AEC rats) were used, and infection studies were performed by spraying lentiviral pseudoviruses containing SARS-CoV-2 spike protein and the GFP gene on the tracheae. After infection, immunohistochemical analyses revealed the existence of GFP-positive-infected transplanted cells in the epithelial and submucosal layers. In this study, a SARS-CoV-2 infection animal model including human cells was established mimicking infection through respiration, and we demonstrated that the hiPSC-AEC rat could be used as an animal model for basic research and the development of therapeutic methods for human-specific respiratory infectious diseases.