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Bovine parainfluenza virus type 3 (BPIV3) is a leading cause of respiratory illness in cattle and a primary component of the bovine respiratory disease complex (BRDC), resulting in significant economic losses. Understanding the mechanisms of BPIV3 infection, particularly the entry process, is essential for developing effective control measures. Identifying specific host factors that viruses exploit during their life cycle can reveal critical vulnerabilities for potential antiviral targets. We established a genome-wide CRISPR/Cas9 knockout screen in bovine cells to identify host factors involved in viral infections. Our screen identified several key host factors required for BPIV3 infection, including the sialic acid transporter SLC35A1 and the Sm-like protein LSM12. Further mechanistic analysis revealed that these factors played critical roles at distinct stages of the BPIV3 entry process. These findings not only advance our understanding of how BPIV3 infects host cells but also identify potential host targets for inhibiting infection and developing novel antiviral strategies.

Primate-specific genes (PSGs), important contributors to the origin of adaptive evolutionary novelties, are abundantly expressed in the testis. However, the specific roles of PSGs in the male reproductive system of humans and other primates are largely unknown. Here, we employed whole-exome sequencing and identified deleterious variants of TFDP3, an X-linked PSG, in eight infertile men with oligoasthenoteratozoospermia. All of the male subjects harboring TFDP3 variants presented dramatic reductions in sperm concentration, motility, and abnormal sperm morphology. Furthermore, Tfdp3-knockdown (KD) in the testes of cynomolgus monkeys confirmed the important role of TFDP3 in normal spermatogenesis in primates. Consistently, dramatic decreases in sperm concentration, motility, and abnormal sperm morphology were also observed in Tfdp3-KD male cynomolgus monkeys. More importantly, further functional studies revealed that TFDP3 deficiency activated E2F1 induced apoptosis and thus led to decreased sperm count and motility. In addition, five of the eight couples underwent intra-cytoplasmic sperm injection treatment and achieved a successful pregnancy, indicating a potentially good outcome of assisted reproduction for those with TFDP3 deficiency-mediated oligoasthenoteratozoospermia. Collectively, our genetic analyses and experimental observations in humans and cynomolgus monkeys highlight the crucial role of TFDP3, an inhibitor of apoptosis, in normal spermatogenesis. These findings expand the spectrum of pathogenic variants for oligoasthenoteratozoospermia-associated male infertility and also reveal the special significance of primate-specific TFDP3 for the human male reproductive system, thus providing important guidance for genetic counseling and the clinical diagnosis of male infertility.

Sequence-specific gene knockdown technologies are crucial for fundamental research and therapeutic applications. RNA interference and CRISPR interference, while extensively utilized for gene expression manipulation, face limitations due to their ectopic or transient expression. In this study, we developed a generalizable and efficient method to downregulate gene expression in human 293T cells by introducing de novo upstream ATGs (uATGs) of genes using CRISPR-Cas9-mediated genome editing. Through CRISPR library screening, in-depth sequencing, and flow cytometry analysis, we validated that the introduction of uATGs served as an effective method to suppress protein expression. Our findings further revealed that this strategy can be tailored to diminish endogenous gene expression in tumor cells without affecting the mRNA transcription levels. Importantly, by introducing a uATG into the 5', untranslated region (UTR) of the Uox gene, we successfully established a Uox-knockdown (KD) mouse model of hyperuricemia associated with metabolic disorders. This model demonstrated hyperuricemia, with serum uric acid levels that exceeded 400 µmol L^-1, along with renal dysfunction, as indicated by elevated serum creatinine and blood urea nitrogen levels. Examination of the kidneys from 8-week-old Uox-KD mice revealed abnormal histopathological characteristics, including partial dilation of Bowman's capsules and renal tubules, focal nephron collapse and necrosis, and lymphocytic infiltration. In addition, the mice exhibited lipid and glucose metabolism disorders, all while maintaining a normal lifespan. This spontaneous hyperuricemia model has potential as a valuable tool for long-term studies on hyperuricemia and gout. Taken together, we present an efficient approach for the constant suppression of specific gene expression in mammalian cells and the development of a Uox-KD mouse model of hyperuricemia via CRISPR-Cas9-mediated uATG introduction. This offers broad implications for fundamental research and therapeutic applications.

As studies aimed at clarifying the evolution of cognition in animals accumulate, some non-human primates are widely considered to be the most cognitively advanced species after humans. Cognitive processes related to social interaction and emotional responsiveness may also play a role in primates' responses to death and dying; however, how social complexity might influence animals' understanding of death remains under-explored. Here, we connect evolutionary development and social systems to death cognition, and establish a five-dimensional framework for comparative purposes, based on existing thanatological studies. Of 135 species included in the analysis, dolphins, elephants and chimpanzees-species with highly complex societies-were shown to have a more advanced death understanding than other taxonomically close species. Focusing on a multilevel society primate, the golden snub-nosed monkey (Rhinopithecus roxellana), we present five death-related case studies involving behavioral, physiological, and social responses to dead conspecifics. Despite evolving on an older phylogenetic branch than that leading to modern hominids, these monkeys display prolonged dead-infant carrying, care/caretaking, silence and compassion, and revisitation of corpses at levels comparable to those seen in chimpanzees, more than other, non-multilevel society primates. In addition to introducing a novel approach to the comparative and evolutionary study of death-related behavior, we suggest that social complexity can exert a modulatory influence on phylogenetic factors that constrain the baseline cognitive architecture, allowing more nuanced expressions of death cognition.

Selecting first-line treatments for advanced hepatocellular carcinoma (HCC) requires carefully balancing the survival benefits against the risk of serious adverse events (SAEs). However, conventional evaluation methods often fail to incorporate multiple clinical endpoints into a single unified framework. To address this limitation, we developed the Efficacy-Safety Integrated Ranking Algorithm (ESIRA), a quantitative framework that generates composite treatment rankings by assigning adjustable weights to efficacy and safety outcomes and calculating the Euclidean distance of each regimen to an ideal efficacy-safety profile. In a training cohort comprising 4,179 patients from eight randomized trials, treatment rankings varied markedly depending on the weighting scheme. Nivolumab plus ipilimumab ranked highest when the safety weight exceeded 0.7, whereas sintilimab plus bevacizumab and camrelizumab plus apatinib rose sharply in ranking when the efficacy weight exceeded 0.8. Sensitivity analysis identified 0.7:0.3 as the optimal efficacy-to-safety weight ratio, under which nivolumab plus ipilimumab achieved the highest composite Q-value (Q is the ranking function with higher values indicating higher ranks) (Q 0.60, 95%CI 0.54-0.68; P=0.192). The robustness of ESIRA was supported by internal consistency analyses within the training cohort. When efficacy weights were below 0.2, the rankings showed strong concordance with the safety endpoint (Spearman's correlation coefficient, Spearman's ρ>0.95), while prioritizing efficacy (weight>0.8) resulted in increasing correlations with efficacy endpoints (Spearman's ρ>0.9). In addition, ESIRA rankings were reproduced in two independent validation cohorts, further demonstrating consistency across heterogeneous datasets. By quantitatively integrating multiple clinical outcomes, ESIRA provides a structured approach to treatment selection that may better capture the trade-offs encountered in real-world clinical decision-making for advanced HCC.

Phylogenomic analyses are instrumental in enhancing our comprehension of evolutionary relationships across the tree of life. Among eukaryotes, ciliates are one of the most diverse groups of single-cell eukaryotes that play important roles in microbial food webs and have been used as important model organisms in a wide range of studies. However, evolutionary relationships within ciliates remain contentious due to low resolution of limited molecular markers and/or limited taxa. To provide a more comprehensive understanding of the evolutionary relationships within the clade Ciliophora, we sequenced the genomes and/or transcriptomes of 52 ciliate species from 10 classes. Combining analyses of these with publicly available data, we generated a dataset that comprises 190 ciliate species spanning 49 orders, encompassing 16 of the 17 recognized classes within the phylum Ciliophora, thereby achieving nearly complete class-level representation of this diverse group. Among these species, 74 were analyzed for the first time from a phylogenomic perspective. We provided an updated classification of Ciliophora, comprising the class Mesodiniea and two major clades, i.e., Postciliodesmatophora and Intramacronucleata, with Intramacronucleata comprising two main clades (CONthreeP and SLAOMP) and Protocruziea. We also evaluated the effect of missing data and provided datasets-a 200 core-gene family list and multiple sequence alignments-to facilitate future phylogenomic analyses. Furthermore, based on the highly reliable phylogenomic tree, we estimated that the phylum Ciliophora originated approximately 1,052 million years ago (Mya) at the beginning of the Meso-proterozoic period. The robust phylogenomic framework presented here not only facilitates a deeper exploration of ciliate evolution but also provides a reference for future phylogenetic and taxonomic studies.