Comparison of deep learning-assisted blinking analysis system and Lipiview interferometer in dry eye patients: a cross-sectional study.

BACKGROUND: Abnormal blinking pattern is associated with ocular surface diseases. However, blink is difficult to analyze due to the rapid movement of eyelids. Deep learning machine (DLM) has been proposed as an optional tool for blinking analysis, but its clinical practicability still needs to be proven. Therefore, the study aims to compare the DLM-assisted Keratograph 5M (K5M) as a novel method with the currently available Lipiview in the clinic and assess whether blinking parameters can be applied in the diagnosis of dry eye disease (DED). METHODS: Thirty-five DED participants and 35 normal subjects were recruited in this cross-sectional study. DED questionnaire and ocular surface signs were evaluated. Blinking parameters including number of blinks, number of incomplete blinking (IB), and IB rate were collected from the blinking videos recorded by the K5M and Lipiview. Blinking parameters were individually collected from the DLM analyzed K5M videos and Lipiview generated results. The agreement and consistency of blinking parameters were compared between the two devices. The association of blinking parameters to DED symptoms and signs were evaluated via heatmap. RESULTS: In total, 140 eyes of 70 participants were included in this study. Lipiview presented a higher number of IB and IB rate than those from DLM-assisted K5M (P ≤ 0.006). DLM-assisted K5M captured significant differences in number of blinks, number of IB and IB rate between DED and normal subjects (P ≤ 0.035). In all three parameters, DLM-assisted K5M also showed a better consistency in repeated measurements than Lipiview with higher intraclass correlation coefficients (number of blinks: 0.841 versus 0.665; number of IB: 0.750 versus 0.564; IB rate: 0.633 versus 0.589). More correlations between blinking parameters and DED symptoms and signs were found by DLM-assisted K5M. Moreover, the receiver operating characteristic analysis showed the number of IB from K5M exhibiting the highest area under curve of 0.773. CONCLUSIONS: DLM-assisted K5M is a useful tool to analyze blinking videos and detect abnormal blinking patterns, especially in distinguishing DED patients from normal subjects. Large sample investigations are therefore warranted to assess its clinical utility before implementation.

A bi-allelic REC114 loss-of-function variant causes meiotic arrest and nonobstructive azoospermia.

Nonobstructive azoospermia (NOA), the most severe manifestation of male infertility, lacks a comprehensive understanding of its genetic etiology. Here, a bi-allelic loss-of-function variant in REC114 (c.568C > T: p.Gln190*) were identified through whole exome sequencing (WES) in a Chinese NOA patient. Testicular histopathological analysis and meiotic chromosomal spread analysis were conducted to assess the stage of spermatogenesis arrested. Co-immunoprecipitation (Co-IP) and Western blot (WB) were used to investigate the influence of variant in vitro. In addition, our results revealed that the variant resulted in truncated REC114 protein and impaired interaction with MEI4, which was essential for meiotic DNA double-strand break (DSB) formation. As far as we know, this study presents the first report that identifies REC114 as the causative gene for male infertility. Furthermore, our study demonstrated indispensability of the REC114-MEI4 complex in maintaining DSB homoeostasis, and highlighted that the disruption of the complex due to the REC114 variant may underline the mechanism of NOA.

A texture-aware U-Net for identifying incomplete blinking from eye videography.

Accurate identification of incomplete blinking from eye videography is critical for the early detection of eye disorders or diseases (e.g., dry eye). In this study, we develop a texture-aware neural network based on the classical U-Net (termed TAU-Net) to accurately extract palpebral fissures from each frame of eye videography for assessing incomplete blinking. We introduced three different convolutional blocks based on element-wise subtraction operations to highlight subtle textures associated with target objects and integrated these blocks with the U-Net to improve the segmentation of palpebral fissures. Quantitative experiments on 1396 frame images showed that the developed network achieved an average Dice index of 0.9587 and a Hausdorff distance (HD) of 4.9462 pixels when applied to segment palpebral fissures. It outperformed the U-Net and its several variants, demonstrating a promising performance in identifying incomplete blinking based on eye videography.

Germline FOXJ2 overexpression causes male infertility via aberrant autophagy activation by LAMP2A upregulation.

Spermatogenesis is a complex biological process that produces haploid spermatozoa and requires precise regulation by many tissue-specific factors. In this study, we explored the role and mechanism of Fork head box J2 (FOXJ2, which is highly expressed in spermatocytes) in the regulation of spermatogenesis using a germline-specific conditional Foxj2 knock-in mouse model (Stra8-Cre; Foxj2 tg/tg mouse). Foxj2 overexpression in mouse testes led to spermatogenesis failure, which started at the initiation of meiosis, and resulted in male infertility. Lysosomes and autophagy-related genes were upregulated in Stra8-cre; Foxj2 tg/tg mouse testes and the number of autolysosomes in the spermatocytes in Stra8-cre; Foxj2 tg/tg mice was increased. Chromatin immunoprecipitation-PCR and Dual-luciferase reporter assays showed that Lamp2 (encoding lysosome-associated membrane protein-2) was a target of FOXJ2. Foxj2 overexpression increased the expression levels of Lamp2a and Hsc70 (70-kDa cytoplasmic heat shock protein) in the Stra8-cre; Foxj2 tg/tg mouse testes. Our results suggested that Foxj2 overexpression in the germ cells of mouse testes affects chaperone-mediated autophagy by upregulating LAMP2A, leading to spermatogenesis failure at the initiation of meiosis, thus resulting in male infertility. Our findings provide a new insight into the function of FOXJ2 in spermatogenesis and the significance of autophagy regulation in spermatogenesis.

The efficacy of different alveolar recruitment maneuvers in holmium laser lithotripsy surgery under general anesthesia using a laryngeal mask.

BACKGROUND: Alveolar recruitment maneuvers (ARMs) is an important part of lung-protective ventilation strategies (LPVSs), but the optimal duration and interval Remain unclear. METHODS: Patients:252 patients who underwent holmium laser lithotripsy surgery and meet inclusion criteria were included and randomized into three groups based on the duration and frequency of ARMs (Regular, one 30 s ARM (RARMs); Improved and intermittent, three 10s ARMs (IARMs); and Control (C), no ARMs). INTERVENTIONS: Groups R and I received ARMs at 20 cmH2O pressures every 30 min. All patients received the same anesthesia and mechanical ventilation. MEASUREMENTS: Outcomes included heart rate and mean arterial pressure changes during ARMs and postoperative pulmonary complications (PPCs) within the first 7 postoperative days. MAIN RESULTS: Incidences of PPCs in groups R(7.1%) and I (5.0%)were slightly lower than those in group C (8.9%).This indicated the potential to reduce lung injury. Heart rate and mean arterial pressure fluctuations during ARMs were significantly higher in groups R and I than in group C (P < 0.01). The rate of blood pressure decrease was significantly higher in group R than in group I (P < 0.01). CONCLUSIONS: IARMs can reduce cycle fluctuations than RARMs in patients Undergoing holmium laser lithotripsy surgery with laryngeal mask general anesthesia. Low tidal volume ventilation and low PEEP combined with ARM did not significantly reduce the incidence of PPCs in healthy lung patients, but tended to reduce lung injury. TRIAL REGISTRATION: The study was registered on the Chinese Clinical Trial Registry. ( ChiCTR2000030815 ,15/03/2020). This study was approved by the ethics committee of Chengdu Fifth People's Hospital with approval number(2020-005(Study)-1).

Impact of Incomplete Blinking Analyzed Using a Deep Learning Model With the Keratograph 5M in Dry Eye Disease.

Purpose: To establish a deep learning model (DLM) for blink analysis, and investigate whether blink video frame sampling rate influences the accuracy of analysis. Methods: This case-controlled study recruited 50 dry eye disease (DED) participants and 50 normal subjects. Blink videos recorded by a Keratograph 5M, symptom questionnaires, and ocular surface assessments were collected. After processing the blink images as datasets, further training and evaluation of DLM was performed. Blink videos of 30 frames per second (FPS) under white light, eight FPS extracted from white light videos, and eight FPS under infrared light were processed by DLM to generate blink profiles, allowing comparison of blink parameters, and their association with DED symptoms and signs. Results: The blink parameters based on 30 FPS video presented higher sensitivity and accuracy than those based on eight FPS. The average relative interpalpebral height (IPH), the frequency and proportion of incomplete blinking (IB) were much higher in DED participants than in normal controls (P < 0.001). The IB frequency was closely associated with DED symptoms and signs (|R| ≥ 0.195, P ≤ 0.048), as was IB proportion and the average IPH (R ≥ 0.202, P ≤ 0.042). Conclusions: DLM is a powerful tool for analyzing blink videos with high accuracy and sensitivity, and a frame rate ≥ 30 FPS is recommended. The IB frequency is indicative of DED. Translational Relevance: The system of DLM-based blink analysis is of great potential for the assessment of IB and diagnosis of DED.

[Impact of overexpressed FOXJ2 on mouse spermatogenesis and its action mechanism].

OBJECTIVE: To analyze the phenotype of the male reproductive system in the germline-specific conditional Foxj2 knock-in mouse model (Stra8-cre; Foxj2tg/+), identify a target gene of the transcription factor FOXJ2, and investigate the effect of the overexpression of Foxj2 on mouse spermatogenesis and its action mechanism. METHODS: Based on the Cre-loxP recombination system, we generated a germline-specific conditional Foxj2 knock-in mouse model (Stra8-cre; Foxj2tg/+). We determined male fertility by counting the number of pups per litter and the fertilization rate after intracytoplasmic sperm injection (ICSI), observed the morphology of the testes and epididymides by HE staining, examined the sperm quality by computer assisted sperm analysis (CASA), detected the expression and localization of Cx43 in the testis by RT-qPCR, Western blot and immunohistochemistry, and verified the binding site of FOXJ2 to the Cx43 promoter using ChIP-PCR and dual luciferase reporter assay. RESULTS: The number of pups per litter and fertilization rate after ICSI were lower in the Stra8-cre; Foxj2tg/+ male mice than in the controls, and so were the size and weight of the testis. HE staining exhibited obvious exfoliation of germ cells and dramatically decreased spermatocytes and spermatids in the seminiferous tubules of the Stra8-cre; Foxj2tg/+ mice. Moreover, sperm concentration in the cauda epididymides was reduced, and the transcription and expression levels of Cx43 in the testis were increased. ChIP-PCR and dual luciferase reporter assay showed direct binding of FOXJ2 to the Cx43 promoter in the testis. CONCLUSIONS: Overexpressed FOXJ2 may lead to spermatogenic failure and subfertility in Stra8-cre; Foxj2tg/+ male mice by upregulating the expression of Cx43.

Spinal CCK contributes to somatic hyperalgesia induced by orofacial inflammation combined with stress in adult female rats.

In some chronic primary pain conditions such as temporomandibular disorder (TMD) and fibromyalgia syndrome (FMS), mild or chronic stress enhances pain. TMD and FMS often occur together, but the underlying mechanisms are unclear. The purpose of this study was to investigate the role of cholecystokinin (CCK) in the spinal cord in somatic hyperalgesia induced by orofacial inflammation combined with stress. Somatic hyperalgesia was detected by the thermal withdrawal latency and mechanical withdrawal threshold. The expression of CCK1 receptors, CCK2 receptors, ERK1/2 and p-ERK1/2 in the spinal cord was examined by Western blot. After the stimulation of orofacial inflammation combined with 3 day forced swim, the expression of CCK2 receptors and p-ERK1/2 protein in the L4-L5 spinal dorsal horn increased significantly, while the expression of CCK1 receptors and ERK1/2 protein remained unchanged. Intrathecal injection of the CCK2 receptor antagonist YM-022 or mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor PD98059 blocked somatic hyperalgesia induced by orofacial inflammation combined with stress. Intrathecal administration of the MEK inhibitor blocked somatic sensitization caused by the CCK receptor agonist CCK8. The CCK2 receptor antagonist YM-022 significantly reduced the expression of p-ERK1/2. These data indicate that upregulation of CCK2 receptors through the MAPK pathway contributes to somatic hyperalgesia in this comorbid pain model. Thus, CCK2 receptors and MAPK pathway may be potential targets for the treatment of TMD comorbid with FMS.

Control of multiciliogenesis by miR-34/449 in the male reproductive tract through enforcing cell cycle exit.

Multiciliated cells (MCCs) are terminally differentiated postmitotic cells that possess hundreds of motile cilia on their apical surface. Defects in cilia formation are associated with ciliopathies that affect many organs. In this study, we tested the role and mechanism of the miR-34/449 family in the regulation of multiciliogenesis in EDs using an miR-34b/c-/-; miR-449-/- double knockout (dKO) mouse model. MiR-34b/c and miR-449 depletion led to a reduced number of MCCs and abnormal cilia structure in the EDs starting from postnatal day (P)14. However, abnormal MCC differentiation in the dKO EDs could be observed as early as P7. RNA-seq analyses revealed that the aberrant development of MCCs in the EDs of dKO mice was associated with the upregulation of genes involved in cell cycle control. Using a cyclin-dependent kinase inhibitor to force cell cycle exit promoted MCC differentiation, and partially rescued the defective multiciliogenesis in the EDs of dKO mice. Taken together, our results suggest that miR-34b/c and miR-449 play an essential role in multiciliogenesis in EDs by regulating cell cycle exit.

[Effects of miR-34b/c and miR-449 on the morphology and function of efferent ductules in mice: A transcriptomics-based analysis].

OBJECTIVE: To investigate the role of miR-34b/c and miR-449 in maintaining the normal structure and function of efferent ductules and explore the molecular mechanism of infertility in miR-34b/c－/－ and miR-449－/－ dKO mice. METHODS: We observed the morphology of mouse efferent ductules by HE staining and analyzed the gene expressions in the efferent ductules of the wild-type and miR-34b/c－/－ and miR-449－/－ dKO mice by RNA sequencing. Then we screened the possible target genes of these two miRNA clusters and analyzed them along with the differentially expressed genes, followed by verification of the sequencing results by qRT-PCR. RESULTS: Compared with the wild-type, the dKO mice showed morphologically abnormal efferent ductules and significantly decreased expressions of the genes involved in the formation of cilia and related to the transportation of water, ion and protein in the efferent ductules. CONCLUSIONS: The deletion of miR-34b/c and miR-449 led to morphological abnormality of efferent ductules and dysfunction of aberrant cilia motility and reabsorption in the efferent ductules of dKO mice, resulting in infertility.

VceC Mediated IRE1 Pathway and Inhibited CHOP-induced Apoptosis to Support Brucella Replication in Goat Trophoblast Cells.

The effectors of the type IV secretion system (T4SS) of bacteria play important roles in mediating bacterial intracellular proliferation and manipulating host-related pathway responses to bacterial infection. Brucella Spp. inhibit the apoptosis of host cells to benefit their own intracellular proliferation. However, the underlying mechanisms between T4SS effectors and Brucella-inhibited apoptosis in goat trophoblast cells remain unclear. Here, based on Brucella suis vaccine strain 2, the VceC was deleted by allelic exchange. We show that ΔVceC was able to infect and proliferate to high titers in goat trophoblast cells (GTCs) and increase C/EBP-homologous protein (CHOP)-mediated apoptosis. GRP78 expression decreased upon ΔVceC infection. In addition, we discovered that the inositolrequiring enzyme 1 (IRE1) pathway was inhibited in this process. Changing endoplasmic reticulum (ER) stress affected Brucella intracellular replication in GTCs. The replication of ΔVceC was more sensitive under the different ERstress conditions in the GTC line after treatment with ER stress inhibitors 4 phenyl butyric acid (4-PBA) or ER stress activator Tm. Together, our findings show that VceC has a protective effect on the intracellular persistence of Brucella infection, and inhibits ER stress-induced apoptosis in the CHOP pathway. The present work provides new insights for understanding the mechanism of VceC in the establishment of chronic Brucella infection.

Ginsenoside Rg3 Suppresses Proliferation and Induces Apoptosis in Human Osteosarcoma.

Osteosarcoma is the most common primary malignancy of bone in children and the elderly. Recently, more and more researches have demonstrated that Ginsenoside Rg3 (Rg3) is involved in chemotherapy resistance in many cancer, making it a promising Chinese herbal monomer for oncotherapy. In this study, we investigated the efficacy of Rg3 in human osteosarcoma cell lines (MG-63, U-2OS, and SaOS-2). Cell proliferation was measured by CCK8 assay. The migration of cells was examined using the scratch assay method. Quantification of apoptosis was assessed further by flow cytometry. In addition, the expression of apoptosis-related genes (caspase9, caspase3, Bcl2, and Bax) were investigated using RT-PCR. We further investigated the protein level expression of Bcl 2, cleaved-caspase3, and PI3K/AKT/mTOR signaling pathway factors by Western blot assay. Our results revealed that Rg3 inhibited the proliferation and migration of human osteosarcoma cells and induced apoptosis in a concentration- and time-dependent manner. Western blot results showed that Rg3 reduced the protein expression of Bcl2 and PI3K/AKT/mTORbut increased the levels of cleaved-caspase3. Therefore, we hypothesized Rg3 inhibits the proliferation of osteosarcoma cell line and induces their apoptosis by affecting apoptosis-related genes (Bcl2, caspase3) as well as the PI3K/AKT/mTOR signaling pathway. To conclude, Rg3 is a new therapeutic agent against osteosarcoma.

Brucella induces unfolded protein response and inflammatory response via GntR in alveolar macrophages.

Brucella is an intracellular bacterium that causes the zoonosis brucellosis worldwide. Alveolar macrophages (AM) constitute the main cell target of inhaled Brucella. Brucella thwarts immune surveillance and evokes endoplasmic reticulum (ER) stress to replicate in macrophages via virulence factors. The GntR regulators family was concentrated as an important virulence factor in controlling virulence and intracellular survival of Brucella. However, the detailed underlying mechanism for the host-pathogen interaction is poorly understood. In this study the BSS2_II0438 mutant (ΔGntR) was constructed. The type IV secretion system (T4SS) virulence factor genes (VirB2, VirB6, and VirB8) were down-expression in ΔGntR. ΔGntR could infect and proliferate to high titers in GAMs without a significant difference compared with the parental strain. ΔGntR infection increased the expression of ER stress marker genes GRP78, ATF6, and PERK in the early stages of its intracellular cycle but decreased the expression of these genes in the late stages. ΔGntR increased greatly the number of Brucella CFUs in the inactive ER stress state in GAMs. Meanwhile, ΔGntR infection increased the levels of IFN-γ, IL-1ß, and TNF-α, indicating ΔGntR could induce the secretion of inflammatory but not anti-inflammatory cytokines IL-10. Taken together, our results clarified the role of the GntR in B. suis. S2 virulence expression and elucidated that GntR is potentially involved in the signaling pathway of the Brucella-induced UPR and inflammatory response in GAMs.

Mechanisms for the reaction of thiophene and methylthiophene with singlet and triplet molecular oxygen.

Mechanisms for the reaction of thiophene and 2-methylthiophene with molecular oxygen on both the triplet and singlet potential energy surfaces (PESs) have been investigated using ab initio methods. Geometries of various stationary points involved in the complex reaction routes are optimized at the MP2/6-311++G(d, p) level. The barriers and energies of reaction for all product channels were refined using single-point calculations at the G4MP2 level of theory. For thiophene, CCSD(T) single point energies were also determined for comparison with the G4MP2 energies. Thiophene and 2-methylthiophene were shown to react with O(2) via two types of mechanisms, namely, direct hydrogen abstraction and addition/elimination. The barriers for reaction with triplet oxygen are all significantly large (i.e., >30 kcal mol(-1)), indicating that the direct oxidation of thiophene by ground state oxygen might be important only in high temperature processes. Reaction of thiophene with singlet oxygen via a 2 + 4 cycloaddition leading to endoperoxides is the most favorable channel. Moreover, it was found that alkylation of the thiophene ring (i.e., methyl-substituted thiophene) is capable of lowering the barrier height for the addition pathway. The implication of the current theoretical results may shed new light on the initiation mechanisms for combustion of asphaltenes.