Initiation of Parental Genome Reprogramming in Fertilized Oocyte by Splicing Kinase SRPK1-Catalyzed Protamine Phosphorylation.

The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes.

A Translation-Activating Function of MIWI/piRNA during Mouse Spermiogenesis.

Spermiogenesis is a highly orchestrated developmental process during which chromatin condensation decouples transcription from translation. Spermiogenic mRNAs are transcribed earlier and stored in a translationally inert state until needed for translation; however, it remains largely unclear how such repressed mRNAs become activated during spermiogenesis. We previously reported that the MIWI/piRNA machinery is responsible for mRNA elimination during late spermiogenesis in preparation for spermatozoa production. Here we unexpectedly discover that the same machinery is also responsible for activating translation of a subset of spermiogenic mRNAs to coordinate with morphological transformation into spermatozoa. Such action requires specific base-pairing interactions of piRNAs with target mRNAs in their 3' UTRs, which activates translation through coupling with cis-acting AU-rich elements to nucleate the formation of a MIWI/piRNA/eIF3f/HuR super-complex in a developmental stage-specific manner. These findings reveal a critical role of the piRNA system in translation activation, which we show is functionally required for spermatid development.

Ubiquitination-Deficient Mutations in Human Piwi Cause Male Infertility by Impairing Histone-to-Protamine Exchange during Spermiogenesis.

Genetic studies have elucidated critical roles of Piwi proteins in germline development in animals, but whether Piwi is an actual disease gene in human infertility remains unknown. We report germline mutations in human Piwi (Hiwi) in patients with azoospermia that prevent its ubiquitination and degradation. By modeling such mutations in Piwi (Miwi) knockin mice, we demonstrate that the genetic defects are directly responsible for male infertility. Mechanistically, we show that MIWI binds the histone ubiquitin ligase RNF8 in a Piwi-interacting RNA (piRNA)-independent manner, and MIWI stabilization sequesters RNF8 in the cytoplasm of late spermatids. The resulting aberrant sperm show histone retention, abnormal morphology, and severely compromised activity, which can be functionally rescued via blocking RNF8-MIWI interaction in spermatids with an RNF8-N peptide. Collectively, our findings identify Piwi as a factor in human infertility and reveal its role in regulating the histone-to-protamine exchange during spermiogenesis.

Non-O blood types are associated with a greater risk of large artery atherosclerosis stroke and dysregulation of cholesterol metabolism: an observational study.

BACKGROUND: Previous research on ABO blood types and stroke has been controversial, predominantly suggesting heightened risk of stroke in non-O blood types. Nonetheless, investigations into the correlation and underlying mechanisms between ABO blood groups and stroke subtypes, especially within Chinese cohorts, remain limited. METHODS: The ABO blood types of 9,542 ischaemic stroke (IS) patients were inferred using two ABO gene loci (c.261G > del; c.802G > A). The healthy population was derived from the 1000 Genomes Project. Patients were classified by the causative classification system (CCS). Volcano plot and gene ontology (GO) analysis were employed to explore protein differential expression among blood types. Additionally, HT29 and SW480 cell lines with downregulated ABO expression were generated to evaluate its impact on cholesterol uptake and efflux. RESULTS: A greater proportion of stroke patients had non-O blood types (70.46%) than did healthy individuals (61.54%). Notable differences in blood type distributions were observed among stroke subtypes, with non-O blood type patients mainly classified as having large artery atherosclerosis (LAA). Clinical baseline characteristics, such as the low-density lipoprotein cholesterol level, activated partial thromboplastin time and thrombin time, varied significantly among blood types. A volcano plot revealed 17 upregulated and 42 downregulated proteins in the O blood type. GO term analysis indicated that downregulated proteins were primarily associated with lipid metabolism pathways. In vitro experiments revealed that reducing ABO gene expression decreased cholesterol uptake and increased cholesterol efflux. CONCLUSIONS: This study revealed that the non-O blood type increased the risk of LAA stroke through cholesterol metabolism.

Noncanonical functions of PIWIL1/piRNAs in animal male germ cells and human diseases†.

PIWI proteins and PIWI-interacting RNAs (piRNAs) are specifically expressed in animal germlines and play essential roles during gametogenesis in animals. The primary function of PIWI/piRNAs is known to silence transposable elements for protecting genome integrity in animal germlines, while their roles beyond silencing transposons are also documented by us and others. In particular, we show that mouse PIWIL1 (MIWI)/piRNAs play a dual role in regulating protein-coding genes in mouse spermatids through interacting with different protein factors in a developmental stage-dependent manner, including translationally activating a subset of AU-rich element-containing mRNAs in round spermatids and inducing massive mRNA degradation in late spermatids. We further show that MIWI is eliminated through the ubiquitin-26S proteasome pathway during late spermiogenesis. By exploring the biological function of MIWI ubiquitination by APC/C, we identified ubiquitination-deficient mutations in human PIWIL1 of infertile men and further established their causative role in male infertility in mouse model, supporting PIWIL1 as a human male infertility-relevant gene. Additionally, we reported that PIWIL1, aberrantly induced in human tumors, functions as an oncoprotein in a piRNA-independent manner in cancer cells. In the current review, we summarize our latest findings regarding the roles and mechanisms of PIWIL1 and piRNAs in mouse spermatids and human diseases, and discuss the related works in the field.

RBFox2-miR-34a-Jph2 axis contributes to cardiac decompensation during heart failure.

Heart performance relies on highly coordinated excitation-contraction (EC) coupling, and defects in this critical process may be exacerbated by additional genetic defects and/or environmental insults to cause eventual heart failure. Here we report a regulatory pathway consisting of the RNA binding protein RBFox2, a stress-induced microRNA miR-34a, and the essential EC coupler JPH2. In this pathway, initial cardiac defects diminish RBFox2 expression, which induces transcriptional repression of miR-34a, and elevated miR-34a targets Jph2 to impair EC coupling, which further manifests heart dysfunction, leading to progressive heart failure. The key contribution of miR-34a to this process is further established by administrating its mimic, which is sufficient to induce cardiac defects, and by using its antagomir to alleviate RBFox2 depletion-induced heart dysfunction. These findings elucidate a potential feed-forward mechanism to account for a critical transition to cardiac decompensation and suggest a potential therapeutic avenue against heart failure.

Differences in driver genes between smoking-related and non-smoking-related lung cancer in the Chinese population.

Recently, non-smoking-related lung cancer was classified as an independent disease entity because it is different from tobacco-associated lung cancer. Non-smoking-related lung cancer occurs more often in women than men, and the predominant histological type is adenocarcinoma (ADC) rather than squamous cell carcinoma. Most of the driver gene alterations that have been identified in ADC in never-smokers include epidermal growth factor receptor mutations, KRAS mutations, echinoderm microtubule-associated protein like 4/anaplastic lymphoma kinase fusion, and ROS1 fusion, among others. Meanwhile, significant progress has been made in the treatment of ADC. However, in comparison with ADC, no such available molecular targets exist for smoking-associated lung cancer, for which treatment strategies are limited. Next-generation sequencing has been widely applied to the discovery of more genetic profiles of lung cancers. This review summarizes the differences between smoking-related and non-smoking-related lung cancer as follows: different somatic mutation burdens, C:G→A:T transversions, common and novel driver genes, and treatment strategies. Overall, smoking-related lung cancer is more complicated than non-smoking-related lung cancer. Furthermore, we review the prevalence of driver genes in smoking-associated and non-smoking-associated lung cancers in the Chinese population.

Human testis-expressed sequence 101 is limitedly distributed in germinal epithelium of testis and disappears in seminoma.

BACKGROUND: Testis-expressed sequence 101 (TEX101) was found to be highly expressed in testis and involved in acrosome reaction in previous studies. Recently, the metastasis suppressor function of TEX101 in cancer was disclosed, but the comprehensive investigation of its expression has rarely been reported. In this study, the expression features of TEX101 in normal human organs and seminoma were systematically analyzed. RESULTS: Immunohistochemistry demonstrated intense staining of TEX101 in human testis tissues; however, its expression in 27 other types of normal human organs, including the ovary, was negligible. Higher expression of TEX101 was observed in the spermatocytes and spermatids of the testis, but relatively lower staining was detected in spermatogonia. Western blotting showed a single TEX101 band of 38 kDa in human testis, but it did not correspond to the predicted molecular weight of its mature form at 21 KDa. Furthermore, we examined seminoma tissues by immunohistochemistry and found that none of the 36 samples expressed TEX101. CONCLUSIONS: Our data confirmed TEX101 to be a testis protein that could be related to the maturation process of male germ cells. The lack of TEX101 in seminoma indicated its potential role in tumor progression. This characteristic expression of TEX101 could provide a valuable reference for understanding its biological functions.

WNT16B from ovarian fibroblasts induces differentiation of regulatory T cells through ß-catenin signal in dendritic cells.

Treatment for cancer can induce a series of secreted factors into the tumor microenvironment, which can affect cancer progression. Wingless-type MMTV (mouse mammary tumor virus) integration site 16B (WNT16B) is a new member of the WNT family and has been reported to play growth-related roles in previous studies. In this study, we found WNT16B could be expressed and secreted into the microenvironment by human ovarian fibroblasts after DNA damage-associated treatment, including chemotherapy drugs and radiation. We also demonstrated that fibroblast-derived WNT16B could result in accumulation of ß-catenin in dendritic cells and secretion of interleukin-10 (IL-10) and transforming growth factor beta (TGF-ß), which contributed to the differentiation of regulatory T cells in a co-culture environment. These results shed light on the roles of WNT16B in immune regulation, especially in regard to cancer treatment.

Small RNAs: An expanding world with therapeutic promises.

Small non-coding RNAs (sncRNAs), such as microRNAs (miRNAs), small interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs), and transfer RNA (tRNA)-derived small RNAs (tsRNAs), play essential roles in regulating various cellular and developmental processes. Over the past three decades, researchers have identified novel sncRNA species from various organisms. These molecules demonstrate dynamic expression and diverse functions, and they are subject to intricate regulation through RNA modifications in both healthy and diseased states. Notably, certain sncRNAs in gametes, particularly sperm, respond to environmental stimuli and facilitate epigenetic inheritance. Collectively, the in-depth understanding of sncRNA functions and mechanisms has accelerated the development of small RNA-based therapeutics. In this review, we present the recent advances in the field, including new sncRNA species and the regulatory influences of RNA modifications. We also discuss the current limitations and challenges associated with using small RNAs as either biomarkers or therapeutic drugs.

The PIWI-specific insertion module helps load longer piRNAs for translational activation essential for male fertility.

PIWI-clade proteins harness piRNAs of 24-33 nt in length. Of great puzzles are how PIWI-clade proteins incorporate piRNAs of different sizes and whether the size matters to PIWI/piRNA function. Here we report that a PIWI-Ins module unique in PIWI-clade proteins helps define the length of piRNAs. Deletion of PIWI-Ins in Miwi shifts MIWI to load with shorter piRNAs and causes spermiogenic failure in mice, demonstrating the functional importance of this regulatory module. Mechanistically, we show that longer piRNAs provide additional complementarity to target mRNAs, thereby enhancing the assembly of the MIWI/eIF3f/HuR super-complex for translational activation. Importantly, we identify a c.1108C>T (p.R370W) mutation of HIWI (human PIWIL1) in infertile men and demonstrate in Miwi knock-in mice that this genetic mutation impairs male fertility by altering the property of PIWI-Ins in selecting longer piRNAs. These findings reveal a critical role of PIWI-Ins-ensured longer piRNAs in fine-tuning MIWI/piRNA targeting capacity, proven essential for spermatid development and male fertility.

Expression of CD40 and growth-inhibitory activity of CD40 agonist in ovarian carcinoma cells.

The CD40 receptor is a member of the tumour necrosis factor receptor family and is widely expressed on various cell types. The antitumour activity of CD40 agonist antibody has been observed in B-cell-derived malignancies, but its activity on ovarian cancer remains unclear. However, in this paper, we first confirmed that the anti-CD40 agonist antibody could inhibit the growth of ovarian cancer cells and induce apoptosis. This study investigated the expression of CD40 by ovarian carcinoma tissues and cell lines, at the same time, we evaluated the effect of a recombinant soluble human CD40L (rshCD40L) and an anti-CD40 agonist antibody on cell growth and apoptosis. Flow cytometry and immunohistochemistry assay demonstrated that CD40 was expressed on ovarian carcinoma cell lines and primary ovarian carcinoma cells derived from ascites, as well as on ovarian carcinoma tissues. The growth inhibition of rshCD40L and the anti-CD40 agonist antibody on ovarian carcinoma cells was examined by MTT assay, and the proportion of apoptotic tumour cells was analysed by flow cytometry and Hoechst staining. Our study showed that CD40 was expressed on all ovarian carcinoma cell lines and was examined in 86.2% (162/188) of ovarian cancer tissue samples, but not in normal ovarian tissues (n = 20). Treatment with rshCD40L or anti-CD40 agonist antibody significantly inhibited ovarian carcinoma cell growth and induced apoptosis. Theses results suggest that CD40 is expressed on ovarian carcinoma cells, moreover, that rshCD40L and anti-CD40 agonist antibody have therapeutic potential to inhibit human ovarian cancer growth.

A fully human CD19/CD3 bi-specific antibody triggers potent and specific cytotoxicity by unstimulated T lymphocytes against non-Hodgkin's lymphoma.

The use of a bi-specific antibody (BsAb) is an attractive and specific approach to cancer therapy. We have constructed a fully human recombinant single chain Fv BsAb against CD19 and CD3 that was an effective treatment in an animal model of non-Hodgkin's lymphoma (NHL). The CD19/CD3 BsAb was expressed in CHO cells and purified by Ni-column chromatography. Flow cytometry revealed that the CD19/CD3 BsAb specifically bound to both CD19 and CD3-positive cells. In vitro, the CD19/CD3 BsAb could stimulate T cell proliferation and induce the lysis of cultured Raji cells in the presence of unstimulated T lymphocytes. In vivo, the CD19/CD3 BsAb efficiently inhibited tumour growth in SCID mice of NHL, and the survival time of the mice was significantly prolonged. Therefore, our CD19/CD3 BsAb is a useful tool that could be a suitable candidate for treatment of NHL.

LLPS of FXR1 drives spermiogenesis by activating translation of stored mRNAs.

Postmeiotic spermatids use a unique strategy to coordinate gene expression with morphological transformation, in which transcription and translation take place at separate developmental stages, but how mRNAs stored as translationally inert messenger ribonucleoproteins in developing spermatids become activated remains largely unknown. Here, we report that the RNA binding protein FXR1, a member of the fragile X-related (FXR) family, is highly expressed in late spermatids and undergoes liquid-liquid phase separation (LLPS) to merge messenger ribonucleoprotein granules with the translation machinery to convert stored mRNAs into a translationally activated state. Germline-specific Fxr1 ablation in mice impaired the translation of target mRNAs and caused defective spermatid development and male infertility, and a phase separation-deficient FXR1L351P mutation in Fxr1 knock-in mice produced the same developmental defect. These findings uncover a mechanism for translational reprogramming with LLPS as a key driver in spermiogenesis.

Polyclonal rabbit anti-murine plasmacytoma cell globulins induce myeloma cells apoptosis and inhibit tumour growth in mice.

Multiple myelomas (MMs) are etiologically heterogeneous and there are limited treatment options; indeed, current monoclonal antibody therapies have had limited success, so more effective antibodies are urgently needed. Polyclonal antibodies are a possible alternative because they target multiple antigens simultaneously. In this study, we produced polyclonal rabbit anti-murine plasmacytoma cell immunoglobulin (PAb) by immunizing rabbits with the murine plasmacytoma cell line MPC-11. The isolated PAb bound to plasma surface antigens in several MM cell lines, inhibited their proliferation as revealed by MTT assay, and induce apoptosis as indicated by flow cytometry, microscopic observation of apoptotic changes in morphology, and DNA fragmentation on agarose gels. The cytotoxicity of PAb on MPC-11 cell lines was both dose-dependent and time-dependent; PAb exerted a 50% inhibitory effect on MPC-11 cell viability at a concentration of 200 µg/ml in 48 h. Flow cytometry demonstrated that PAb treatment significantly increased the number of apoptotic cells (48.1%) compared with control IgG (8.3%). Apoptosis triggered by PAb was confirmed by activation of caspase-3, -8, and -9. Serial intravenous or intraperitoneal injections of PAb inhibited tumour growth and prolonged survival in mice bearing murine plasmacytoma, while TUNEL assay demonstrated that PAb induced statistically significant apoptosis (P < 0.05) compared to control treatments. We conclude that PAb is an effective agent for in vitro and in vivo induction of apoptosis in multiple myeloma and that exploratory clinical trials may be warranted.

Small Noncoding RNAs in Reproduction and Infertility.

Infertility has been reported as one of the most common reproductive impairments, affecting nearly one in six couples worldwide. A large proportion of infertility cases are diagnosed as idiopathic, signifying a deficit in information surrounding the pathology of infertility and necessity of medical intervention such as assisted reproductive therapy. Small noncoding RNAs (sncRNAs) are well-established regulators of mammalian reproduction. Advanced technologies have revealed the dynamic expression and diverse functions of sncRNAs during mammalian germ cell development. Mounting evidence indicates sncRNAs in sperm, especially microRNAs (miRNAs) and transfer RNA (tRNA)-derived small RNAs (tsRNAs), are sensitive to environmental changes and mediate the inheritance of paternally acquired metabolic and mental traits. Here, we review the critical roles of sncRNAs in mammalian germ cell development. Furthermore, we highlight the functions of sperm-borne sncRNAs in epigenetic inheritance. We also discuss evidence supporting sncRNAs as promising biomarkers for fertility and embryo quality in addition to the present limitations of using sncRNAs for infertility diagnosis and treatment.

Periplocin inhibits growth of lung cancer in vitro and in vivo by blocking AKT/ERK signaling pathways.

Periplocin is one of cardenolides isolated from cortex periplocae which is used for treatment of rheumatoid arthritis and reinforcement of bones and tendons in traditional medicine. Here, we investigated the anti-tumor activity of periplocin against lung cancer cells bothin vitro and in vivo, and explored its anti-cancer mechanism. Periplocin inhibited the growth of lung cancer cells and induced their apoptosis in time- and dose-dependent manners by cell cycle arrest in G0/G1 phase. Periplocin exhibited anti-tumor activity both in human (A549) and mouse (LL/2) lung cancer xenograft models. Immunohistochemical analysis revealed that intratumoral angiogenesis was significantly suppressed. Furthermore, anti-cancer activity mediated by periplocin was associated with decreased level of phosphorylated AKT and ERK both in vitro and in vivo, which were important for cell growth and survival. Moreover, periplocin induced apoptosis by downregulating Bcl-2 and upregulating Bax, leading to activation of caspase-3 and caspase-9. These findings suggested that periplocin could inhibit the growth of lung cancer both in vitro and in vivo, which could be attributed to the inhibition of proliferation and the induction of apoptosis signaling pathway, such as AKT and ERK. These observations provide further evidence on the anti-tumor effect of periplocin, and it may be of importance to further explore its potential role as a therapeutic agent for cancer.

Identification, characterization, and effects of Xenopus laevis PNAS-4 gene on embryonic development.

Apoptosis plays an important role in embryonic development. PNAS-4 has been demonstrated to induce apoptosis in several cancer cells. In this study, we cloned Xenopus laevis PNAS-4 (xPNAS-4), which is homologous to the human PNAS-4 gene. Bioinformatics analysis for PNAS-4 indicated that xPNAS-4 shared 87.6% identity with human PNAS-4 and 85.5% with mouse PNAS-4. The phylogenetic tree of PNAS-4 protein was also summarized. An analysis of cellular localization using an EGFP-fused protein demonstrated that xPNAS-4 was localized in the perinuclear region of the cytoplasm. RT-PCR analysis revealed that xPNAS-4, as a maternally expressed gene, was present in all stages of early embryo development. Whole-mount in situ hybridization showed that xPNAS-4 was mainly expressed in ectoderm and mesoderm. Furthermore, microinjection of xPNAS-4 mRNA in vivo caused developmental defects manifesting as a small eye phenotype in the Xenopous embryos, and as a small eye or one-eye phenotype in developing zebrafish embryos. In addition, embryos microinjected with xPNAS-4 antisense morpholino oligonucleotides (MOs) exhibited a failure of head development and shortened axis.

Knockout of glutathione peroxidase 5 down-regulates the piRNAs in the caput epididymidis of aged mice.

The mammalian epididymis not only plays a fundamental role in the maturation of spermatozoa, but also provides protection against various stressors. The foremost among these is the threat posed by oxidative stress, which arises from an imbalance in reactive oxygen species and can elicit damage to cellular lipids, proteins, and nucleic acids. In mice, the risk of oxidative damage to spermatozoa is mitigated through the expression and secretion of glutathione peroxidase 5 (GPX5) as a major luminal scavenger in the proximal caput epididymidal segment. Accordingly, the loss of GPX5-mediated protection leads to impaired DNA integrity in the spermatozoa of aged Gpx5-/- mice. To explore the underlying mechanism, we have conducted transcriptomic analysis of caput epididymidal epithelial cells from aged (13 months old) Gpx5-/- mice. This analysis revealed the dysregulation of several thousand epididymal mRNA transcripts, including the downregulation of a subgroup of piRNA pathway genes, in aged Gpx5-/- mice. In agreement with these findings, we also observed the loss of piRNAs, which potentially bind to the P-element-induced wimpy testis (PIWI)-like proteins PIWIL1 and PIWIL2. The absence of these piRNAs was correlated with the elevated mRNA levels of their putative gene targets in the caput epididymidis of Gpx5-/- mice. Importantly, the oxidative stress response genes tend to have more targeting piRNAs, and many of them were among the top increased genes upon the loss of GPX5. Taken together, our findings suggest the existence of a previously uncharacterized somatic piRNA pathway in the mammalian epididymis and its possible involvement in the aging and oxidative stress-mediated responses.