Structures of sperm flagellar doublet microtubules expand the genetic spectrum of male infertility.

Sperm motility is crucial for successful fertilization. Highly decorated doublet microtubules (DMTs) form the sperm tail skeleton, which propels the movement of spermatozoa. Using cryo-electron microscopy (cryo-EM) and artificial intelligence (AI)-based modeling, we determined the structures of mouse and human sperm DMTs and built an atomic model of the 48-nm repeat of the mouse sperm DMT. Our analysis revealed 47 DMT-associated proteins, including 45 microtubule inner proteins (MIPs). We identified 10 sperm-specific MIPs, including seven classes of Tektin5 in the lumen of the A tubule and FAM166 family members that bind the intra-tubulin interfaces. Interestingly, the human sperm DMT lacks some MIPs compared with the mouse sperm DMT. We also discovered variants in 10 distinct MIPs associated with a subtype of asthenozoospermia characterized by impaired sperm motility without evident morphological abnormalities. Our study highlights the conservation and tissue/species specificity of DMTs and expands the genetic spectrum of male infertility.

Identification of five hub genes as monitoring biomarkers for breast cancer metastasis in silico.

BACKGROUND: Breast cancer is one of the most common endocrine cancers among females worldwide. Distant metastasis of breast cancer is causing an increasing number of breast cancer-related deaths. However, the potential mechanisms of metastasis and candidate biomarkers remain to be further explored. RESULTS: The gene expression profiles of GSE102484 were downloaded from the Gene Expression Omnibus (GEO) database. Weighted gene co-expression network analysis (WGCNA) was used to screen for the most potent gene modules associated with the metastatic risk of breast cancer, and a total of 12 modules were identified based on the analysis. In the most significant module (R2 = 0.68), 21 network hub genes (MM > 0.90) were retained for further analyses. Next, protein-protein interaction (PPI) networks were used to further explore the biomarkers with the most interactions in gene modules. According to the PPI networks, five hub genes (TPX2, KIF2C, CDCA8, BUB1B, and CCNA2) were identified as key genes associated with breast cancer progression. Furthermore, the prognostic value and differential expression of these genes were validated based on data from The Cancer Genome Atlas (TCGA) and Kaplan-Meier (KM) Plotter. Receiver operating characteristic (ROC) curve analysis revealed that the mRNA expression levels of these five hub genes showed excellent diagnostic value for breast cancer and adjacent tissues. Moreover, these five hub genes were significantly associated with worse distant metastasis-free survival (DMFS) in the patient cohort based on KM Plotter. CONCLUSION: Five hub genes (TPX2, KIF2C, CDCA8, BUB1B, and CCNA2) associated with the risk of distant metastasis were extracted for further research, which might be used as biomarkers to predict distant metastasis of breast cancer.

CRISPR/dCas9-mediated transcriptional improvement of the biosynthetic gene cluster for the epothilone production in Myxococcus xanthus.

BACKGROUND: The CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites. RESULTS: In this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects. CONCLUSIONS: Our results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in microorganisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites.

Noninvasive prenatal diagnosis of common aneuploidies by semiconductor sequencing.

Massively parallel sequencing (MPS) of cell-free fetal DNA from maternal plasma has revolutionized our ability to perform noninvasive prenatal diagnosis. This approach avoids the risk of fetal loss associated with more invasive diagnostic procedures. The present study developed an effective method for noninvasive prenatal diagnosis of common chromosomal aneuploidies using a benchtop semiconductor sequencing platform (SSP), which relies on the MPS platform but offers advantages over existing noninvasive screening techniques. A total of 2,275 pregnant subjects was included in the study; of these, 515 subjects who had full karyotyping results were used in a retrospective analysis, and 1,760 subjects without karyotyping were analyzed in a prospective study. In the retrospective study, all 55 fetal trisomy 21 cases were identified using the SSP with a sensitivity and specificity of 99.94% and 99.46%, respectively. The SSP also detected 16 trisomy 18 cases with 100% sensitivity and 99.24% specificity and 3 trisomy 13 cases with 100% sensitivity and 100% specificity. Furthermore, 15 fetuses with sex chromosome aneuploidies (10 45,X, 2 47,XYY, 2 47,XXX, and 1 47,XXY) were detected. In the prospective study, nine fetuses with trisomy 21, three with trisomy 18, three with trisomy 13, and one with 45,X were detected. To our knowledge, this is the first large-scale clinical study to systematically identify chromosomal aneuploidies based on cell-free fetal DNA using the SSP and provides an effective strategy for large-scale noninvasive screening for chromosomal aneuploidies in a clinical setting.

High prevalence of thalassemia in migrant populations in Guangdong Province, China.

BACKGROUND: The objectives of this study were to estimate the prevalence of thalassemia and to analyze the need for public health services for migrant populations in different cities in Guangdong Province, China. METHODS: A cross-sectional survey was conducted in 21 cities of Guangdong Province. Twenty-three types of a- and ß-globin gene mutations were detected in a total of 14,230 pregnant women and 14,249 husbands. RESULTS: There was a 16.45% prevalence of thalassemia among the 28,479 individuals, and the prevalences of α-, ß-, and combined α-/ß- thalassemia were 12.03%, 3.80%, and 0.63%, respectively. Compared with the native city residents in the province, the migrants from within the province and the immigrants from outside the province had lower prevalences of thalassemia, but the prevalence values were >11%. CONCLUSIONS: The prevalence values for thalassemia gene mutations were high in all three population groups studied in Guangdong Province. The results indicate that all segments of the Guangdong population should be screened for thalassemia.

The novel HLA-A*11:448 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-A*11:448 differs from HLA-A*11:01:01:01 by one nucleotide in exon 5.

The novel HLA-B*46:01:42 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-B*46:01:42 differs from HLA-B*46:01:01:01 by one nucleotide in exon 5.

The novel HLA-C*03:652 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-C*03:652 differs from the HLA-C*03:04:01:01 by one nucleotide in exon 3.

The novel HLA-DPB1*05:01:20 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-DPB1*05:01:20 differs from HLA-DPB1*05:01:01:01 by one nucleotide in exon 3.

The novel HLA-B*48:01:13 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-B*48:01:13 differs from HLA-B*48:01:01:01 by one nucleotide in exon 5.

The novel HLA-C*08:273 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-C*08:273 differs from HLA-C*08:01:01:01 by one nucleotide in exon 2.

The novel HLA-B*38:103N allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-B*38:103N differs from HLA-B*38:02:01:01 by one nucleotide in exon 3.

Novel mutations in LRRC23 cause asthenozoospermia in a nonconsanguineous family.

ABSTRACT: The cause of asthenozoospermia (AZS) is not well understood because of its complexity and heterogeneity. Although some gene mutations have been identified as contributing factors, they are only responsible for a small number of cases. Radial spokes (RSs) are critical for adenosine triphosphate-driven flagellar beating and axoneme stability, which is essential for flagellum motility. In this study, we found novel compound heterozygous mutations in leucine-rich repeat-containing protein 23 ( LRRC23 ; c.1018C>T: p.Q340X and c.881_897 Del: p.R295Gfs*32) in a proband from a nonconsanguineous family with AZS and male infertility. Diff-Quik staining and scanning electron microscopy revealed no abnormal sperm morphology. Western blotting and immunofluorescence staining showed that these mutations suppressed LRRC23 expression in sperm flagella. Additionally, transmission electron microscopy showed the absence of RS3 in sperm flagella, which disrupts stability of the radial spoke complex and impairs motility. Following in vitro fertilization and embryo transfer, the proband's spouse achieved successful pregnancy and delivered a healthy baby. In conclusion, our study indicates that two novel mutations in LRRC23 are associated with AZS, but successful fertility outcomes can be achieved by in vitro fertilization-embryo transfer techniques.

The novel HLA-B*27:267 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-B*27:267 differs from HLA-B*27:04:01 by one nucleotide in exon 2.

Exploration of KIR genes and hematological-related diseases in Chinese Han population.

The function of natural killer (NK) cells has previously been implicated in hematopoietic-related diseases. Killer immunoglobulin-like receptors (KIR) play an important role in NK cells after hematopoietic stem cell transplantation. To explore the immunogenetic predisposition of hematological-related diseases, herein, a multi-center retrospective study in China was conducted, analyzing and comparing 2519 patients with hematopathy (mainly, acute lymphoblastic leukemia, acute myeloid leukemia, aplastic anemia, and myelodysplastic syndrome) to 18,108 individuals without known pathology. Genotyping was performed by polymerase chain reaction with specific sequence primers (PCR-SSP). As a result, we discovered four genes including KIR2DL5 (OR: 0.74, 95% CI 0.59-0.93; Pc = 0.0405), 2DS1 (OR: 0.74, 95% CI 0.59-0.93; Pc = 0.0405), 2DS3 (OR: 0.58, 95% CI 0.41-0.81; Pc = 0.0180), and 3DS1 (OR: 0.74, 95% CI 0.58-0.94; Pc = 0.0405) to be protective factors that significantly reduce the risk of aplastic anemia. Our findings offer new approaches to immunotherapy for hematological-related diseases. As these therapies mature, they are promising to be used alone or in combination with current treatments to help to make blood disorders a manageable disease.

Isolation and Aflatoxin B1-Degradation Characteristics of a Microbacterium proteolyticum B204 Strain from Bovine Faeces.

Aflatoxin B1 (AFB1) is one of the most harmful mycotoxins, raising serious global health and economic problems. Searching for biological approaches for effective and safe AFB1 degradation is imminent. In our study, Microbacterium proteolyticum B204 isolated from bovine faeces degraded 77% of AFB1 after 24 h, becoming the first reported bacteria from the Microbacterium family to possess AFB1 degradation characteristics. Temperature variation showed little effect on its degradation ratio, demonstrating high thermostability of 75% and 79% after boiling and sterilization, respectively. We suppose that the components playing a key role during this process were proteins, considering the decreased degradation rate caused by Proteinase K. Cell viability detection on HepG2 cells indicated that the degradation products were much less toxic than pure AFB1. Furthermore, B204 cell-free culture supernatant also degraded AFB1-contaminated food, such as peanuts, corn and cheese. These results suggested that this strain with AFB1 degradation properties could be a prospective candidate for application in the food and feed industries.

Influence of Social Support Network and Perceived Social Support on the Subjective Wellbeing of Mothers of Children With Autism Spectrum Disorder.

This study explored the relations between the social support network of mothers of children with autism spectrum disorder (ASD), perceived social support, and their subjective wellbeing. The participants were mothers of children with ASD in Shanghai. Their social support network structure was explored via the nomination method. Perceived social support was measured using the Revised Social Provisions Scale for Autism (R-SPS-A), and the mothers' subjective wellbeing was assessed using the Index of Wellbeing, Index of General Affect. A significant correlation was observed between the subjective wellbeing of mothers of children with ASD and perceived social support. Meanwhile, perceived social support was significantly correlated with the effectiveness of overall social support. Finally, perceived social support was also significantly correlated with the network size of social support. Moreover, the effectiveness of social support was significantly associated with the network size of social support and was highly significantly associated with the degree of intimacy of social support. Furthermore, the network size of instrumental support has a significant influence on all perceived social support subdimensions. Overall, social support effectiveness plays an important role in the social support network mechanism on perceived social support and subjective wellbeing in China.

Differential requirements of IQUB for the assembly of radial spoke 1 and the motility of mouse cilia and flagella.

The T-shaped radial spoke (RS) is a protein complex attached to the A microtubule of the outer doublet microtubules, and it extends toward the central pair (CP). It modulates the beat frequency, amplitude, and waveform of the flagella and cilia by serving as a mechanochemical signal transducer between the CP and dyneins. In humans, RS defects cause primary ciliary dyskinesia, but the structural components of triple RSs (RS1, RS2, and RS3) in mammals remain to be elucidated in detail. Here, we introduce a mouse model that lacked the entire RS1 in sperm flagella, due to the deletion of Iqub, while the tracheal cilia possessed intact triple RSs. Furthermore, the absence of IQUB only resulted in male infertility, owing to the sperm motility defect. Based on the mouse model, the RS1 compositions are identified in sperm flagella. In summary, this study elucidates the RS1 components and function in mammalian flagella.

Characterization of human cytomegalovirus UL145 and UL136 genes in low-passage clinical isolates from infected Chinese infants.

BACKGROUND: Human cytomegalovirus (HCMV) is a leading cause of morbidity and mortality in immunocompromised individuals. The unique long b' (ULB') region of HCMV contains at least 19 open reading frames (ORFs); however, little is known about the function of UL145 and UL136. We characterized UL145 and UL136 in low-passage clinical isolates from Chinese infants. MATERIAL/METHODS: The clinical strains of HCMV were recovered from the urine from HCMV-infected infants. Human embryonic lung fibroblasts (HELFs) were infected with clinical isolates of HCMV, and the viral DNA and mRNA for UL145 and UL136 were analyzed by polymerase chain reaction (PCR) and sequencing techniques. We also predicted the structure and function of UL145 and UL136 proteins. RESULTS: Sixty-two Chinese infants infected with HCMV were recruited into this study and the clinical isolates were recovered from the urine. Two strains among the low-passage isolates, D2 and D3, were obtained. The UL145 and UL136 sequences were deposited with GenBank under accession numbers of DQ180367, DQ180381, DQ180377, and DQ180389. The mRNA expression of both UL145 and UL136 was confirmed by reverse transcription (RT-PCR) assays. UL145 was predicted to contain 1 protein kinase C phosphorylation site, 2 casein kinase II phosphorylation sites and a zinc finger structure. UL136 was predicted to contain a protein kinase C phosphorylation site, N-myristoylation site, cAMP- and cGMP-dependent protein kinase phosphorylation site and tyrosine kinase II phosphorylation site. Both UL145 and UL136 are highly conserved. CONCLUSIONS: UL145 may act as an intranuclear regulating factor by direct binding to DNA, while UL136 may be a membrane receptor involving signal transduction.