UBR4 deficiency causes male sterility and testis abnormal in Drosophila.

Introduction: It has been established that UBR4 encodes E3 ubiquitin ligase, which determines the specificity of substrate binding during protein ubiquitination and has been associated with various functions of the nervous system but not the reproductive system. Herein, we explored the role of UBR4 on fertility with a Drosophila model. Methods: Different Ubr4 knockdown flies were established using the UAS/GAL4 activating sequence system. Fertility, hatchability, and testis morphology were studied, and bioinformatics analyses were conducted. Our results indicated that UBR4 deficiency could induce male sterility and influent egg hatchability in Drosophila. Results: We found that Ubr4 deficiency affected the testis during morphological analysis. Proteomics analysis indicated 188 upregulated proteins and 175 downregulated proteins in the testis of Ubr4 knockdown flies. Gene Ontology analysis revealed significant upregulation of CG11598 and Sfp65A, and downregulation of Pelota in Ubr4 knockdown flies. These proteins were involved in the biometabolic or reproductive process in Drosophila. These regulated proteins are important in testis generation and sperm storage promotion. Bioinformatics analysis verified that UBR4 was low expressed in cryptorchidism patients, which further supported the important role of UBR4 in male fertility. Discussion: Overall, our findings suggest that UBR4 deficiency could promote male infertility and may be involved in the protein modification of UBR4 by upregulating Sfp65A and CG11598, whereas downregulating Pelota protein expression.

Efficient strategies based on behavioral and electrophysiological methods for epilepsy-related gene screening in the Drosophila model.

Introduction: With the advent of trio-based whole-exome sequencing, the identification of epilepsy candidate genes has become easier, resulting in a large number of potential genes that need to be validated in a whole-organism context. However, conducting animal experiments systematically and efficiently remains a challenge due to their laborious and time-consuming nature. This study aims to develop optimized strategies for validating epilepsy candidate genes using the Drosophila model. Methods: This study incorporate behavior, morphology, and electrophysiology for genetic manipulation and phenotypic examination. We utilized the Gal4/UAS system in combination with RNAi techniques to generate loss-of-function models. We performed a range of behavioral tests, including two previously unreported seizure phenotypes, to evaluate the seizure behavior of mutant and wild-type flies. We used Gal4/UAS-mGFP flies to observe the morphological alterations in the brain under a confocal microscope. We also implemented patch-clamp recordings, including a novel electrophysiological method for studying synapse function and improved methods for recording action potential currents and spontaneous EPSCs on targeted neurons. Results: We applied different techniques or methods mentioned above to investigate four epilepsy-associated genes, namely Tango14, Klp3A, Cac, and Sbf, based on their genotype-phenotype correlation. Our findings showcase the feasibility and efficiency of our screening system for confirming epilepsy candidate genes in the Drosophila model. Discussion: This efficient screening system holds the potential to significantly accelerate and optimize the process of identifying epilepsy candidate genes, particularly in conjunction with trio-based whole-exome sequencing.

Evaluation of RT-LAMP Assay for Rapid Detection of SARS-CoV-2.

OBJECTIVE: To evaluate the accuracy of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in community or primary-care settings. METHOD: We systematically searched the Web of Science, Embase, PubMed, and Cochrane Library databases. We conducted quality evaluation using ReviewManager software (version 5.0). We then used MetaDisc software (version 1.4) and Stata software (version 12.0) to build forest plots, along with a Deeks funnel plot and a bivariate boxplot for analysis. RESULT: Overall, the sensitivity, specificity, and diagnostic odds ratio were 0.79, 0.97, and 328.18, respectively. The sensitivity for the subgroup with RNA extraction appeared to be higher, at 0.88 (0.86-0.90), compared to the subgroup without RNA extraction, at 0.50 (0.45-0.55), with no significant difference in specificity. CONCLUSION: RT-LAMP assay exhibited high specificity regarding current SARS-CoV-2 infection. However, its overall sensitivity was relatively moderate. Extracting RNA was found to be beneficial in improving sensitivity.

Dynamic pattern of histone H3 core acetylation in human early embryos.

After fertilization, highly differentiated sperm and oocyte are reprogrammed to totipotent embryo, which subsequently cleavages and develops into an individual through spatial-temporal differentiation. Histone modifications play critical roles to coordinate with other reprogramming events in early stages of embryogenesis. However, most of studies focus on modifications at N-terminus of histones, those at nucleosome core were not well understood. Here, we characterize the three key acetylation events in the histone H3 core, H3K56/K64/K122ac, in early human embryos. The three residues localize at DNA entry-exit position of the nucleosome. Globally, H3K56ac, H3K64ac and H3K122ac were detectable throughout preimplantation stages, with H3K64ac levels being relatively stronger and H3K122ac levels being much weaker. Besides, H3K56ac level had a peak at two-cell stage. Moreover, we found that LINEs also peak at two-cell stage, and H3K56ac was enriched at young LINE-1 in human ESCs, supporting that H3K56ac is an important driving force for young LINE-1 activation in human preimplantation embryos. Our results suggest that acetylation in the nucleosome core of histone H3 is dynamic and various during preimplantation development, and may drive diverse chromatin remodeling events in this developmental window.

Rybp orchestrates spermatogenesis via regulating meiosis and sperm motility in mice.

Ring1 and Yin Yang 1-Binding Protein (RYBP) is a member of non-canonical polycomb repressive complex 1 to mediate monoubiquitination of histone H2A at lysine 119. It plays an important role in development, but its role in reproduction remains illusive. In this study, we used Rybp conditional knockout mouse model to genetically ablate Rybp in male germ cells. We found that Rybp deficiency during spermatogenesis led to smaller testes, loss of germline cells, disturbed meiosis, increased apoptosis of spermatocytes, decreased sperm motility, and reduced global H3K9me3, without impacting retrotransposon expression. Meanwhile, we depleted Rybp during oogenesis, but oocyte maturation and preimplantation development were normal. Our findings demonstrate that RYBP plays important roles in spermatogenesis through regulating meiosis and sperm motility.

H3.3 impedes zygotic transcriptional program activated by Dux.

During development, fertilization triggers totipotency establishment, featured by zygotic genome activation/embryonic genome activation (ZGA/EGA). Mouse embryonic stem cells (mESCs) occasionally cycle through a two-cell (2C)-like status with activated expression of Dux and its targeted ZGA genes. Here, we demonstrate that deficiency of histone variant H3.3 dramatically stimulates expression of ZGA genes in mESCs. Our analysis revealed that H3.3 directly associates with Dux locus and inhibits Dux expression, therefore it is an important upstream regulator of Dux. Our finding is further supported by transcriptome change in early mouse embryos with H3.3 knockdown. We suggest that proper H3.3 level in early embryos is important to orchestrate ZGA activity for totipotency establishment.

Expert consensus for PVCR in severe, rigid and angular spinal deformity treatment: The Kunming consensus.

In the past decades, an increasing number of surgeons started using posterior vertebral column resection (PVCR) to treat severe, rigid and angular spinal deformities. Little high-level evidence is available to guide surgical treatment. The aims of our study were to identify important surgical strategies and key technical points of Chinese experts who have extensive experience in the management of severe, rigid and angular spinal deformities using PVCR, and to standardize and unify the current core concepts. Workgroups of consensus were formed by selecting nationwide representing experts and comprehensive consultations. Eight task forces for major issues were established, then retrieval of literature, collection of expert opinions and writing of review articles were carried out. A modified Delphi process was chosen in round-table forum with three face-to-face meetings. Consensus was reached with items graded more than seven points including: indications and contraindications of PVCR; review PVCR in the evolution of spinal osteotomies; The corrective mechanism and safety of spinal cord; monitoring and responses of spinal cord crisis; characteristics and therapeutic outcome of pulmonary function; management of bleeding during PVCR; relationship of pedicle screw insertion and spinal cord safety; and analysis of non-neurologic complications and prevention strategies. In conclusion, The essential properties regarding PVCR procedure are tightly linked with various factors such as medical and surgical indication, range and level of vertebral column resection, strategies of correction, corrective efficiency and control of neurological risk. PVCR is used mainly for severe, rigid spinal deformity that is not manageable by other osteotomy techniques.