Applications of single-cell RNA sequencing in spermatogenesis and molecular evolution.

Spermatogenic cell heterogeneity is determined by the complex process of spermatogenesis differentiation. However, effectively revealing the regulatory mechanisms underlying mammalian spermatogenic cell development and differentiation via traditional methods is difficult. Advances in technology have led to the emergence of many single-cell transcriptome sequencing protocols, which have partially addressed these challenges. In this review, we detail the principles of 10x Genomics technology and summarize the methods for downstream analysis of single-cell transcriptome sequencing data. Furthermore, we explore the role of single-cell transcriptome sequencing in revealing the heterogeneity of testicular ecological niche cells, delineating the establishment and disruption of testicular immune homeostasis during human spermatogenesis, investigating abnormal spermatogenesis in humans, and, ultimately, elucidating the molecular evolution of mammalian spermatogenesis.

Transcutaneous cervical vagus nerve stimulation improved motor cortex excitability in healthy adults: a randomized, single-blind, self-crossover design study.

Purpose: To investigate the effect of transcutaneous cervical vagus nerve stimulation (tcVNS) on motor cortex excitability in healthy adults. Method: Twenty eight healthy subjects were assigned to receive real and sham tcVNS for 30 min. The interval between the real and sham conditions was more than 24 h, and the sequence was random. The central and peripheral motor-evoked potential (MEP) of the right first dorsal interosseous (FDI) muscle was measured by transcranial magnetic stimulation (TMS) before and after stimulation. MEP latency, MEP amplitude and rest motor threshold (rMT) were analyzed before and after stimulation. Results: MEP amplitude, MEP latency and rMT had significant interaction effect between time points and conditions (p < 0.05). After real stimulation, the MEP amplitude was significantly increased (p < 0.001). MEP latency (p < 0.001) and rMT (p = 0.006) was decreased than that of baseline. The MEP amplitude on real condition was higher than that of sham stimulation after stimulation (p = 0.027). The latency after the real stimulation was significantly shorter than that after sham stimulation (p = 0.005). No significantly difference was found in rMT after stimulation between real and sham conditions (p > 0.05). Conclusion: tcVNS could improve motor cortex excitability in healthy adults.

Transcription factor Dmrt1 triggers the SPRY1-NF-κB pathway to maintain testicular immune homeostasis and male fertility.

Bacterial or viral infections, such as Brucella, mumps virus, herpes simplex virus, and Zika virus, destroy immune homeostasis of the testes, leading to spermatogenesis disorder and infertility. Of note, recent research shows that SARS-CoV-2 can infect male gonads and destroy Sertoli and Leydig cells, leading to male reproductive dysfunction. Due to the many side effects associated with antibiotic therapy, finding alternative treatments for inflammatory injury remains critical. Here, we found that Dmrt1 plays an important role in regulating testicular immune homeostasis. Knockdown of Dmrt1 in male mice inhibited spermatogenesis with a broad inflammatory response in seminiferous tubules and led to the loss of spermatogenic epithelial cells. Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) revealed that Dmrt1 positively regulated the expression of Spry1, an inhibitory protein of the receptor tyrosine kinase (RTK) signaling pathway. Furthermore, immunoprecipitation-mass spectrometry (IP-MS) and co-immunoprecipitation (Co-IP) analysis indicated that SPRY1 binds to nuclear factor kappa B1 (NF-κB1) to prevent nuclear translocation of p65, inhibit activation of NF-κB signaling, prevent excessive inflammatory reaction in the testis, and protect the integrity of the blood-testis barrier. In view of this newly identified Dmrt1- Spry1-NF-κB axis mechanism in the regulation of testicular immune homeostasis, our study opens new avenues for the prevention and treatment of male reproductive diseases in humans and livestock.

SeUneter: Channel attentive U-Net for instance segmentation of the cervical spine MRI medical image.

In recent years, cervical spondylosis has become one of the most common chronic diseases and has received much attention from the public. Magnetic resonance imaging (MRI) is the most widely used imaging modality for the diagnosis of degenerative cervical spondylosis. The manual identification and segmentation of the cervical spine on MRI makes it a laborious, time-consuming, and error-prone process. In this work, we collected a new dataset of 300 patients with a total of 600 cervical spine images in the MRI T2-weighted (T2W) modality for the first time, which included the cervical spine, intervertebral discs, spinal cord, and spinal canal information. A new instance segmentation approach called SeUneter was proposed for cervical spine segmentation. SeUneter expanded the depth of the network structure based on the original U-Net and added a channel attention module to the double convolution of the feature extraction. SeUneter could enhance the semantic information of the segmentation and weaken the characteristic information of non-segmentation to the screen for important feature channels in double convolution. In the meantime, to alleviate the over-fitting of the model under insufficient samples, the Cutout was used to crop the pixel information in the original image at random positions of a fixed size, and the number of training samples in the original data was increased. Prior knowledge of the data was used to optimize the segmentation results by a post-process to improve the segmentation performance. The mean of Intersection Over Union (mIOU) was calculated for the different categories, while the mean of the Dice similarity coefficient (mDSC) and mIOU were calculated to compare the segmentation results of different deep learning models for all categories. Compared with multiple models under the same experimental settings, our proposed SeUneter's performance was superior to U-Net, AttU-Net, UNet++, DeepLab-v3+, TransUNet, and Swin-Unet on the spinal cord with mIOU of 86.34% and the spinal canal with mIOU of 73.44%. The SeUneter matched or exceeded the performance of the aforementioned segmentation models when segmenting vertebral bodies or intervertebral discs. Among all models, SeUneter achieved the highest mIOU and mDSC of 82.73% and 90.66%, respectively, for the whole cervical spine.

Low-intensity transcranial ultrasound stimulation facilitates hand motor function and cortical excitability: A crossover, randomized, double blind study.

Objective: Transcranial ultrasound stimulation (TUS) is a new form of non-invasive brain stimulation. Low-intensity TUS is considered highly safe. We aimed to investigate the effect of low-intensity TUS on hand reaction responses and cortical excitability in healthy adults. Methods: This study used a crossover, randomized, and double-blind design. A total of 20 healthy participants were recruited for the study. All the participants received TUS and sham stimulation on separate days in random order. The finger tapping test (tapping score by using a tablet) and motor evoked potential (MEP) were assessed before and after stimulation, and discomfort levels were assessed using a visual analog scale (VAS) score. Results: No significant differences in tapping score or MEP amplitude between the two experimental conditions were registered before stimulation. After stimulation, tapping scores were increased regardless of the specific treatment, and the real stimulation condition receiving TUS (90.4 ± 11.0 points) outperformed the sham stimulation condition (86.1 ± 8.4 points) (p = 0.002). The MEP latency of real TUS (21.85 ± 1.33 ms) was shorter than that of sham TUS (22.42 ± 1.43 ms) (p < 0.001). MEP amplitude of real TUS (132.18 ± 23.28 µV) was higher than that of sham TUS (114.74 ± 25.5 µV, p = 0.005). There was no significant difference in the discomfort score between the two conditions (p = 0.163). Conclusion: Transcranial ultrasound stimulation (TUS) can decrease the hand reaction response time and latency of the MEP, enhance the excitability of the motor cortex, and improve hand motor function in healthy individuals without obvious discomfort.

Single-cell RNA sequencing reveals atlas of dairy goat testis cells.

Single-cell RNA sequencing (scRNA-seq) is useful for exploring cell heterogeneity. For large animals, however, little is known regarding spermatogonial stem cell (SSC) self-renewal regulation, especially in dairy goats. In this study, we described a high-resolution scRNA-seq atlas derived from a dairy goat. We identified six somatic cell and five spermatogenic cell subtypes. During spermatogenesis, genes with significantly changed expression were mainly enriched in the Notch, TGF-ß, and Hippo signaling pathways as well as the signaling pathway involved in the regulation of stem cell pluripotency. We detected and screened specific candidate marker genes ( TKTL1 and AES) for spermatogonia. Our study provides new insights into goat spermatogenesis and the development of testicular somatic cells.

Dmrt1 regulates the immune response by repressing the TLR4 signaling pathway in goat male germline stem cells.

Double sex and mab-3-related transcription factor 1 (Dmrt1), which is expressed in goat male germline stem cells (mGSCs) and Sertoli cells, is one of the most conserved transcription factors involved in sex determination. In this study, we highlighted the role of Dmrt1 in balancing the innate immune response in goat mGSCs. Dmrt1 recruited promyelocytic leukemia zinc finger (Plzf), also known as zinc finger and BTB domain-containing protein 16 (Zbtb16), to repress the Toll-like receptor 4 (TLR4)-dependent inflammatory signaling pathway and nuclear factor (NF)-κB. Knockdown of Dmrt1 in seminiferous tubules resulted in widespread degeneration of germ and somatic cells, while the expression of proinflammatory factors were significantly enhanced. We also demonstrated that Dmrt1 stimulated proliferation of mGSCs, but repressed apoptosis caused by the immune response. Thus, Dmrt1 is sufficient to reduce inflammation in the testes, thereby establishing the stability of spermatogenesis and the testicular microenvironment.

Preparation of SnO2 nanotubes via a template-free electrospinning process.

A facile and environmentally friendly template-free method is developed for the fabrication of SnO2 nanotubes via electrospinning and precisely controlled heat treatment method. It is revealed that the as-spun solid SnO2 precursor fibers gradually transformed into hollow-structured nanotubes when the temperature was controlled precisely from 200 °C to 600 °C. It was confirmed, that this remarkable structural evolution corporate the respective thermal decomposition of polyvinyl butyral (PVB) at the surface and inside of the fibers. The formation mechanism of the nanotubes has been clarified by systematically investigating the morphology, phase structure, chemical state, and decomposition of the organic compounds during the heat treatment. The as-prepared SnO2 nanotubes exhibit a high specific surface area of 32.91 m2 g-1 and a porous structure with pore sizes of 2 nm and 10-25 nm. The SnO2 nanotubes were assembled as a photosensor, which demonstrates a fast response upon UV light illumination at 254 nm. From this discovery, it is expected that a new method for fabricating nanotubes will be established and the development of materials with a higher functionality will be promoted.

[Adsorption and Interaction of Cu2+ and Pb2+ on BS-12 Amphoteric Modified Bentonites].

Amphoteric modification can simultaneously improve the adsorption of organic pollutants and heavy metals on clay minerals. Study of the adsorption and interaction of multiple heavy metals on amphoteric modified soils is therefore of practical significance. Here, bentonite-(CK) and 150BS-12-modified bentonites (150BS-12) were characterized both before and after metal ion adsorption using Fourier-transform infrared spectroscopy (FTIR). The equilibrium adsorption characteristics and differences of Cu2+ and Pb2+ in single and binary systems were studied by batch methods. The interaction mechanism of the metals on modified bentonites is also discussed. The results showed that the adsorption capacity of Cu2+ and Pb2+ in single and binary systems was ranked, in descending order, as 150BS-12 > 100BS-12 > 50BS-12 > CK, and that the adsorption isotherm could be described by the Langmuir and Freundlich equations. Although the modification of BS-12 was more beneficial for the adsorption of Cu2+, the adsorption capacity and selectivity coefficient of Pb2+ on BS-12-modified bentonites were larger than for Cu2+. Cu2+ and Pb2+ had a mutually antagonistic effect on each other; modification with BS-12 enhanced the effect of Cu2+ on Pb2+ and weakened the effect of Pb2+ on Cu2+, which was always stronger than the effect of Cu2+ on Pb2+. Increases in temperature and pH, and a reduction in ionic strength, can increase the adsorption of Cu2+ and Pb2+; however, the influence of these factors varied between the two metals, which also affected the interaction between the metals.

The transcriptional regulation of an antimicrobial peptide hepcidin1 in Oryzias melastigma upon EE2 exposure involved in a new pathway with a novel transcriptional regulatory element HepERE.

17α-ethinylestradiol (EE2) exerts endocrine disrupting effect and immunotoxic effect on marine animals, including modulation of hepcidin expression. The antimicrobial peptide hepcidin displays a crucial role in innate immunity in fish against invading pathogens. It is known that the transcription of hepcidin in mammals is individually regulated by many stimuli, including inflammation, iron overload, anemia or hypoxia, through several distinct molecular pathways. The canonical mechanism for endocrine disrupting effects is mediated by an estrogen receptor (ER) and estrogen responsive element (ERE), whereas the underlying mechanism for immunotoxic effect is still unclear. In this study, a hepcidin from Oryzias melastigma (OM-hep1) was found to be down-regulated upon EE2 exposure and was associated with ERα. Unlike the revealed signaling pathways for hepcidin regulation in mammals, it was revealed by promoter activity analysis that the OM-hep1 transcription was not associated with canonical immune-associated and hormone-associated regulatory elements, known as the nuclear factor κB (NF-κB), signal transducer and activator of transcription 3 (STAT3), ERE and estrogen-related receptor responsive element (ERRE). Further analysis through a series of base mutations revealed a short fragment from -315 to -289 bp on the OM-hep1 promoter with high activity. This fragment was composed of a putative ERE-like element (23 bases) plus an adjacent down-streamed four bases motif GTGT. Replacement of either of the core bases (GGTCA) of ERE-like or GTGT motif showed non-activity and non-response to EE2 exposure, thus a new hepcidin-associated element named as HepERE was revealed. Evidences from electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) assay demonstrated that the EE2-mediated down-regulation of OM-hep1 expression was associated with ERα binding to HepERE but not classical ERE. Taken together, a novel signaling pathway was revealed and the regulatory mechanism associated with the ERα and HepERE element on immunomodulation of OM-hep1 expression upon EE2 exposure was first reported here.

[Animal model analysis on transient ischemic attack based on clinical symptom characteristics of Chinese and Western medicine].

Based on the clinical symptom characteristics of transient ischemic attack in Chinese and Western medicines, the existing models of transient ischemic attack were summarized and analyzed. Then the advantages and disadvantages of each model, the diagnostic criteria of traditional Chinese and Western medicine and clinical symptoms compliance were analyzed to put forward the evaluation method and improvement method of the corresponding animal models. It was found that there were many modeling methods of transient ischemic attack, but they can not reflect the transience, reversibility, recurrence and other typical characteristics of the disease, with significant differences with clinical symptoms. Moreover, there is lack of reasonable quantitative criteria for the success of the animal model. By combining the existing single factor animal models, a composite animal model that was more closely related to the clinical symptoms of transient ischemic attack was established to replicate an animal model that was more compatible with the characteristics of clinical symptoms. It is the future development directions of the transient ischemic attack animal models to establish reasonable quantitative standards, reflect the causes of Chinese and Western medicine symptoms and improving a series of systematic and complete model evaluation methods.