[Delivery of epididymis-specific mRNAs from mouse epididymosomes into N2a and TM4 cells].

OBJECTIVE: To investigate whether mouse epididymis-specific mRNAs Adam7 and Crisp1 can be delivered into N2a and TM4 cells, and to provide an experimental basis for exploring the function of epididymal mRNAs. METHODS: Using RT-PCR, we detected the presence of epididymis-specific genes (Adam7, Crisp1, Defb22, Wfdc2, and Wfdc9) in the testis, epididymis, epididymosome and sperm of adult male BALB/c mice as well as in the human testis, seminal vesicles and sperm. We isolated epididymosomes of BALB/c mice by low-speed centrifugation, filtration and ultracentrifugation, fluorescently labeled them by PKH26, co-incubated them for 1 hour with the N2a and TM4 cells after 24 hours of starvation culture, and observed whether they were fused with the N2a and TM4 cells and ingested using the epididymosomes without PKH26 labeling, PKH26 dye without epididymosomes, and non- epididymosome or -PKH26 dye as controls. Then we detected the epididymis-specific genes in the N2a and TM4 cells after 1-hour co-incubation by RT-PCR. RESULTS: Adam7 and Crisp1 were present in the mouse epididymis, epididymosomes and sperm, and in the human seminal vesicles and sperm as well, but not in the testes of either the mice or men. PKH26 and Hoechst33258 fluorescence double-labeling showed that the mouse epididymosomes were fused with the N2a and TM4 cells and ingested; RT-PCR revealed the mRNAs of Adam7 and Crisp1 in the N2a and TM4 cells after 1-hour co-incubation; and Western blot exhibited the CRISP1 protein in the N2a and TM4 cells incubated with epididymosomes. CONCLUSION: Epididymosomes can deliver epididymis-specific mRNAs Adam7 and Crisp1 into N2a and TM4 cells, where Crisp1 may be translated into proteins, though their function and significance need to be further studied.

Is differences in embryo morphokinetic development significantly associated with human embryo sex?†.

To date, very few studies have reported on the relationship between live birth gender and embryo development kinetics. This study included 1735 women undergoing in vitro fertilization or intracytoplasmic sperm injection by using a time-lapse system. Finally, a total of 228 qualified patients with 100% implantation and known live birth information were included in the analysis. There were 174 male live births and 134 female live births. The time to 3 (t3), 4 (t4), and 5 (t5) cell development of male embryos was significantly shorter/earlier than female embryos (P < 0.05). The duration of the second cell cycle (cc2) in male embryos was significantly shorter than female embryos (P = 0.002). Multivariate logistic regression showed that only t3 had a significant correlation with live birth gender; the odds ratio (OR) was 0.786, 95% confidence interval (CI) was 0.625-0.988 (P < 0.05). When morphokinetic parameters were divided into groups based on quartiles, embryos within the sex ranges were observed to have significantly different proportions of male and female live births (P < 0.05). The results showed that t3 (<14 h) was the most relevant parameter related to live birth gender (OR 2.452, 95% CI 1.071-5.612, P = 0.03). These findings support the idea that embryo morphokinetic parameters were affected by the sex of the embryo. Currently, embryologists use embryo morphokinetics to establish models of development, in order to improve accurate selection of viable embryos. Thus, this factor needs to be considered when embryologists use embryo morphokinetics to select embryos.

A Possible Role for Long Interspersed Nuclear Elements-1 (LINE-1) in Huntington's Disease Progression.

BACKGROUND Recent studies have shown that increased mobilization of Long Interspersed Nuclear Elements-1 (L1) can promote the pathophysiology of multiple neurological diseases. However, its role in Huntington's disease (HD) remains unknown. MATERIAL AND METHODS R6/2 mice - a common mouse model of HD - were used to evaluate changes in L1 mobilization. Pyrosequencing was used to evaluate methylation content changes. L1-ORF1 and L1-ORF2 expression analysis were evaluated by RT-PCR and immunoblotting. Changes in pro-survival signaling were evaluated by L1-ORF overexpression studies and validated in the mouse model by immunohistochemistry and immunoblotting. RESULTS We found an increased mobilization of L1 elements in the caudate genome of R6/2 mice (p<0.05) - a common mouse model of HD - but not in wild-type mice. Subsequent pyrosequencing and expression analysis showed that the L1 elements were hypomethylated and their respective ORFs were overexpressed in the affected tissues. In addition, a significant decrease in the pro-survival proteins such as the phosphoproteins of AKT target proteins, mTORC1 activity, and AMPK alpha levels was observed with the increase in the expression L1-ORF2. CONCLUSIONS These findings indicate that hyperactive retrotransposition of L1 triggers a downstream signaling pathway affecting the neuronal survival pathways via downregulation of mTORC1 activity and AMPKalpha and increasing apoptosis in neurons.

Structural dynamics control the MicroRNA maturation pathway.

MicroRNAs (miRNAs) are crucial gene expression regulators and first-order suspects in the development and progression of many diseases. Comparative analysis of cancer cell expression data highlights many deregulated miRNAs. Low expression of miR-125a was related to poor breast cancer prognosis. Interestingly, a single nucleotide polymorphism (SNP) in miR-125a was located within a minor allele expressed by breast cancer patients. The SNP is not predicted to affect the ground state structure of the primary transcript or precursor, but neither the precursor nor mature product is detected by RT-qPCR. How this SNP modulates the maturation of miR-125a is poorly understood. Here, building upon a model of RNA dynamics derived from nuclear magnetic resonance studies, we developed a quantitative model enabling the visualization and comparison of networks of transient structures. We observed a high correlation between the distances between networks of variants with that of their respective wild types and their relative degrees of maturation to the latter, suggesting an important role of transient structures in miRNA homeostasis. We classified the human miRNAs according to pairwise distances between their networks of transient structures.

MicroRNA-277 modulates the neurodegeneration caused by Fragile X premutation rCGG repeats.

Fragile X-associated tremor/ataxia syndrome (FXTAS), a late-onset neurodegenerative disorder, has been recognized in older male fragile X premutation carriers and is uncoupled from fragile X syndrome. Using a Drosophila model of FXTAS, we previously showed that transcribed premutation repeats alone are sufficient to cause neurodegeneration. MiRNAs are sequence-specific regulators of post-transcriptional gene expression. To determine the role of miRNAs in rCGG repeat-mediated neurodegeneration, we profiled miRNA expression and identified selective miRNAs, including miR-277, that are altered specifically in Drosophila brains expressing rCGG repeats. We tested their genetic interactions with rCGG repeats and found that miR-277 can modulate rCGG repeat-mediated neurodegeneration. Furthermore, we identified Drep-2 and Vimar as functional targets of miR-277 that could modulate rCGG repeat-mediated neurodegeneration. Finally, we found that hnRNP A2/B1, an rCGG repeat-binding protein, can directly regulate the expression of miR-277. These results suggest that sequestration of specific rCGG repeat-binding proteins could lead to aberrant expression of selective miRNAs, which may modulate the pathogenesis of FXTAS by post-transcriptionally regulating the expression of specific mRNAs involved in FXTAS.

Retrotransposon activation contributes to fragile X premutation rCGG-mediated neurodegeneration.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder associated with fragile X premutation carriers. Previous studies have shown that fragile X rCGG repeats are sufficient to cause neurodegeneration and that the rCGG-repeat-binding proteins Pur α and heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 could modulate rCGG-mediated neuronal toxicity. Mobile genetic elements or their remnants populate the genomes, and the activities of these elements are tightly controlled for the fitness of host genomes in different organisms. Here we provide both biochemical and genetic evidence to show that the activation of a specific retrotransposon, gypsy, can modulate rCGG-mediated neurodegeneration in an FXTAS Drosophila model. We find that one of the rCGG-repeat-binding proteins, hnRNP A2/B1, is involved in this process via interaction with heterochromatin protein 1. Knockdown of gypsy RNA by RNAi could suppress the neuronal toxicity caused by rCGG repeats. These data together point to a surprisingly active role for retrotransposition in neurodegeneration.

Fragile X premutation RNA is sufficient to cause primary ovarian insufficiency in mice.

Spontaneous 46,XX primary ovarian insufficiency (POI), also known as 'premature menopause' or 'premature ovarian failure', refers to ovarian dysfunction that results in a range of abnormalities, from infertility to early menopause as the end stage. The most common known genetic cause of POI is the expansion of a CGG repeat to 55-199 copies (premutation) in the 5' untranslated region in the X-linked fragile X mental retardation 1 (FMR1) gene. POI associated with the FMR1 premutation is referred to as fragile X-associated POI (FXPOI). Here, we characterize a mouse model carrying the human FMR1 premutation allele and show that FMR1 premutation RNA can cause a reduction in the number of growing follicles in ovaries and is sufficient to impair female fertility. Alterations in selective serum hormone levels, including FSH, LH and 17ß-estradiol, are seen in this mouse model, which mimics findings in humans. In addition, we also find that LH-induced ovulation-related gene expression is specifically altered. Finally, we show that the FMR1 premutation allele can lead to reduced phosphorylation of Akt and mTOR proteins. These results together suggest that FMR1 premutation RNA could cause the POI associated with FMR1 premutation carriers, and the Akt/mTOR pathway may serve as a therapeutic target for FXPOI.

Emerging gene editing in industrial microbiology beyond CRISPR-Cas9.

The CRISPR-Cas9 system has been widely applied for industrial microbiology but is not effective in certain microorganisms. This forum explores the strategies aimed at overcoming these challenges, including the use of the Cas12a system, Cas9 variants, and non-CRISPR techniques, to provide more effective strategies for expanding applications in microbial engineering.

Novel compound heterozygous mutations in DNAH1 cause primary infertility in Han Chinese males with multiple morphological abnormalities of the sperm flagella.

This study aimed to identify genetic causes responsible for multiple morphological abnormalities of the sperm flagella (MMAF) in the Han Chinese population. Three primary infertile males with completely immobile sperm and MMAF were enrolled. Whole-exome sequencing and Sanger sequencing were performed to identify disease-causing genes. Subsequently, morphological and ultrastructural analyses of sperm flagella were investigated. The probable impact of genetic variants on protein function was analyzed by online bioinformatic tools and immunofluorescence assay. Three patients with dynein axonemal heavy chain 1 (DNAH1) gene compound heterozygous variations were identified. DNAH1 c.7435C>T, p.R2479X and c.10757T>C, p.F3586S were identified in the patient from Family 1, c.11726_11727delCT, p.P3909fs and c.12154delC, p.L4052fs were found in the patient from Family 2, and c.10627-3C>G and c.11726_11727delCT, p.P3909fs existed in the patient from Family 3. Four of these variations have not been reported, and all the mutations showed pathogenicity by functional effect predictions. The absence of the center pair and disorganization of the fibrous sheath were present in sperm flagella at the ultrastructural level. Moreover, the expression of DNAH1 was absent in spermatozoa from the participants, validating the pathogenicity of the variants. All three couples have undergone intracytoplasmic sperm injection (ICSI), and two couples of them became pregnant after the treatment. In conclusion, the newly identified DNAH1 mutations can expand the mutational and phenotypic spectrum of MMAF genes and provide a theoretical basis for genetic diagnosis in MMAF patients. It is recommended to conduct genetic screening in male infertility patients with MMAF and provide rational genetic counseling, and ICSI might be an optimal strategy to help with fertilization and conception for patients with DNAH1 mutations.

Engineering Komagataella phaffii to biosynthesize cordycepin from methanol which drives global metabolic alterations at the transcription level.

Cordycepin has the potential to be an alternative to the disputed herbicide glyphosate. However, current laborious and time-consuming production strategies at low yields based on Cordyceps militaris lead to extremely high cost and restrict its application in the field of agriculture. In this study, Komagataella phaffii (syn. Pichia pastoris) was engineered to biosynthesize cordycepin from methanol, which could be converted from CO2. Combined with fermentation optimization, cordycepin content in broth reached as high as 2.68 ± 0.04 g/L within 168 h, around 15.95 mg/(L·h) in productivity. Additionally, a deaminated product of cordycepin was identified at neutral or weakly alkaline starting pH during fermentation. Transcriptome analysis found the yeast producing cordycepin was experiencing severe inhibition in methanol assimilation and peroxisome biogenesis, responsible for delayed growth and decreased carbon flux to pentose phosphate pathway (PPP) which led to lack of precursor supply. Amino acid interconversion and disruption in RNA metabolism were also due to accumulation of cordycepin. The study provided a unique platform for the manufacture of cordycepin based on the emerging non-conventional yeast and gave practical strategies for further optimization of the microbial cell factory.

Emotions, COVID-19 related thoughts and satisfaction with life during the critical period from control to relaxation.

Introduction: In the context of declining mortality rates and increasing infectivity, it has become unavoidable for the majority of individuals to experience a COVID-19 infection at some point. This study aimed to investigate the psychological well-being of the general population during China's transition period from strict control measures to relaxed policies in COVID-19 prevention and control, as well as the impact of COVID-19 related thoughts on emotion and life satisfaction during widespread infections. Methods: A cross-sectional study was conducted involving a sample size of 1578 participants. Participants completed self-report questionnaires assessing positive and negative emotions, thoughts about COVID-19, and satisfaction with life. Demographic characteristics such as sex, age, and education level were controlled for in the analysis. Results: The findings revealed that individuals who had been infected with COVID-19 (specifically the Omicron variant BA.5.2 or BF.7) reported lower levels of positive emotions compared to those who were uninfected or had recovered from the infection. There was a significant relationship between COVID-19-related thoughts, emotions, and life satisfaction. Positive COVID-19 related thoughts were found to mediate the relationship between negative emotions and satisfaction with life. Discussion: This study represents a comprehensive examination conducted in China, focusing on assessing the impact of the COVID-19 pandemic on the general population during the critical transition period from control to relaxation. Throughout this period, the number of infections experienced fluctuations, initially rising but eventually declining over a one-month span. In such a momentous historical period, maintaining a positive perspective on COVID-19 and its management becomes paramount in enhancing the emotional well-being, life satisfaction and overall well-being of individuals.

Stable isotope labeling with amino acids in Drosophila for quantifying proteins and modifications.

Drosophila melanogaster is a common animal model for genetics studies, and quantitative proteomics studies of the fly are emerging. Here, we present in detail the development of a procedure to incorporate stable isotope-labeled amino acids into the fly proteome. In the method of stable isotope labeling with amino acids in Drosophila melanogaster (SILAC fly), flies were fed with SILAC-labeled yeast grown with modified media, enabling near complete labeling in a single generation. Biological variation in the proteome among individual flies was evaluated in a series of null experiments. We further applied the SILAC fly method to profile proteins from a model of fragile X syndrome, the most common cause of inherited mental retardation in human. The analysis identified a number of altered proteins in the disease model, including actin-binding protein profilin and microtubulin-associated protein futsch. The change of both proteins was validated by immunoblotting analysis. Moreover, we extended the SILAC fly strategy to study the dynamics of protein ubiquitination during the fly life span (from day 1 to day 30), by measuring the level of ubiquitin along with two major polyubiquitin chains (K48 and K63 linkages). The results show that the abundance of protein ubiquitination and the two major linkages do not change significantly within the measured age range. Together, the data demonstrate the application of the SILAC principle in D. melanogaster, facilitating the integration of powerful fly genomics with emerging proteomics.

MiR-3666 serves as a tumor suppressor in ovarian carcinoma by down-regulating AK4 via targeting STAT3.

As a result of metastasis and high recurrence, ovarian carcinoma (OC) is one of the most frequent gynecological carcinomas affecting women up to now. In spite of advances in OC treatments, the molecular mechanisms underlying OC progression are still needed to be deeply understood. MicroRNAs (miRNAs) with aberrant expressions are widely known to regulate target genes so as to mediate diverse biological activities of tumor cells. In the present study, we inspected the expression profile and latent mechanism of miR-3666 in OC. First of all, our research revealed the down-regulated miR-3666 in OC cells. Furthermore, miR-3666 up-regulation could repress cell proliferation and migration as well as induce cell apoptosis in OC. In addition, we unmasked that miR-3666 targeted STAT3 (signal transducer and activator of transcription 3) and further down-regulated STAT3 expression. Moreover, adenylate kinase 4 (AK4) was transcriptionally enhanced by STAT3, and then miR-3666 restrained AK4 expression by mediating STAT3. In the end, rescue experiments depicted that miR-3666 suppressed the development of OC via STAT3-mediated AK4. We uncovered that miR-3666 inhibited the tumorigenesis and even development of OC via suppressing STAT3/AK4 axis, offering a novel biomarker and therapeutic target for OC.

Effects of mutant huntingtin on mGluR5-mediated dual signaling pathways: implications for therapeutic interventions.

Glutamate excitotoxicity is thought to play an important role in Huntington's disease (HD), which is caused by a polyglutamine expansion in the HD protein huntingtin (htt). Overactivation of group I metabotropic glutamate receptors (mGluRs), which include mGluR1 as well as mGluR5 and are coupled via phospholipase C to the inositol phosphate pathway, is found to be involved in mutant htt-mediated neurotoxicity. However, activation of mGluR5 also leads to neuronal protection. Here, we report that mutant htt can activate both mGluR5-mediated ERK and JNK signaling pathways. While increased JNK signaling causes cell death, activation of ERK signaling pathway is protective against cell death. Expression of mutant htt in cultured cells causes greater activation of JNK than ERK. These findings suggest that selective inhibition of the JNK signaling pathway may offer an effective therapeutic approach for reducing htt-mediated excitotoxicity.

The Effect of Intrauterine Administration of Human Chorionic Gonadotropin (hCG) Before Embryo Transfer During Assisted Reproductive Cycles: a Meta-Analysis of Randomized Controlled Trials.

The fertility success rates of clinical and laboratory-assisted reproductive techniques (ART) remain low, despite major advances. The aim of this study was to conduct a systematic literature review and assess whether the intrauterine administration of human chorionic gonadotropin (hCG) before embryo transfer (ET) improved the clinical outcomes of sub-fertile women undergoing assisted reproduction. The electronic databases PUBMED, EMBASE and Web of Science were systematically searched for randomized controlled trials (RCTs) published from inception to June 2018. The trial data were independently extracted and analyzed using risk ratios (RRs) and 95% confidence intervals (CIs) according to a random- or fixed-effect model (as appropriate), and a meta-analysis was conducted using Review Manager 5.2 software. The meta-analysis included 3241 patients from 12 RCTs, and the combined results demonstrated that intrauterine hCG injection significantly improved the rates of clinical (RR = 1.33; 95% CI: 1.12 - 1.58) and ongoing pregnancy (RR = 1.87; 95% CI: 1.54 - 2.27), compared with controls. However, intrauterine hCG injection had no significant effect on the implantation rate (RR = 1.30; 95% CI: 0.89 - 1.90), abortion rate (RR = 1.06; 95% CI: 0.78 - 1.44), ectopic pregnancy rate (RR = 0.77; 95% CI: 0.17 - 3.42) or live birth rate (RR = 0.99; 95% CI: 0.60 - 1.63). In a subgroup analysis, the intrauterine injection of > 500 IU hCG led to a significant increase in the implantation rate (RR = 1.64; 95% CI: 1.04 - 2.61) relative to controls. Furthermore, the subgroup of women with cleavage-stage ETs who received an intracavity injection of hCG (IC-hCG) exhibited increases in the implantation, clinical pregnancy and ongoing pregnancy rates, compared to women with cleavage-stage ETs and no IC-hCG. The current evidence indicates that intrauterine hCG administration before ET provides an advantage in terms of the clinical pregnancy and ongoing pregnancy rates.

Alterations of testis-specific promoter methylation in cell-free seminal deoxyribonucleic acid of idiopathic nonobstructive azoospermic men with different testicular phenotypes.

OBJECTIVE: To explore the feasibility of quantification of testicular DNA methylation from cell-free seminal DNA (cfsDNA), and analyze promoter methylation alterations in men with idiopathic nonobstructive azoospermia (NOA). DESIGN: Comparison between testicular DNA and paired cfsDNA, and among NOA patients with different testicular phenotypes. SETTING: Academic research institute and andrology practice. PATIENT(S): Eighty-eight idiopathic NOA patients with different testicular phenotypes and 24 normozoospermic men. INTERVENTION(S): Testicular biopsies and semen analysis. MAIN OUTCOME MEASURE(S): Five testis-specific methylated promoters were selected. Promoter methylation was quantified using MethyLight in testicular DNA and paired cfsDNA, and the mRNA level was determined by real-time quantitative polymerase chain reaction. RESULT(S): Correlations of methylation of the selected five promoters between testicular DNA and paired cfsDNA were observed; and promoter methylation was negatively related to the messenger RNA level in testis. The cfsDNA methylation of these promoters showed different dynamic changes among the subtypes of NOA and normozoospermia. Among them, CCNA1 and DMRT1 promoter methylation in the hypospermatogenesis group was higher than in other groups and showed diagnostic potential for the patient with hypospermatogenesis. CONCLUSION(S): Cell-free seminal DNA could be a novel, noninvasive biomarker for the detection of testicular epigenetic aberrations. Epigenetic information in cfsDNA related to spermatogenesis may serve to predict successful testicular sperm retrieval in NOA patients.

Atypical ubiquitination by E3 ligase WWP1 inhibits the proteasome-mediated degradation of mutant huntingtin.

Huntington's disease (HD) is caused by the expansion of CAG trinucleotide repeats in exon 1 of HD gene encoding huntingtin (Htt), which is characterized by aggregation and formation of mutant Htt containing expanded polyglutamine (polyQ) repeats. Dysfunction of the ubiquitin-proteasome system (UPS) plays a critical role in the pathogenesis of HD. As the linkage mediator between ubiquitin and specific target proteins, E3 ubiquitin ligases have been suggested to be involved in mHtt degradation and HD pathology. However, the potential involvement of the E3 ligase WWP1 in HD has not been explored. The present study determined whether WWP1 is involved in the development of HD in both in vivo and in vitro models. The results showed that in contrast to several other E3 ligases, expression of WWP1 is enhanced in mice and N2a cells expressing mutant Htt (160Q) and co-localized with mHtt protein aggregates. In addition, expression of WWP1 positively regulates mutan Htt levels, aggregate formation, and cell toxicity. Further analysis revealed that WWP1 ubiquitinated mHtt at an atypical position of Lys-63, which may have inhibited degradation of mutant Htt through the ubiquitin-proteasome pathway. In conclusion, these results suggested that the E3 ligase WWP1 is involved in the pathogenesis of HD; therefore, it may be a novel target for therapeutic intervention.

Tet-mediated covalent labelling of 5-methylcytosine for its genome-wide detection and sequencing.

5-methylcytosine is an epigenetic mark that affects a broad range of biological functions in mammals. The chemically inert methyl group prevents direct labelling for subsequent affinity purification and detection. Therefore, most current approaches for the analysis of 5-methylcytosine still have limitations of being either density-biased, lacking in robustness and consistency, or incapable of analysing 5-methylcytosine specifically. Here we present an approach, TAmC-Seq, which selectively tags 5-methylcytosine with an azide functionality that can be further labelled with a biotin for affinity purification, detection and genome-wide mapping. Using this covalent labelling approach, we demonstrate high sensitivity and specificity for known methylated loci, as well as increased CpG dinucleotide coverage at lower sequencing depth as compared with antibody-based enrichment, providing an improved efficiency in the 5-methylcytosine enrichment and genome-wide profiling.

Role of noncoding RNAs in trinucleotide repeat neurodegenerative disorders.

Increasingly complex networks of noncoding RNAs are being found to play important and diverse roles in the regulation of gene expression throughout the genome. Many lines of evidence are linking mutations and dysregulations of noncoding RNAs to a host of human diseases, and noncoding RNAs have been implicated in the molecular pathogenesis of some neurodegenerative disorders. The expansion of trinucleotide repeats is now recognized as a major cause of neurological disorders. Here we will review our current knowledge of the proposed mechanisms behind the involvement of noncoding RNAs in the molecular pathogenesis of neurodegenerative disorders, particularly the sequestration of specific RNA-binding proteins, the regulation of antisense transcripts, and the role of the microRNA pathway in the context of known neurodegenerative disorders caused by the expansion of trinucleotide repeats.

GRP78 counteracts cell death and protein aggregation caused by mutant huntingtin proteins.

The ER-localized chaperone glucose-regulated protein (GRP78) protects neurons against excitotoxicity and apoptosis. Here we show that overexpressing GRP78 protects N2a cells against mutant huntingtin proteins, reduces formation of mutant huntingtin aggregates, inhibits caspase-12 activation and blocks cell death. Our data suggest that GRP78 may be a promising therapeutic target for the treatment of Huntington's disease.