Reprogramming of Meiotic Chromatin Architecture during Spermatogenesis.

Chromatin organization undergoes drastic reconfiguration during gametogenesis. However, the molecular reprogramming of three-dimensional chromatin structure in this process remains poorly understood for mammals, including primates. Here, we examined three-dimensional chromatin architecture during spermatogenesis in rhesus monkey using low-input Hi-C. Interestingly, we found that topologically associating domains (TADs) undergo dissolution and reestablishment in spermatogenesis. Strikingly, pachytene spermatocytes, where synapsis occurs, are strongly depleted for TADs despite their active transcription state but uniquely show highly refined local compartments that alternate between transcribing and non-transcribing regions (refined-A/B). Importantly, such chromatin organization is conserved in mouse, where it remains largely intact upon transcription inhibition. Instead, it is attenuated in mutant spermatocytes, where the synaptonemal complex failed to be established. Intriguingly, this is accompanied by the restoration of TADs, suggesting that the synaptonemal complex may restrict TADs and promote local compartments. Thus, these data revealed extensive reprogramming of higher-order meiotic chromatin architecture during mammalian gametogenesis.

Homozygous mutations in C14orf39/SIX6OS1 cause non-obstructive azoospermia and premature ovarian insufficiency in humans.

Human infertility is a multifactorial disease that affects 8%-12% of reproductive-aged couples worldwide. However, the genetic causes of human infertility are still poorly understood. Synaptonemal complex (SC) is a conserved tripartite structure that holds homologous chromosomes together and plays an indispensable role in the meiotic progression. Here, we identified three homozygous mutations in the SC coding gene C14orf39/SIX6OS1 in infertile individuals from different ethnic populations by whole-exome sequencing (WES). These mutations include a frameshift mutation (c.204_205del [p.His68Glnfs∗2]) from a consanguineous Pakistani family with two males suffering from non-obstructive azoospermia (NOA) and one female diagnosed with premature ovarian insufficiency (POI) as well as a nonsense mutation (c.958G>T [p.Glu320∗]) and a splicing mutation (c.1180-3C>G) in two unrelated Chinese men (individual P3907 and individual P6032, respectively) with meiotic arrest. Mutations in C14orf39 resulted in truncated proteins that retained SYCE1 binding but exhibited impaired polycomplex formation between C14ORF39 and SYCE1. Further cytological analyses of meiosis in germ cells revealed that the affected familial males with the C14orf39 frameshift mutation displayed complete asynapsis between homologous chromosomes, while the affected Chinese men carrying the nonsense or splicing mutation showed incomplete synapsis. The phenotypes of NOA and POI in affected individuals were well recapitulated by Six6os1 mutant mice carrying an analogous mutation. Collectively, our findings in humans and mice highlight the conserved role of C14ORF39/SIX6OS1 in SC assembly and indicate that the homozygous mutations in C14orf39/SIX6OS1 described here are responsible for infertility of these affected individuals, thus expanding our understanding of the genetic basis of human infertility.

Three-Dimensional Fe Single-Atom Catalyst for High-Performance Cathode of Zn-Air Batteries.

Designing cost-effective and highly active oxygen reduction reaction (ORR) catalysts is critical for the development of Zn-air batteries (ZABs). Iron-nitrogen-carbon (Fe-N-C) catalysts with single-atom Fe-Nx active sites are considered as one of the most promising alternatives to noble Pt but are hindered by unsatisfactory activity and durability. Herein, a NaCl template-assisted in situ pyrolysis technique is utilized to massively fabricate Fe-N-C single-atom catalysts (SACs) anchored on the three-dimensional open-pore carbon networks (denoted as 3D SAFe). The 3D SAFe catalyst exhibits ultrahigh activity with a half-wave potential of 0.90 V (vs RHE), benefiting from the enhanced mass diffusion and the increased amount of effective Fe-N4 sites. Consequently, the ZABs assembled with 3D SAFe deliver high peak power density up to 156 mW cm-2 and outstanding durability of 80 h, suggesting the application potential of the 3D SAFe catalyst. This work inspires the rational design and synthesis of highly efficient SACs for ZABs.

Whole-exome sequencing of consanguineous families with infertile men and women identifies homologous mutations in SPATA22 and MEIOB.

STUDY QUESTION: Can whole-exome sequencing (WES) reveal pathogenic mutations in two consanguineous Pakistani families with infertile patients? SUMMARY ANSWER: A homozygous spermatogenesis associated 22 (SPATA22) frameshift mutation (c.203del), which disrupts the interaction with meiosis specific with OB-fold (MEIOB), and a MEIOB splicing mutation (c.683-1G>A) that led to loss of MEIOB protein cause familial infertility. WHAT IS KNOWN ALREADY: MEIOB and SPATA22, direct binding partners and functional collaborators, form a meiosis-specific heterodimer that regulates meiotic recombination. The protein stability and the axial localization of MEIOB and SPATA22 depend on each other. Meiob and Spata22 knockout mice have the same phenotypes: mutant spermatocytes can initiate meiotic recombination but are unable to complete DSB repair, leading to crossover formation failure, meiotic prophase arrest, and sterility. STUDY DESIGN, SIZE, DURATION: We performed WES for the patients and controls in two consanguineous Pakistani families to screen for mutations. The pathogenicity of the identified mutations was assessed by in vitro assay and mutant mouse model. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two consanguineous Pakistani families with four patients (three men and one woman) suffering from primary infertility were recruited. SPATA22 and MEIOB mutations were screened from the WES data, followed by functional verification in cultured cells and mice. MAIN RESULTS AND THE ROLE OF CHANCE: A homozygous SPATA22 frameshift mutation (c.203del) was identified in a patient with non-obstructive azoospermia (NOA) from a consanguineous Pakistani family and a homozygous MEIOB splicing mutation (c.683-1G>A) was identified in two patients with NOA and one infertile woman from another consanguineous Pakistani family. The SPATA22 mutation destroyed the interaction with MEIOB. The MEIOB splicing mutation induced Exon 9 skipping, which causes a 32aa deletion in the oligonucleotide-binding domain without affecting the interaction between MEIOB and SPATA22. Furthermore, analyses of the Meiob mutant mice modelling the patients' mutation revealed that the MEIOB splicing mutation leads to loss of MEIOB proteins, abolished SPATA22 recruitment on chromosome axes, and meiotic arrest due to meiotic recombination failure. Thus, our study suggests that SPATA22 and MEIOB may both be causative genes for human infertility. LIMITATIONS, REASONS FOR CAUTION: As SPATA22 and MEIOB are interdependent and essential for meiotic recombination, screening for mutations of SPATA22 and MEIOB in both infertile men and women in larger cohorts is important to further reveal the role of the SPATA22 and MEIOB heterodimer in human fertility. WIDER IMPLICATIONS OF THE FINDINGS: These findings provide direct clinical and functional evidence that mutations in SPATA22 and MEIOB can cause meiotic recombination failure, supporting a role for these mutations in human infertility and their potential use as targets for genetic diagnosis of human infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Developmental Program of China (2018YFC1003900, 2018YFC1003700, and 2019YFA0802600), the National Natural Science Foundation of China (31890780, 31630050, 32061143006, 82071709, and 31871514), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19000000). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

LncRNA GAS5 sponges miR-362-5p to promote sensitivity of thyroid cancer cells to 131 I by upregulating SMG1.

This study aims to investigate the role of lncRNA growth arrest-specific transcript 5 (GAS5)/miR-362-5p/suppressor of morphogenesis in the genitalia 1 (SMG1) axis in 131 I-resistance in thyroid cancer (TC). GAS5, miR-362-5p, and SMG1 expression in TC tissues was assessed and the 131 I-resistant TC cells were established, which were treated with altered GAS5, miR-362-5p, and SMG1. The proliferation and apoptosis of 131 I-resistant TC cells were detected, and the expression of Akt/mTOR signaling pathway-related proteins was assessed. Binding relations between GAS5 and miR-362-5p, and miR-362-5p and SMG1 were confirmed. The role of GAS5 in 131 I-resistant TC cell growth in vivo was observed. GAS5 was downregulated and miR-362-5p was upregulated in TC tissues and 131 I-resistant cells. The 131 I-resistant TC cells had enhanced proliferation and repressed apoptosis, and the Akt/mTOR signaling pathway was activated. Overexpressed GAS5 strengthened 131 I sensitivity and suppressed TC cell growth, while upregulated miR-362-5p had an opposite effect. MiR-362-5p upregulation reversed the effect of GAS5, and SMG1 overexpression eliminated the impact of miR-362-5p upregulation on 131 I-resistant TC cells. GAS5 competitively binds to miR-362-5p and SMG1 is targeted by miR-362-5p. GAS5 sponges miR-362-5p to promote sensitivity of TC cells to 131 I by upregulating SMG1 and inactivating Akt/mTOR signaling pathway.

Npat-dependent programmed Sertoli cell proliferation is indispensable for testis cord development and germ cell mitotic arrest.

As the major somatic cell type, Sertoli cells undergo active proliferation and play essential roles to establish testis cord at fetal stage. They also function to maintain germ cell development throughout the life of testicular development. However, the significance of Sertoli cell number for testis cord development and gonocyte fate is still unclear. Nuclear protein ataxia-telangiectasia (NPAT, also known as p220), a substrate of cyclin E/cyclin-dependent kinase 2, is well known as a regulator of cell proliferation through regulating histone expression. To study the role of NPAT during Sertoli cell development, we generated a mouse strain carrying conditional floxed Npat alleles, when crossing with anti-Müllerian hormone-cre, leading to the specific deletion of Npat in Sertoli cells. Npat disruption in Sertoli cells inhibited the programmed proliferation of fetal Sertoli cells resulting in disruption of developing testis cords, and subsequent postnatal mutant testes were severely hypoplastic. Germ cells, which are presumed to be in quiescent status during perinatal stage, exited G0 phase arrest and re-enter mitotic cell cycle prematurely. Of particular note, some germ cells possessed the meiotic signal in Npat-deficient testes. Our data thus indicates that the function of Npat-dependent Sertoli cells is essential at multiple steps in testis development, and this study also identifies Sertoli cells as a major regulator of germ cell development, which are required to maintain a local growth niche to repress premature mitosis and meiosis of gonocytes.-Jiang, X., Yin, S., Fan, S., Bao, J., Jiao, Y., Ali, A., Iqbal, F., Xu, J., Zhang, Y., Shi, Q. Npat-dependent programmed Sertoli cell proliferation is indispensable for testis cord development and germ cell mitotic arrest.

A novel recombination protein C12ORF40/REDIC1 is required for meiotic crossover formation.

During meiosis, at least one crossover must occur per homologous chromosome pair to ensure normal progression of meiotic division and accurate chromosome segregation. However, the mechanism of crossover formation is not fully understood. Here, we report a novel recombination protein, C12ORF40/REDIC1, essential for meiotic crossover formation in mammals. A homozygous frameshift mutation in C12orf40 (c.232_233insTT, p.Met78Ilefs*2) was identified in two infertile men with meiotic arrest. Spread mouse spermatocyte fluorescence immunostaining showed that REDIC1 forms discrete foci between the paired regions of homologous chromosomes depending on strand invasion and colocalizes with MSH4 and later with MLH1 at the crossover sites. Redic1 knock-in (KI) mice homozygous for mutation c.232_233insTT are infertile in both sexes due to insufficient crossovers and consequent meiotic arrest, which is also observed in our patients. The foci of MSH4 and TEX11, markers of recombination intermediates, are significantly reduced numerically in the spermatocytes of Redic1 KI mice. More importantly, our biochemical results show that the N-terminus of REDIC1 binds branched DNAs present in recombination intermediates, while the identified mutation impairs this interaction. Thus, our findings reveal a crucial role for C12ORF40/REDIC1 in meiotic crossover formation by stabilizing the recombination intermediates, providing prospective molecular targets for the clinical diagnosis and therapy of infertility.

Preliminary evaluation of a small interfering RNA molecular probe targeting murine double minute 2 in breast cancer.

INTRODUCTION: Murine double minute 2 (MDM2) is an oncogene that is important in tumorigenesis, tumor metastasis and chemotherapy resistance. We aimed to synthesize a molecular imaging probe, 99m Tc-HYNIC-siRNA 1489, which could specifically bind to MDM2. The [ 99m Tc]HYNIC-siRNA 1489 molecular probe provided an effective way of assessing MDM2 expression via single-photon emission computed tomography. METHOD: Three siRNAs were designed, and their inhibitory efficiencies were determined using western blots and qRT-PCR. The selected siRNA was labeled with the radionuclide technetium-99m ( 99m Tc) through the chelator HYNIC. The bioactivity and properties of [ 99m Tc]HYNIC-siRNA 1489 were evaluated prior to imaging in mice. Imaging and biodistribution of the probe were used to assess its targeting ability. RESULTS: SiRNA 1489, which was labeled with 99m Tc, displayed a strong inhibitory effect in Michigan Cancer Foundation-7 cell lines. The radiochemical purity of [ 99m Tc]HYNIC-siRNA 1489 was stable at various temperatures in phosphate-buffered serum and bovine serum. The tumor/muscle ratio in mice injected with [ 99m Tc]HYNIC-siRNA 1489 was higher than that in those injected with the negative control, [ 99m Tc]HYNIC-NC siRNA. The percentage injected dose per gram (%ID/g) of the tumors injected with 99m Tc-HYNIC-siRNA 1489 was greater than that of the control group. CONCLUSION: The [ 99m Tc]HYNIC-siRNA 1489 was taken up by the tumor, which had a high level of MDM2. The probe exhibited a sufficient retention time in the tumor. This probe may be an effective strategy for evaluating MDM2 expression and achieving early diagnosis in breast cancer.

Molecular imaging and treatment of PSMA-positive prostate cancer with 99mTc radiolabeled aptamer-siRNA chimeras.

INTRODUCTION: Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer (PCa). The aptamer (Apt) A10-3.2 can be used as a specific ligand for the early diagnosis and targeted treatment of PCa. siRNA-Apt has been used to therapeutically target PSMA-positive PCa. We aimed to synthesize a new type of molecular probe to facilitate the integration of diagnosis and treatment for PSMA-positive PCa. METHODS: Chimeras were obtained by covalent linking PSMA Apt-A10-3.2 and the MDM2 siRNA. SHNH, a bifunctional chelating agent, was used to couple 99mTc with chimeras to synthesize a new molecular probe. Labeling efficiency, radiochemical purity, and stability were confirmed using a γ-well counter and Whatman paper No.1. SPECT imaging and biodistribution studies were performed on BALB/c mice bearing 22Rv1 or PC-3 xenografts. Tumor inhibition and cytotoxicity of Chimeras were evaluated. LNCaP, 22RV1, and PC-3 PCa cell lines were used for in vitro and in vivo experiments. RESULTS: [99mTc]Tc-chimeras showed high labeling efficiency (61.47% ± 2.85%, n = 3), radiochemical purity (>95%), and stability. Biodistribution studies and SPECT imaging with 99mTc-chimeras in mice bearing 22Rv1 xenografts demonstrated a high T/M ratio (4.63 ± 0.68, n = 3) and a high T/B ratio (3.61 ± 0.7, n = 3) at 2 h post-injection. 99mTc-chimeras showed rapid renal clearance. Compared with the PBS group, tumor growth in the chimera group was significantly inhibited (P < 0.01, n = 4), but there was no significant difference in body weight (p > 0.05, n = 4). H&E staining showed no obvious liver or kidney damage. CONCLUSIONS: Our study proved that [99mTc]Tc-Aptamer-siRNA chimeras could be used to diagnose and treat PSMA-positive PCa in vivo.

A nomogram model based on MRI and radiomic features developed and validated for the evaluation of lymph node metastasis in patients with rectal cancer.

PURPOSE: The aim of this study was to develop and validate a nomogram model to evaluate lymph node metastasis (LNM) in patients with rectal cancer (RC). METHODS: A total of 162 patients with RC were included in the study. The MRI reported model, the Radscore model, and the Complex model were constructed using the logistics regression (LR) algorithm. The DeLong test and decision curve analysis (DCA) were used to compare the prediction performance and clinical utility of these models. The nomogram model was constructed to visualize the prediction results of the best model. Model performance was evaluated in the training and validation groups, and the calibration curve and Hosmer-Lemeshow goodness of fit test were used to evaluate the calibration. RESULT: All three models constructed by the LR algorithm were good at identifying LNM. The DeLong test and the DCA results showed that the Complex model outperformed the MRI reported model and the Radscore model in relation to their predictive performance and clinical utility. The nomogram of the Complex model had an area under the curve (AUC) of 0.902 (95% confidence interval (CI) 0.848-0.957) in the training group and an AUC of 0.891 (95% CI 0.799-0.983) in the validation group. Meanwhile, the nomogram showed good calibration. CONCLUSION: The nomogram model constructed based on T2WI radiomics and MRI reported had good diagnostic efficacies for LNM in patients with RC, and provided a new auxiliary method for accurate and individualized clinical management.

The Machine Learning Model for Distinguishing Pathological Subtypes of Non-Small Cell Lung Cancer.

Purpose: Machine learning models were developed and validated to identify lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) using clinical factors, laboratory metrics, and 2-deoxy-2[18F]fluoro-D-glucose ([18F]F-FDG) positron emission tomography (PET)/computed tomography (CT) radiomic features. Methods: One hundred and twenty non-small cell lung cancer (NSCLC) patients (62 LUAD and 58 LUSC) were analyzed retrospectively and randomized into a training group (n = 85) and validation group (n = 35). A total of 99 feature parameters-four clinical factors, four laboratory indicators, and 91 [18F]F-FDG PET/CT radiomic features-were used for data analysis and model construction. The Boruta algorithm was used to screen the features. The retained minimum optimal feature subset was input into ten machine learning to construct a classifier for distinguishing between LUAD and LUSC. Univariate and multivariate analyses were used to identify the independent risk factors of the NSCLC subtype and constructed the Clinical model. Finally, the area under the receiver operating characteristic curve (AUC) values, sensitivity, specificity, and accuracy (ACC) was used to validate the machine learning model with the best performance effect and Clinical model in the validation group, and the DeLong test was used to compare the model performance. Results: Boruta algorithm selected the optimal subset consisting of 13 features, including two clinical features, two laboratory indicators, and nine PEF/CT radiomic features. The Random Forest (RF) model and Support Vector Machine (SVM) model in the training group showed the best performance. Gender (P=0.018) and smoking status (P=0.011) construct the Clinical model. In the validation group, the SVM model (AUC: 0.876, ACC: 0.800) and RF model (AUC: 0.863, ACC: 0.800) performed well, while Clinical model (AUC:0.712, ACC: 0.686) performed moderately. There was no significant difference between the RF and Clinical models, but the SVM model was significantly better than the Clinical model. Conclusions: The proposed SVM and RF models successfully identified LUAD and LUSC. The results indicate that the proposed model is an accurate and noninvasive predictive tool that can assist clinical decision-making, especially for patients who cannot have biopsies or where a biopsy fails.

A clinical study on relationship between visualization of cardiac fibroblast activation protein activity by Al18F-NOTA-FAPI-04 positron emission tomography and cardiovascular disease.

Objective: FAP plays a vital role in myocardial injury and fibrosis. Although initially used to study imaging of primary and metastatic tumors, the use of FAPI tracers has recently been studied in cardiac remodeling after myocardial infarction. The study aimed to investigate the application of FAPI PET/CT imaging in human myocardial fibrosis and its relationship with clinical factors. Materials and methods: Retrospective analysis of FAPI PET/CT scans of twenty-one oncological patients from 05/2021 to 03/2022 with visual uptake of FAPI in the myocardium were applying the American Heart Association 17-segment model of the left ventricle. The patients' general data, echocardiography, and laboratory examination results were collected, and the correlation between PET imaging data and the above data was analyzed. Linear regression models, Kendall's TaU-B test, the Spearman test, and the Mann-Whitney U test were used for the statistical analysis. Results: 21 patients (60.1 ± 9.4 years; 17 men) were evaluated with an overall mean LVEF of 59.3 ± 5.4%. The calcific plaque burden of LAD, LCX, and RCA are 14 (66.7%), 12 (57.1%), and 9 (42.9%). High left ventricular SUVmax correlated with BMI (P < 0.05) and blood glucose level (P < 0.05), and TBR correlated with age (P < 0.05). A strong correlation was demonstrated between SUVmean and CTnImax (r = 0.711, P < 0.01). Negative correlation of SUVmean and LVEF (r = -0.61, P < 0.01), SUVmax and LVEF (r = -0.65, P < 0.01) were found. ROC curve for predicting calcified plaques by myocardial FAPI uptake (SUVmean) in LAD, LCX, and RCA territory showed AUCs were 0.786, 0.759, and 0.769. Conclusion: FAPI PET/CT scans might be used as a new potential method to evaluate cardiac fibrosis to help patients' management further. FAPI PET imaging can reflect the process of myocardial fibrosis. High FAPI uptakes correlate with cardiovascular risk factors and the distribution of coronary plaques.

A TOP6BL mutation abolishes meiotic DNA double-strand break formation and causes human infertility.

Meiosis is pivotal for sexual reproduction and fertility. Meiotic programmed DNA double-strand breaks (DSBs) initiate homologous recombination, ensuring faithful chromosome segregation and generation of gametes. However, few studies have focused on meiotic DSB formation in human reproduction. Here, we report four infertile siblings born to a consanguineous marriage, with three brothers suffering from non-obstructive azoospermia and one sister suffering from unexplained infertility with normal menstrual cycles and normal ovary sizes with follicular activity. An autosomal recessive mutation in TOP6BL was found co-segregating with infertility in this family. Investigation of one male patient revealed failure in programmed meiotic DSB formation and meiotic arrest prior to pachytene stage of prophase I. Mouse models carrying similar mutations to that in patients recapitulated the spermatogenic abnormalities of the patient. Pathogenicity of the mutation in the female patient was supported by observations in mice that meiotic programmed DSBs failed to form in mutant oocytes and oocyte maturation failure due to absence of meiotic recombination. Our study thus illustrates the phenotypical characteristics and the genotype-phenotype correlations of meiotic DSB formation failure in humans.