A Comprehensive Genetic Study of Microtubule-Associated Gene Clusters for Male Infertility in a Taiwanese Cohort.

Advanced reproductive technologies are utilized to identify the genetic mutations that lead to spermatogenic impairment, and allow informed genetic counseling to patients to prevent the transmission of genetic defects to offspring. The purpose of this study was to identify potential single nucleotide polymorphisms (SNPs) associated with male infertility. Genetic variants that may cause infertility are identified by combining the targeted next-generation sequencing (NGS) panel and whole exome sequencing (WES). The validation step of Sanger sequencing adds confidence to the identified variants. Our analysis revealed five distinct affected genes covering seven SNPs based on the targeted NGS panel and WES data: SPATA16 (rs16846616, 1515442, 1515441), CFTR (rs213950), KIF6 (rs2273063), STPG2 (r2903150), and DRC7 (rs3809611). Infertile men have a higher mutation rate than fertile men, especially those with azoospermia. These findings strongly support the hypothesis that the dysfunction of microtubule-related and spermatogenesis-specific genes contributes to idiopathic male infertility. The SPATA16, CFTR, KIF6, STPG2, and DRC7 mutations are associated with male infertility, specifically azoospermia, and a further examination of this genetic function is required.

Erythropoietin in Optic Neuropathies: Current Future Strategies for Optic Nerve Protection and Repair.

Erythropoietin (EPO) is known as a hormone for erythropoiesis in response to anemia and hypoxia. However, the effect of EPO is not only limited to hematopoietic tissue. Several studies have highlighted the neuroprotective function of EPO in extra-hematopoietic tissues, especially the retina. EPO could interact with its heterodimer receptor (EPOR/ßcR) to exert its anti-apoptosis, anti-inflammation and anti-oxidation effects in preventing retinal ganglion cells death through different intracellular signaling pathways. In this review, we summarized the available pre-clinical studies of EPO in treating glaucomatous optic neuropathy, optic neuritis, non-arteritic anterior ischemic optic neuropathy and traumatic optic neuropathy. In addition, we explore the future strategies of EPO for optic nerve protection and repair, including advances in EPO derivates, and EPO deliveries. These strategies will lead to a new chapter in the treatment of optic neuropathy.

Heme activation by DNA: isoguanine pentaplexes, but not quadruplexes, bind heme and enhance its oxidative activity.

Guanine-rich, single-stranded, DNAs and RNAs are able to fold to form G-quadruplexes that are held together by guanine base quartets. G-quadruplexes are known to bind ferric heme [Fe(III)-protoporphyrin IX] and to strongly activate such bound hemes toward peroxidase (1-electron oxidation) as well as oxygenase/peroxygenase (2-electron oxidation) activities. However, much remains unknown about how such activation is effected. Herein, we investigated whether G-quadruplexes were strictly required for heme activation or whether related multi-stranded DNA/RNA structures such as isoguanine (iG) quadruplexes and pentaplexes could also bind and activate heme. We found that iG-pentaplexes did indeed bind and activate heme comparably to G-quadruplexes; however, iG-quadruplexes did neither. Earlier structural and computational studies had suggested that while the geometry of backbone-unconstrained iG-quintets templated by cations such as Na(+) or NH4 (+) was planar, that of iG-quartets deviated from planarity. We hypothesize that the binding as well as activation of heme by DNA or RNA is strongly supported by the planarity of the nucleobase quartet or quintet that interacts directly with the heme.

Detection of G-quadruplex DNA in mammalian cells.

It has been proposed that guanine-rich DNA forms four-stranded structures in vivo called G-quadruplexes or G4 DNA. G4 DNA has been implicated in several biological processes, but tools to study G4 DNA structures in cells are limited. Here we report the development of novel murine monoclonal antibodies specific for different G4 DNA structures. We show that one of these antibodies designated 1H6 exhibits strong nuclear staining in most human and murine cells. Staining intensity increased on treatment of cells with agents that stabilize G4 DNA and, strikingly, cells deficient in FANCJ, a G4 DNA-specific helicase, showed stronger nuclear staining than controls. Our data strongly support the existence of G4 DNA structures in mammalian cells and indicate that the abundance of such structures is increased in the absence of FANCJ. We conclude that monoclonal antibody 1H6 is a valuable tool for further studies on the role of G4 DNA in cell and molecular biology.

An Essential Role of cAMP Response Element Binding Protein in Ginsenoside Rg1-Mediated Inhibition of Na+/Glucose Cotransporter 1 Gene Expression.

The Na(+)/glucose cotransporter 1 (SGLT1) is responsible for glucose uptake in intestinal epithelial cells. It has been shown that the intestinal SGLT1 level is significantly increased in diabetic individuals and positively correlated with the pathogenesis of diabetes. The development of targeted therapeutics that can reduce the intestinal SGLT1 expression level is, therefore, important. In this study, we showed that ginsenoside Rg1 effectively decreased intestinal glucose uptake through inhibition of SGLT1 gene expression in vivo and in vitro. Transient transfection analysis of the SGLT1 promoter revealed an essential cAMP response element (CRE) that confers the Rg1-mediated inhibition of SGLT1 gene expression. Chromatin immunoprecipitation assay and targeted CRE-binding protein (CREB) silencing demonstrated that Rg1 reduced the promoter binding of CREB and CREB binding protein associated with an inactivated chromatin status. In addition, further studies showed that the epidermal growth factor receptor (EGFR) signaling pathway also plays an essential role in the inhibitory effect of Rg1; taken together, our study demonstrates the involvement of the EGFR-CREB signaling pathway in the Rg1-mediated downregulation of SGLT1 expression, which offers a potential strategy in the development of antihyperglycemic and antidiabetic treatments.

DNA mechatronic devices switched by K⁺ and by Sr²âº are structurally, topologically, and electronically distinct.

DNAs and RNAs that fold via the formation of guanine quartets form G-quadruplexes that are often highly diverse in terms of architecture and topology. G-quadruplexes are specifically stabilized by metal cations such as K(+) and Sr(2+), but not Li(+). DNA duplexes that incorporate two separated clusters of G•G mismatches ("P-duplexes") can function as electronic switches, capable of toggling reversibly from a poorly conductive conformer (E) with only Li(+) in the solution to a G-quadruplex incorporating conformer of higher conductivity (C) in the presence of K(+). Herein, we report results from fluorescence energy transfer, circular dichroism, charge conduction, and chemical footprinting experiments, which cumulatively demonstrate that P-duplex E↔C transitions are genuinely mechatronic, with causally coupled mechanical and electronic states. We show, further, that the K(+) - and the Sr(2+)-fuelled E↔C switching of a given P-duplex are structurally, topologically, and electronically distinct from each other. A single DNA P-duplex can thus exist in at least three distinguishable mechatronic states in aqueous solution.

Mechatronic DNA devices driven by a G-quadruplex-binding platinum ligand.

Contractile duplexes are DNA double helices that incorporate two strategically placed patches of guanine-guanine (G·G) base mismatches. Such duplexes are cation-driven mechatronic devices, able to toggle between states with distinct mechanical and charge conduction properties. In aqueous lithium chloride solution contractile duplexes have an extended (E) and poorly conductive conformation; however, potassium ions drive them to a relatively conductive and structurally contracted (C) conformation, via intramolecular G-quadruplex formation. Here, we report that even in the absence of K(+) ions, a known G-quadruplex binding ligand, Pt-PIP [phenylphenanthroimidazole ethylenediamine platinum(II)] efficiently promotes the E→C transition, while a poor binder, Pt-bpy [bipyridine ethylenediamine platinum(II)], does not promote this transition. An examination of E→C transitions within two different designs for DNA contractile helices found an unexpected complexity: the formation of distinct C states, both electrically conductive, but possessing dissimilar DNA topologies. Ligand-driven DNA mechatronic devices such as these may constitute prototypes for electronic biosensors that identify G-quadruplex binding ligands.

A twisting electronic nanoswitch made of DNA.

Single-stranded DNAs and RNAs that are rich in the nucleobase guanine form four-stranded G-quadruplexes, which are held together by hydrogen-bonded guanine quartets. In aqueous solution, both DNA duplexes and G-quadruplexes are modest conductors of electrical charge. A tight, topologically constrained DNA construct called twDNA is now reported, in which a core of four guanine-rich single strands structurally and electronically links together four DNA double helices. The addition and removal of K(+) or Sr(2+) cations promote alternative conformers of twDNA, which have strikingly distinct electronic properties. Unlike DNA mechano-electronic switches that require large conformational changes, twDNA requires only modest twisting/untwisting structural attenuations to achieve electronic switching.

Activation of the ROCK/MYLK Pathway Affects Complex Molecular and Morphological Changes of the Trabecular Meshwork Associated With Ocular Hypertension.

Purpose: The Rho-associated protein kinase and myosin light chain kinase (ROCK/MYLK) pathway undeniably plays a pivotal role in the pathophysiology of primary open-angle glaucoma (POAG). In our study, we utilized both ocular hypertension (OHT) rabbit models and clinical investigations to gain invaluable insights that propel the development of novel treatments targeting proteins and genes associated with the trabecular meshwork (TM), thereby offering promising avenues for the management of POAG. Methods: Following microbead injections into the anterior chamber of the ocular cavity of rabbits, we observed elevated histiocyte numbers and immune scores for MYLK-4/ pMLC-2, alongside a reduction in the void space within the TM. Notably, treatment was performed with 0.1% ITRI-E-(S)-4046, a compound with dual kinase inhibitor (highly specific inhibitor of ROCK1/2 and MYLK4), significantly reduced intraocular pressure (IOP; P < 0.05) and expanded the void space within the TM (P < 0.0001) compared with OHT rabbits. In clinical investigations, we utilized whole transcriptome sequencing to analyze gene expression specifically related to the TM, obtained from patients (5 early-onset and 5 late-onset) undergoing trabeculectomy. Results: Our findings revealed 103 differential expression genes (DEGs) out of 265 molecules associated with the Rho family GTPase pathway, exhibiting a P value of 1.25E-10 and a z-score of -2.524. These results underscore significant differences between the early-onset and late-onset POAG and highlight the involvement of the ROCK/MYLK pathway. Conclusions: These findings underscore the critical involvement of the ROCK/MYLK pathway in both OHT-related and different onsets of POAG, providing valuable insights into the TM-related molecular mechanisms underlying the disease.

Investigation of the Protective Effect of Extracellular Vesicle miR-124 on Retinal Ganglion Cells Using a Photolabile Paper-Based Chip.

Purpose: Photolabile paper-based chips were developed to isolate extracellular vesicles (EVs) from small-volume samples (less than 30 µL), such as vitreous humor. Putative neuroprotective effects of EVs' microRNAs were investigated by using the paper chip and a rodent model with nonarteritic anterior ischemic optic neuropathy (rNAION). Methods: rNAION was established using laser-induced photoactivation of rose bengal administered intravenously. On days 0, 0.25, 1, 3, and 7 after rNAION induction, CD63-positive EV microRNAs (CD63+-EV miRNAs) in vitreous humor samples were enriched using the paper chip and assessed using microarray and quantitative RT-PCR analyses. The viability and visual function of retinal ganglion cells (RGCs) were further assessed by measuring photopic flash visual evoked potentials (FVEPs). Results: We identified 38 different variations of CD63+-EV miRNAs with more than twofold altered expressions. Among them, M1-related miRNA, mR-31a-5p, and M2-related miRNA, miR-125a-5p, miR-182, miR-181a-5p, and miR-124-3, were capable of coordinating anti-inflammatory reactions during rNAION because of their capacity to activate macrophages. In particular, miR-124, having the most dramatic alteration of gene expression, was synthesized and injected intravitreally. Compared to controls, rats that received miR-124 had shown increased RGC survivability and improved visual function. Conclusions: Our research team has developed a paper-based chip capable of capturing EVs that can be released after UV exposure. The quantity and quality of EV-miRNAs extracted are adequate for microarray and quantitative RT-PCR analyses. Animal studies suggest that miR-124 may play a neuroprotective role in the natural recovery of rNAION and holds the potential to be a novel treatment option.

Assessing the Feasibility of Using Electrochemical Skin Conductance as a Substitute for the Quantitative Sudomotor Axon Reflex Test in the Composite Autonomic Scoring Scale and Its Correlation with Composite Autonomic Symptom Scale 31 in Parkinson's Disease.

The Composite Autonomic Scoring Scale (CASS) is a quantitative scoring system that integrates the sudomotor, the cardiovagal, and the adrenergic subscores, and the Composite Autonomic Symptom Scale 31 (COMPASS 31) is based on a well-established comprehensive questionnaire designed to assess the autonomic symptoms across multiple domains. We tested the hypothesis that electrochemical skin conductance (Sudoscan) can be a substitute for the quantitative sudomotor axon reflex test (QSART) in the sudomotor domain and assessed its correlation with COMPASS 31 in patients with Parkinson's disease (PD). Fifty-five patients with PD underwent clinical assessment and cardiovascular autonomic function tests and completed the COMPASS 31 questionnaire. We compared the modified CASS (integrating the Sudoscan-based sudomotor, adrenergic, and cardiovagal subscores) and CASS subscores (the sum of the adrenergic and cardiovagal subscores). The total weighted score of COMPASS 31 was significantly correlated with both the modified CASS and the CASS subscore (p = 0.007 and p = 0.019). The correlation of the total weighted score of COMPASS 31 increased from 0.316 (CASS subscores) to 0.361 (modified CASS). When we added the Sudoscan-based sudomotor subscore, the case numbers for autonomic neuropathy (AN) increased from 22 (40%, CASS subscores) to 40 (72.7%, modified CASS). The modified CASS not only better reflects the exact autonomic function, but also improves the characterization and quantification of AN in patients with PD. In areas in which a QSART facility is not easily available, Sudoscan could be a time-saving substitution.

Corrigendum: Cell volume restriction by mercury chloride reduces M1-like inflammatory response of bone marrow-derived macrophages.

[This corrects the article DOI: 10.3389/fphar.2022.1074986.].

Quantitative thermal testing as a screening and follow-up tool for diabetic sensorimotor polyneuropathy in patients with type 2 diabetes and prediabetes.

Introduction: The diagnosis and assessment of neuropathy severity of diabetic sensorimotor polyneuropathy (DSPN) are mainly based on clinical neuropathy scores and electrophysiologic studies. This study aimed to determine whether quantitative thermal testing (QTT) can be used as a screening and follow-up tool for DSPN of prediabetes and type 2 diabetes at baseline and at 1-year follow-up. Methods: All patients were assessed using the Toronto Clinical Neuropathy Score (TCNS) and underwent electrophysiological testing, including a nerve conduction study (NCS) and QTT, at baseline and at a 1-year follow-up. The TCNS and the composite scores of nerve conduction were used to assess the severity of DSPN. The DSPN status at the 1-year follow-up was classified as remaining no DSPN, remaining DSPN, regression to no DSPN, or progression to DSPN. Results: Diabetic sensorimotor polyneuropathy was initially diagnosed in 89 patients with prediabetes and type 2 diabetes (22%). The regressed to no DSPN in 29 patients and progressed to DSPN in 20 patients at the 1-year follow-up. TCNS was significantly correlated with composite scores of nerve conduction, hand cold detection threshold (CDT), hand warm detection threshold (WDT), foot CDT, and foot WDT. Stepwise logistic regression demonstrated that the foot CDT (p < 0.0001) was independently associated with the presence of DSPN. The TCNS, composite scores of the nerve conduction, hand WDT, hand CDT, foot WDT, and foot CDT were all statistically significant among the four different DSPN status groups at two different time periods (baseline and the 1-year follow-up). Conclusion: The foot CDT can be used as an initial screening tool for DSPN alternatively. The characteristics of nerve damage after 1 year of DSPN can be progressive or reversible, and the neurological functions of large and small fibers have a parallel trend, which can be objectively measured by NCS and QTT.

RNF128 regulates neutrophil infiltration and myeloperoxidase functions to prevent acute lung injury.

Acute lung injury (ALI) is characterised by severe pulmonary inflammation, alveolar-capillary barrier disruption, and pulmonary oedema. Therefore, establishing effective therapeutic targets for ALI prevention is crucial. The present study reports a novel function of RNF128 in regulating LPS-induced ALI. Severe lung damage and increased immune cell infiltration were detected in RNF128-deficient mice. In vitro experiments revealed that RNF128 inhibits neutrophil activation by binding to myeloperoxidase (MPO) and reducing its levels and activity. Moreover, RNF128 regulates alveolar macrophage activation and neutrophil infiltration by interacting with TLR4, targeting it for degradation, and inhibiting NF-κB activation, hence decreasing pro-inflammatory cytokines. Our results demonstrate for the first time that RNF128 is a negative regulator of MPO and TLR4 in neutrophils and alveolar macrophages, respectively. However, AAV9-mediated RNF128 overexpression alleviated lung tissue damage and reduced inflammatory cell infiltration. Thus, RNF128 is a promising therapeutic candidate for pharmacological interventions in ALI.

ERCC1 polymorphisms and risk of adult glioma in a Chinese population: a hospital-based case-control study.

To examine the associations of two polymorphisms in excision repair cross-complementing rodent repair deficiency complementation group 1 (ERCC1) gene, C8092A (rs3212986) and T19007C (rs11615), with the risk of adult glioma, we performed a hospital-based case-control study with 257 new cases of glioma and 278 controls in Wenzhou, China. Results showed that polymorphisms C8092A and T19007C in ERCC1 gene were not associated with the risk of glioma in a Chinese population. Further studies in Chinese populations with larger sample sizes are still warranted.

Cell volume restriction by mercury chloride reduces M1-like inflammatory response of bone marrow-derived macrophages.

Dysregulation of macrophages in the pro-inflammatory (M1) and anti-inflammatory (M2) sub-phenotypes is a crucial element in several inflammation-related diseases and injuries. We investigated the role of aquaporin (AQP) in macrophage polarization using AQP pan-inhibitor mercury chloride (HgCl2). Lipopolysaccharides (LPSs) induced the expression of AQP-1 and AQP-9 which increased the cell size of bone marrow-derived macrophages. The inhibition of AQPs by HgCl2 abolished cell size changes and significantly suppressed M1 polarization. HgCl2 significantly reduced the activation of the nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) pathways and inhibited the production of IL-1ß. HgCl2 attenuated LPS-induced activation of mitochondria and reactive oxygen species production and autophagy was promoted by HgCl2. The increase in the light chain three II/light chain three I ratio and the reduction in PTEN-induced kinase one expression suggests the recycling of damaged mitochondria and the restoration of mitochondrial activity by HgCl2. In summary, the present study demonstrates a possible mechanism of the AQP inhibitor HgCl2 in macrophage M1 polarization through the restriction of cell volume change, suppression of the p38 MAPK/NFκB pathway, and promotion of autophagy.

Antibody CDR amino acids underlying the functionality of antibody repertoires in recognizing diverse protein antigens.

Antibodies recognize protein antigens with exquisite specificity in a complex aqueous environment, where interfacial waters are an integral part of the antibody-protein complex interfaces. In this work, we elucidate, with computational analyses, the principles governing the antibodies' specificity and affinity towards their cognate protein antigens in the presence of explicit interfacial waters. Experimentally, in four model antibody-protein complexes, we compared the contributions of the interaction types in antibody-protein antigen complex interfaces with the antibody variants selected from phage-displayed synthetic antibody libraries. Evidently, the specific interactions involving a subset of aromatic CDR (complementarity determining region) residues largely form the predominant determinant underlying the specificity of the antibody-protein complexes in nature. The interfacial direct/water-mediated hydrogen bonds accompanying the CDR aromatic interactions are optimized locally but contribute little in determining the epitope location. The results provide insights into the phenomenon that natural antibodies with limited sequence and structural variations in an antibody repertoire can recognize seemingly unlimited protein antigens. Our work suggests guidelines in designing functional artificial antibody repertoires with practical applications in developing novel antibody-based therapeutics and diagnostics for treating and preventing human diseases.

Multifunctional Membrane for Thermal Management Applications.

Space cooling and heating consume a large proportion of global energy, so passive thermal management materials (i.e., without energy input), especially dual-mode materials including cooling and heating bifunctions, are becoming more and more attractive in many areas. Herein, a function-switchable Janus membrane between cooling and heating consisting of a multilayer structure of polyvinylidene fluoride nanofiber/zinc oxide nanosheet/carbon nanotube/Ag nanowire/polydimethylsiloxane was fabricated for comprehensive thermal management applications. In the cooling mode, the high thermal radiation emissivity (89.2%) and sunlight reflectivity (90.6%) of the Janus membrane resulted in huge temperature drops of 8.2-12.6, 9.0-14.0, and 10.9 °C for a substrate, a closed space, and a semiclosed space, respectively. When switching to the heating mode, temperature rises of 3.8-4.6, 4.0-4.8, and 12.5 °C for the substrate, closed space, and semiclosed space, respectively, were achieved owing to the high thermal radiation reflectivity (89.5%) and sunlight absorptivity (74.1%) of the membrane. Besides, the Janus membrane has outstanding comprehensive properties of the membrane, including infrared camouflaging/disguising, electromagnetic shielding (53.1 dB), solvent tolerance, waterproof properties, and high flexibility, which endow the membrane with promising application prospects.

CPAP enhances and maintains chronic inflammation in hepatocytes to promote hepatocarcinogenesis.

Chronic and persistent inflammation is a well-known carcinogenesis promoter. Hepatocellular carcinoma (HCC) is one of the most common inflammation-associated cancers; most HCCs arise in the setting of chronic inflammation and hepatic injury. Both NF-κB and STAT3 are important regulators of inflammation. Centrosomal P4.1-associated protein (CPAP), a centrosomal protein that participates primarily in centrosome functions, is overexpressed in HCC and can increase TNF-α-mediated NF-κB activation and IL-6-induced STAT3 activation. A transgenic (Tg) mouse model with hepatocyte-specific CPAP expression was established to investigate the physiological role of CPAP in hepatocarcinogenesis. Obvious inflammatory cell accumulation and fatty change were observed in the livers of CPAP Tg mice. The alanine aminotransferase (ALT) level and the expression levels of inflammatory genes, such as IL-6, IL-1ß and TNF-α, were higher in CPAP Tg mice than in wild type (WT) mice. High-dose/short-term treatment with diethylnitrosamine (DEN) increased the ALT level, proinflammatory gene expression levels, and STAT3 and NF-κB activation in CPAP Tg mice; low-dose/long-term DEN treatment induced more severe liver tumor formation in CPAP Tg mice than in WT mice. CPAP can increase the expression of chemokine (C-C motif) ligand 16 (CCL-16), an important chemotactic cytokine, in human hepatocytes. CCL-16 expression is positively correlated with CPAP and TNF-α mRNA expression in the peritumoral part of HCC. In summary, these results suggest that CPAP may promote hepatocarcinogenesis through enhancing the inflammation pathway via increasing the expression of CCL-16.