Pulsed Low-Intensity Focused Ultrasound (LIFU) Activation of Ovarian Follicles.

Objective: A biological system's internal morphological structure or function can be changed as a result of the mechanical effect of focused ultrasound. Pulsed low-intensity focused ultrasound (LIFU) has mechanical effects that might induce follicle development with less damage to ovarian tissue. The potential development of LIFU as a non-invasive method for the treatment of female infertility is being considered, and this study sought to explore and confirm that LIFU can activate ovarian follicles. Results: We found a 50% increase in ovarian weight and in the number of mature follicles on the ultrasound-stimulated side with pulsed LIFU and intraperitoneal injection of 10 IU PMSG in 10-day-old rats. After ultrasound stimulation, the PCOS-like rats had a decrease in androgen levels, restoration of regular estrous cycle and increase in the number of mature follicles and corpora lutea, and the ratio of M1 and M2 type macrophages was altered in antral follicles of PCOS-like rats, consequently promoting further development and maturation of antral follicles. Conclusion: LIFU treatment could trigger actin changes in ovarian cells, which might disrupt the Hippo signal pathway to promote follicle formation, and the mechanical impact on the ovaries of PCOS-like rats improved antral follicle development.

Cimifugin Relieves Histamine-Independent Itch in Atopic Dermatitis via Targeting the CQ Receptor MrgprA3.

Background: We previously found that cimifugin has a potent antiallergic inflammatory effect in atopic dermatitis (AD). However, whether cimifugin has an antipruritic effect in AD was unknown. Methods: Mouse scratching behavior tests were performed to verify the proposed antipruritic effect of cimifugin on DNFB- or FITC-mediated AD. Chloroquine (CQ)- and compound 48/80-evoked acute itch models were employed to clarify the effect of cimifugin on histamine-dependent or -independent itch. Intracellular calcium changes were assessed in a primary culture of mouse dorsal root ganglia (DRG) in response to pruritogen exposure with or without cimifugin treatment, including CQ, histamine, allyl-isothiocyanate (AITC), and capsaicin. Molecular docking and microscale thermophoresis (MST) assays were performed to predict and verify the binding ability and modes between cimifugin and the CQ receptor MrgprA3, respectively. Results: We found that cimifugin attenuates itch behaviors effectively in FITC-induced AD. Notably, cimifugin significantly alleviated acute itching behaviors induced by CQ but not compound 48/80 in vivo. Moreover, cimifugin remarkably inhibited CQ-evoked calcium influx in DRG cells but had no obvious effect on histamine-induced calcium influx. Nevertheless, cimifugin did not interfere with either AITC-stimulated TRPA1 activation- or capsaicin-stimulated TRPV1 activation-mediated calcium influx in DRG cells. Molecular docking predicted that CQ and cimifugin might share similar binding abilities and binding modes with MrgprA3. MST assay confirmed cimifugin directly targeting MrgprA3. Conclusion: The present study demonstrates that cimifugin has a potent antipruritic effect in AD with a histamine-independent mechanism via targeting the CQ receptor MrgprA3. Thus, cimifugin is a promising candidate antipruritic agent for AD.

CHARR efficiently estimates contamination from DNA sequencing data.

DNA sample contamination is a major issue in clinical and research applications of whole-genome and -exome sequencing. Even modest levels of contamination can substantially affect the overall quality of variant calls and lead to widespread genotyping errors. Currently, popular tools for estimating the contamination level use short-read data (BAM/CRAM files), which are expensive to store and manipulate and often not retained or shared widely. We propose a metric to estimate DNA sample contamination from variant-level whole-genome and -exome sequence data called CHARR, contamination from homozygous alternate reference reads, which leverages the infiltration of reference reads within homozygous alternate variant calls. CHARR uses a small proportion of variant-level genotype information and thus can be computed from single-sample gVCFs or callsets in VCF or BCF formats, as well as efficiently stored variant calls in Hail VariantDataset format. Our results demonstrate that CHARR accurately recapitulates results from existing tools with substantially reduced costs, improving the accuracy and efficiency of downstream analyses of ultra-large whole-genome and exome sequencing datasets.

Spatiotemporal observations of host-pathogen interactions in mucosa during SARS-CoV-2 infection indicate a protective role of ILC2s.

IMPORTANCE: Our study revealed the spatial interaction between humanized ACE2 and pseudovirus expressing Spike, emphasizing the role of type 2 innate lymphoid cells during the initial phase of viral infection. These findings provide a foundation for the development of mucosal vaccines and other treatment approaches for both pre- and post-infection management of coronavirus disease 2019.

Mfat-1 ameliorates cachexia after hypoxic-ischemic brain damage in mice by protecting the hypothalamus-pituitary-adrenal axis.

AIMS: Cachexia, a metabolic syndrome, affects 21 % of patients suffering from ischemic encephalopathy. However, the specific mechanism and prevention measures are still unclear. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been proven to reduce inflammatory cytokine levels during ischemic events, but whether they have a protective effect against cachexia after hypoxic-ischemic brain damage (HIBD) remains unclear. MAIN METHODS: C57BL/6J wild-type and mfat-1 transgenic male mice were treated with and without HIBD. One day after HIBD, the epididymal white fat, gastrocnemius muscle and hypothalamus were weighed and analyzed the phenotypic changes. RNA sequencing was applied to gastrocnemius muscle to identify differential genes and pathways in HIBD groups. The effect of HPA axis on cachexia post-HIBD was examined via adrenalectomy, dexamethasone (0.1 mg/kg), and corticosterone injection (100 mg/kg). KEY FINDINGS: The results showed that the incidence of cachexia in mfat-1 mice, which produce high proportion of n-3 PUFAs, was significantly lower than that in wild-type mice post-HIBD. Cachexia-related factors, such as inflammation, muscle atrophy and lipid metabolism were significantly improved in mfat-1 HIBD. RNA sequencing revealed that catabolic and proteasome pathways were significantly downregulated. In hypothalamus, inflammatory cytokines, lipid peroxidation levels were reduced. Corticosterone, glucocorticoid receptor, and dexamethasone suppression test all showed that mfat-1 improved the dysfunction of the HPA axis post-HIBD. The present study elucidated for the first time that mfat-1 reduced HIBD-induced hyperactivation of the HPA axis in mice by reducing inflammation and oxidative stress and contributed to the reduction of metabolic imbalance in peripheral tissues. SIGNIFICANCE: Our study provides mechanistic information for the development of intervention strategies to prevent cachexia.

CHARR efficiently estimates contamination from DNA sequencing data.

DNA sample contamination is a major issue in clinical and research applications of whole genome and exome sequencing. Even modest levels of contamination can substantially affect the overall quality of variant calls and lead to widespread genotyping errors. Currently, popular tools for estimating the contamination level use short-read data (BAM/CRAM files), which are expensive to store and manipulate and often not retained or shared widely. We propose a new metric to estimate DNA sample contamination from variant-level whole genome and exome sequence data, CHARR, Contamination from Homozygous Alternate Reference Reads, which leverages the infiltration of reference reads within homozygous alternate variant calls. CHARR uses a small proportion of variant-level genotype information and thus can be computed from single-sample gVCFs or callsets in VCF or BCF formats, as well as efficiently stored variant calls in Hail VDS format. Our results demonstrate that CHARR accurately recapitulates results from existing tools with substantially reduced costs, improving the accuracy and efficiency of downstream analyses of ultra-large whole genome and exome sequencing datasets.

Region-related and layer-specific permeability of the iris vasculature with morphological mechanism: A novel understanding of blood-aqueous barrier.

The permeability of iris blood vessels has an important role in maintaining aqueous humor (AH) homeostasis, contributing to variation in iris volume and probably the pathogenesis of angle closure glaucoma. This study investigates the permeability of the iris microvasculature to plasma-derived protein and correspond it with the morphologic characteristics of vascular mural cells (MCs). Twenty-two enucleated porcine eyes were used in this study. 12 eyes were micro-perfused with vehicle alone as control or with FITC-albumin as a marker of protein leakage and histological sections subsequently made to examine for FITC-albumin presence. The other 10 eyes were immunolabeled via micro-perfusion for αSMA and VE-cadherin to investigate their topographic distribution in the porcine iris vasculature, and to cross correspond with the locations of FITC-albumin deposits. Distribution of FITC-signals exhibited a site-dependent pattern and time-dependent change in the iris. Fluorescence was initially detected around capillaries in the superficial and deep layer of the iris microvascular network. The pupillary region and the iris root retained more fluorescent signal than the iridal ciliary region. At low magnification, αSMA labelling displayed a regional variation which was inversely correlated with vascular permeability. At the cellular level, αSMA labeling corresponded with vascular MCs distribution in the iris vascular network. The correspondence between iris microvascular permeability to FITC-albumin and the pattern of αSMA distribution and MCs coverage adds to the understanding of the elements comprising the blood-aqueous barrier with implications for the bio-mechanics of iris volume change.

Simulating time-to-event data subject to competing risks and clustering: A review and synthesis.

Simulation studies play an important role in evaluating the performance of statistical models developed for analyzing complex survival data such as those with competing risks and clustering. This article aims to provide researchers with a basic understanding of competing risks data generation, techniques for inducing cluster-level correlation, and ways to combine them together in simulation studies, in the context of randomized clinical trials with a binary exposure or treatment. We review data generation with competing and semi-competing risks and three approaches of inducing cluster-level correlation for time-to-event data: the frailty model framework, the probability transform, and Moran's algorithm. Using exponentially distributed event times as an example, we discuss how to introduce cluster-level correlation into generating complex survival outcomes, and illustrate multiple ways of combining these methods to simulate clustered, competing and semi-competing risks data with pre-specified correlation values or degree of clustering.

A systemic review and network meta-analysis of accuracy of intraocular lens power calculation formulas in primary angle-closure conditions.

BACKGROUND: For primary angle-closure and angle-closure glaucoma, the fact that refractive error sometimes deviates from predictions after intraocular lens (IOL) implantation is familiar to cataract surgeons. Since controversy remains in the accuracy of IOL power calculation formulas, both traditional and network meta-analysis on formula accuracy were conducted in patients with primary angle-closure conditions. METHODS: A comprehensive literature search was conducted through Aug 2022, focusing on studies on intraocular lens power calculation in primary angle-closure (PAC) and primary angle-closure glaucoma (PACG). A systemic review and network meta-analysis was performed. Quality of studies were assessed. Primary outcomes were the mean absolute errors (MAE) and the percentages of eyes with a prediction error within ±0.50 diopiters (D) or ±1.00 D (% ±0.50/1.00 D) by different formulas. RESULTS: Six retrospective studies involving 419 eyes and 8 formulas (Barrett Universal II, Kane, SRK/T, Hoffer Q, Haigis, Holladay I, RBF 3.0 and LSF) were included. SRK/T was used as a reference as it had been investigated in all the studies included. Direct comparison showed that none of the involved formula outperformed or was defeated by SRK/T significantly in terms of either MAE or % ±0.50/1.00 D (all P>0.05). Network comparison and ranking possibilities disclosed BUII, Kane, RBF 3.0 with statistically insignificant advantage. No significant publication bias was detected by network funnel plot. CONCLUSIONS: No absolute advantage was disclosed among the formulas involved in this study for PAC/PACG eyes. Further carefully designed studies are warranted to evaluate IOL calculation formulae in this target population. TRAIL REGISTRATION: Registration: PROSEPRO ID: CRD42022326541.

Out of step societal and Darwinian adaptation during evolution is the cause of multiple women's health issues.

During human evolution, major changes in our societal conditions and environment took place without sufficient time for concomitant genetic alterations, leading to out of step adaptation and diseases in women. We first discuss recent societal adaptation mismatch (menstrual bleeding; increases in cancers of reproductive organs, endometriosis; mother's nursing; polycystic ovarian syndrome; transgenerational epigenetic modifications), followed by Darwinian out of step adaptation (labor difficulties; sex chromosomes, human diseases and sex disparity in genomic DNA). We discuss the evolutionary basis of menstrual bleeding, followed by recent increases in cancers of reproductive organs and endometriosis. The importance of breastfeeding by mothers is also emphasized. Earlier onset of menarche, decreased rates of childbirths and breastfeeding resulted in increased number of menstrual cycles in a lifetime, coupled with excess estrogen exposure and incessant ovulation, conditions that increased the susceptibility to mammary and uterine cancers as well as ovarian epithelial cancer and endometriosis. Shorter lactation duration in mothers also contributed to more menstrual cycles. We further discuss the evolutionary basis of the prevalent polycystic ovary syndrome. During the long-term Darwinian evolution, difficulties in childbirth evolved due to a narrowed pelvis, our upright walking and enlarged fetal brain sizes. Because there are 1.5% genomic DNA differences between woman and man, it is of significance to investigate sex-specific human physiology and diseases. In conclusion, understanding out of step adaptation during evolution could allow the prevention and better management of female reproductive dysfunction and diseases.

Three-dimensional visualization of electroacupuncture-induced activation of brown adipose tissue via sympathetic innervation in PCOS rats.

BACKGROUND: Low-frequency electroacupuncture (EA) has been shown to ameliorate obesity and reproductive dysfunctions in patients with polycystic ovary syndrome (PCOS), and further explorations in PCOS-like rats showed that EA could affect white adipose tissue. However, the function and neuromodulation of brown adipose tissue (BAT) in PCOS and after EA treatment have remained unknown. The present study focused on the role of BAT in PCOS-like rats and its relationship with EA and characterized the three-dimensional (3D) innervation of BAT associated with activation molecules. METHODS: Female rats (21 days old) were implanted with dihydrotestosterone or fed with a high fat diet to establish PCOS-like and obesity models, respectively, and then EA treatment at "Guilai" (ST 29) and "Sanyinjiao" (SP 6) was carried out for 4 weeks. In the present study, morphological analysis, 3D imaging, molecular biology, and other experimental techniques were used to study the sympathetic nerves and activity of BAT. RESULTS: PCOS-like rats showed both obvious weight gain and reproductive dysfunction, similar to what was seen in obese rats except for the absence of reproductive dysfunction. The body weight gain was mainly caused by an increase in white adipose tissue, and there was an abnormal decrease in BAT. Because both the lipid metabolism and reproductive disorders could be improved with bilateral EA at "Guilai" (ST 29) and "Sanyinjiao" (SP 6), especially the restoration of BAT, we further investigated the neuromodulation and inflammation in BAT and identified the sympathetic marker tyrosine hydroxylase as one of the key factors of sympathetic nerves. Modified adipo-clearing technology and 3D high-resolution imaging showed that crooked or dispersed sympathetic nerves, but not the twisted vasculature, were reconstructed and associated with the activation of BAT and are likely to be the functional target for EA treatment. CONCLUSION: Our study highlights the significant role of BAT and its sympathetic innervations in PCOS and in EA therapy.

A comparison of analytical strategies for cluster randomized trials with survival outcomes in the presence of competing risks.

While statistical methods for analyzing cluster randomized trials with continuous and binary outcomes have been extensively studied and compared, little comparative evidence has been provided for analyzing cluster randomized trials with survival outcomes in the presence of competing risks. Motivated by the Strategies to Reduce Injuries and Develop Confidence in Elders trial, we carried out a simulation study to compare the operating characteristics of several existing population-averaged survival models, including the marginal Cox, marginal Fine and Gray, and marginal multi-state models. For each model, we found that adjusting for the intraclass correlations through the sandwich variance estimator effectively maintained the type I error rate when the number of clusters is large. With no more than 30 clusters, however, the sandwich variance estimator can exhibit notable negative bias, and a permutation test provides better control of type I error inflation. Under the alternative, the power for each model is differentially affected by two types of intraclass correlations-the within-individual and between-individual correlations. Furthermore, the marginal Fine and Gray model occasionally leads to higher power than the marginal Cox model or the marginal multi-state model, especially when the competing event rate is high. Finally, we provide an illustrative analysis of Strategies to Reduce Injuries and Develop Confidence in Elders trial using each analytical strategy considered.

Electroacupuncture improves metabolic and ovarian function in a rat model of polycystic ovary syndrome by decreasing white adipose tissue, increasing brown adipose tissue, and modulating the gut microbiota.

BACKGROUND: Polycystic ovary syndrome (PCOS) affects 8%-15% of reproductive-age women and is associated with reproductive disorders, abdominal obesity, hyperinsulinemia, insulin resistance, type 2 diabetes, and cardiovascular diseases. Acupuncture, as a traditional physical therapy method, could affect various metabolic disorders such as obesity, hyperplasia, gout, and cardiovascular and cerebrovascular diseases in clinical practice. Moreover, electroacupuncture (EA) has been shown to decrease body weight in rats with PCOS; however, the mechanism of weight loss and the relationship between adipose tissue and gut microbiota remain unclear. OBJECTIVE: To explore the effect and mechanism of EA on white and brown adipose tissues and gut microbiota, and its follow-up effect on reproductive function, in a rat model of PCOS. METHODS: Daily EA treatment was administered at ST29 and SP6 in a dihydrotestosterone (DHT)-induced PCOS-like rat model (PCOS + EA group). Effects of EA on in vivo and in vitro adipose volume and weight, organ weight coefficients, body weight, hormonal profiles, and estrous cyclicity were measured, and compared with untreated PCOS model rats (PCOS group) and healthy rats (control group). Microbial DNA was extracted from the fecal samples to analyze group abundance and diversity. RESULTS: EA improved estrous cyclicity, decreased body weight, decreased visceral and subcutaneous fat content, and increased brown adipose tissue weight. EA also normalized serum DHT and progesterone levels and improved glucose tolerance. There were few significant differences in the composition or diversity of the gut microbiota between control, PCOS, and PCOS + EA groups, except for the relative abundances of Tenericutes at the phylum level and Prevotella_9 at the genus level, which were significantly different in the PCOS group before and after EA treatment. Both are important microflora, strongly related to body weight. CONCLUSION: EA regulated the metabolic disorders and improved reproductive function in this PCOS-like rat model by adjusting visceral fat and brown fat, as well as intestinal flora.

Systematic single-variant and gene-based association testing of thousands of phenotypes in 394,841 UK Biobank exomes.

Genome-wide association studies have successfully discovered thousands of common variants associated with human diseases and traits, but the landscape of rare variations in human disease has not been explored at scale. Exome-sequencing studies of population biobanks provide an opportunity to systematically evaluate the impact of rare coding variations across a wide range of phenotypes to discover genes and allelic series relevant to human health and disease. Here, we present results from systematic association analyses of 4,529 phenotypes using single-variant and gene tests of 394,841 individuals in the UK Biobank with exome-sequence data. We find that the discovery of genetic associations is tightly linked to frequency and is correlated with metrics of deleteriousness and natural selection. We highlight biological findings elucidated by these data and release the dataset as a public resource alongside the Genebass browser for rapidly exploring rare-variant association results.

Recent progress in optical clearing of eye tissues.

The growing need for viewing the detailed 3D structures of various tissues and organs requires advanced tissue processing and imaging techniques. However, light scattering by tissues hinders detailed structural observations. To overcome this, the emerging technique of "tissue optical clearing" has been flourishing in recent decades, providing excellent opportunities for imaging deep, micro-scale structures of various organs, or even of the whole body. In recent years, advanced tissue clearing techniques have been optimized for specific tissues and organs. Among these tissues, the eye is unique owing to its delicate structure and pigmented retinal epithelial cells, calling for more work on making these tissues "transparent". In this review, we searched Medline and Embase for studies published between January 2006 and August 2021 using the terms "tissue optical clearing", "ophthalmology", "eye", and "optical clearing agents", and we reviewed the publications on the optical clearing techniques of eye tissue from 2006 to the present, including both the clearing procedures and the subsequent analytical processes, thus gaining more insight into the application of tissue optical clearing in basic eye research. Furthermore, we discuss the future potential of optical clearing applications in clinical ophthalmology.

Spatial topological analysis of sympathetic neurovascular characteristic of acupoints in Ren meridian using advanced tissue-clearing and near infrared II imaging.

Acupuncture has been used for treating various medical conditions in traditional Chinese medicine. Both manual and electro-acupuncture stimulate specific acupoints to obtain local and systemic biological effects, but the underlying mechanisms remain unclear. Here, we used three-dimensional tissue-clearing technology to study acupoints on the Ren meridian of mice to reveal the distribution, density, branching, and relationships between blood vessels and nerves. Using topological Mapper methods, we found that sympathetic neurovascular networks were denser in the CV 4 acupoint compared with surrounding non-acupoints. Furthermore, high resolution in vivo real-time vascular imaging using the near infrared-II probe LZ-1105 demonstrated increased blood flow in the CV 4 acupoint compared with neighboring non-acupoints after manual or electro-acupuncture. Consistent with earlier findings, our research indicated that acupuncture could enhance local blood flow, and our high-resolution 3D images show for the first time the important role of sympathetic neurovascular networks in the CV 4 acupoint.

Ovarian Innervation Coupling With Vascularity: The Role of Electro-Acupuncture in Follicular Maturation in a Rat Model of Polycystic Ovary Syndrome.

Low-frequency electro-acupuncture (EA) has been shown to restore ovulation in patients with polycystic ovary syndrome (PCOS), and previous animal experiments showed that EA improves ovarian blood flow and angiogenesis. We performed EA for 4 weeks in dihydrotestosterone (DHT)-induced PCOS-like rats and investigated the three-dimensional (3D) ovarian innervation to determine the role of innervation in folliculogenesis and vascularity. Ovarian tissues were made transparent following the CUBIC 3D tissue-clearing protocol and were immunostained using antibodies against platelet endothelial cell adhesion molecule-1 and tyrosine hydroxylase to visualize the ovarian vasculature and innervation, respectively. This was followed by 3D imaging using lightsheet microscopy and analysis using the Imaris software. In control rats, ovarian innervation increased with age, and the neuronal branching started from the ovarian hilum and reached the individual follicles at different follicle stages. At the individual follicle level, each follicle was mainly innervated by one neuronal fiber. Compared with control rats, ovaries from DHT-treated PCOS-like rats had more antral follicles and fewer preovulatory follicles and corpora lutea. Furthermore, PCOS ovaries showed decreased innervation of blood vessels near the hilum and the surrounding individual antral follicles. EA in PCOS-like rats led to increased numbers of preovulatory follicles and corpora lutea together with increased innervation of blood vessels near the hilum. To determine the role of ovarian innervation, we further performed unilateral sectioning of the superior ovarian nerve (SON) in PCOS + EA rats and found that the left sectioned ovary had fewer preovulatory follicles and corpora lutea compared with those in the right non-sectioned ovary. In conclusion, ovarian innervation likely played an important role in folliculogenesis, and EA might restore PCOS pathophysiology by regulating ovarian innervation, at least partially mediated through the SON.