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Summary Multiple morphological abnormalities of the sperm flagella (MMAF)-induced asthenoteratozoospermia is a common cause of male infertility. Previous studies have identified several MMAF-associated genes, highlighting the condition’s genetic heterogeneity. To further define the genetic causes underlying MMAF, we performed whole-exome sequencing in a cohort of 643 Chinese MMAF-affected men. Bi-allelic DNAH10 variants were identified in five individuals with MMAF from four unrelated families. These variants were either rare or absent in public population genome databases and were predicted to be deleterious by multiple bioinformatics tools. Morphological and ultrastructural analyses of the spermatozoa obtained from men harboring bi-allelic DNAH10 variants revealed striking flagellar defects with the absence of inner dynein arms (IDAs). DNAH10 encodes an axonemal IDA heavy chain component that is predominantly expressed in the testes. Immunostaining analysis indicated that DNAH10 localized to the entire sperm flagellum of control spermatozoa. In contrast, spermatozoa from the men harboring bi-allelic DNAH10 variants exhibited an absence or markedly reduced staining intensity of DNAH10 and other IDA components, including DNAH2 and DNAH6. Furthermore, the phenotypes were recapitulated in mouse models lacking Dnah10 or expressing a disease-associated variant, confirming the involvement of DNAH10 in human MMAF. Altogether, our findings in humans and mice demonstrate that DNAH10 is essential for sperm flagellar assembly and that deleterious bi-allelic DNAH10 variants can cause male infertility with MMAF. These findings will provide guidance for genetic counseling and insights into the diagnosis of MMAF-associated asthenoteratozoospermia.

Background and aims Propionic acidemia (PA) is an autosomal recessive metabolic disorder caused by a deficiency of propionyl-CoA carboxylase and mutations in the PCCA and PCCB genes. In this study, we investigated the clinical characteristics of individuals with PA and conducted genetic analyses to provide new genetic evidence for the diagnosis of PA. Materials and methods We conducted whole-exome sequencing and Sanger sequencing in four individuals with PA from three unrelated Chinese families. We also performed a structural analysis of the PCCB protein variants. Couples from the three families included in our study underwent in vitro fertilization with preimplantation genetic testing. Results We found five variants of PCCB. These biallelic variants were inherited from heterozygous parental carriers and were located in the functional domain, absent in human population genome datasets, and predicted to be deleterious. These findings indicate that the variants might be responsible for the clinical features observed in these particular patients with PA. Through successful embryo transfer and implantation, one of the couples fortunately gave birth to a healthy child. Conclusion Overall, our study can expand the mutation spectrum of PCCB and provide useful information for the prenatal diagnosis of PA and genetic counseling for affected individuals.

Asthenoteratospermia is a common cause of male infertility. Recent studies have revealed that CFAP65 mutations lead to severe asthenoteratospermia due to acrosome hypoplasia and flagellum malformations. However, the molecular mechanism underlying CFAP65-associated sperm malformation is largely unclear. Here, we initially examined the role of CFAP65 during spermiogenesis using Cfap65 knockout (Cfap65−/−) mice. The results showed that Cfap65−/− male mice exhibited severe asthenoteratospermia characterized by morphologically defective sperm heads and flagella. In Cfap65−/− mouse testes, hyper-constricted sperm heads were apparent in step 9 spermatids accompanied by abnormal manchette development, and acrosome biogenesis was abnormal in the maturation phase. Moreover, subsequent flagellar elongation was also severely affected and characterized by disrupted assembly of the mitochondrial sheath (MS) in Cfap65−/− male mice. Furthermore, the proteomic analysis revealed that the proteostatic system during acrosome formation, manchette organization and MS assembly was disrupted when CFAP65 was lost. Importantly, endogenous immunoprecipitation and immunostaining experiments revealed that CFAP65 may form a cytoplasmic protein network comprising MNS1, RSPH1, TPPP2, ZPBP1 and SPACA1. Overall, these findings provide insights into the complex molecular mechanisms of spermiogenesis by uncovering the essential roles of CFAP65 during sperm head shaping, acrosome biogenesis and MS assembly.

Background: Before October 1846, surgery and pain were synonymous but not thereafter. Conquering pain must be one of the very few strategies that has potentially affected every human being in the world of all milestones in medicine. Methods: This review article describes how various general anesthetics were discovered historically and how they work in the brain to induce sedative, hypnosis and immobility. Their advantages and disadvantages will also be discussed. Results: Anesthesia is a relatively young field but is rapidly evolving. Currently used general anesthetics are almost invariably effective, but nagging side effects, both short (e.g., cardiac depression) and long (e.g., neurotoxicity) term, have reawakened the call for new drugs. Conclusion: Based on the deepening understanding of historical development and molecular targets and actions of modern anesthetics, novel general anesthetics are being investigated as potentially improved sedative-hypnotics or a key to understand the mechanism of anesthesia.

Spermiogenesis is a complex and tightly regulated process, consisting of acrosomal biogenesis, condensation of chromatin, flagellar assembly, and disposal of extra cytoplasm. Previous studies have reported that sperm flagellar 2 (SPEF2) deficiency causes severe asthenoteratozoospermia owing to spermiogenesis failure, but the underlying molecular mechanism in humans remains unclear. Here, we performed proteomic analysis on spermatozoa from three SPEF2 mutant patients to study the functional role of SPEF2 during sperm tail development. A total of 1262 differentially expressed proteins were detected, including 486 upregulated and 776 downregulated. The constructed heat map of the differentially expressed proteins showed similar trends. Among these, the expression of proteins related to flagellar assembly, including SPEF2, sperm associated antigen 6 (SPAG6), dynein light chain tctex-type 1 (DYNLT1), radial spoke head component 1 (RSPH1), translocase of outer mitochondrial membrane 20 (TOM20), EF-hand domain containing 1 (EFHC1), meiosis-specific nuclear structural 1 (MNS1) and intraflagellar transport 20 (IFT20), was verified by western blot. Functional clustering analysis indicated that these differentially expressed proteins were specifically enriched for terms such as spermatid development and flagellar assembly. Furthermore, we showed that SPEF2 interacts with radial spoke head component 9 (RSPH9) and IFT20 in vitro, which are well-studied components of radial spokes or intra-flagellar transport and are essential for flagellar assembly. These results provide a rich resource for further investigation into the molecular mechanism underlying the role that SPEF2 plays in sperm tail development and could provide a theoretical basis for gene therapy in SPEF2 mutant patients in the future.

Social security systems were successively established in most developing countries in the 1980s and 1990s. To ensure the long-term sustainability of these newly established systems it is essential to carefully monitor the economic impact. Based on the panel data of 21 developing countries from 2000 to 2016, this paper is the first to apply the panel threshold model to empirically analyze the relationship between national health expenditures and economic growth under different levels of human capital. The results show that health expenditure and economic growth have significant interval effects because of the different levels of human capital. Specifically, when human capital levels are low, health expenditure is significantly negatively correlated with economic growth. When human capital is at a medium level, health expenditure has a positive but not significant impact on economic growth. When the level of human capital is high, the positive economic impact of the health expenditure is significantly enhanced. In addition, subgroup analyses indicate that population aging and low fertility aggravate the negative impact of health expenditures on economic growth. This study provides reliable analysis and can be used by developing countries to maintain a long-term sustainable social security system.

Severe high-order polygonal wear has been found on metro train wheels of one metro line in China, which causes considerable vibration of vehicle components and disturbing interior noise. Field tests and numerical simulations were conducted to investigate the formation mechanism and key influencing factors of wheel high-order polygonisation. Wheel out-of-roundness (OOR), vibration behavior of vehicle components, and rail surface irregularities were tested. On-site measurement results indicate that the wheels exhibit 13th- to 16th-order polygonal wear. The dominant wavelengths are about 165–203 mm. The experimental results show that the wheel polygonisation is related to the first-order bending vibration of the wheelset and the P2 resonance generated on ladder sleeper tracks and steel spring floating slab tracks. A long-term wear model was developed to further study the cause of wheel polygonisation, which included a vehicle–track coupled dynamic model, considering the flexibility of the wheelset and track, as well as a wear model. The effects of operating speeds, P2 resonance frequencies for different tracks, and wheelset flexibility on the development of wheel polygonisation were investigated. Based on the experimental and simulation results, it is further believed that the P2 resonance is the main cause of the higher-order polygonal wear of wheels, and the first-order bending vibration of the wheelset can exacerbate the wheel polygonisation.

The widely used Streptococcus pyogenes Cas9 (SpCas9) requires NGG as a protospacer adjacent motif (PAM) for genome editing. Although SpCas9 is a powerful genome-editing tool, its use has been limited on the targetable genomic locus lacking NGG PAM. The SpCas9 variants xCas9 and Cas9-NG have been developed to recognize NG, GAA, and GAT PAMs in human cells. Here, we show that xCas9 cannot recognize NG PAMs in tomato, and Cas9-NG can recognize some of our tested NG PAMs in the tomato and Arabidopsis genomes. In addition, we engineered SpCas9 (XNG-Cas9) based on mutations from both xCas9 and Cas9-NG, and found that XNG-Cas9 can efficiently mutagenize endogenous target sites with NG, GAG, GAA, and GAT PAMs in the tomato or Arabidopsis genomes. The PAM compatibility of XNG-Cas9 is the broadest reported to date among Cas9s (SpCas9 and Cas9-NG) active in plant.

Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New Background Transgenic mouse studies suggest that γ-aminobutyric acid type A (GABAA) receptors containing β3 subunits mediate important effects of etomidate, propofol, and pentobarbital. Zebrafish, recently introduced for rapid discovery and characterization of sedative-hypnotics, could also accelerate pharmacogenetic studies if their transgenic phenotypes reflect those of mammals. The authors hypothesized that, relative to wild-type, GABAA-β3 functional knock-out (β3-/-) zebrafish would show anesthetic sensitivity changes similar to those of β3-/- mice. Methods Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 mutagenesis was used to create a β3-/- zebrafish line. Wild-type and β3-/- zebrafish were compared for fertility, growth, and craniofacial development. Sedative and hypnotic effects of etomidate, propofol, pentobarbital, alphaxalone, ketamine, tricaine, dexmedetomidine, butanol, and ethanol, along with overall activity and thigmotaxis were quantified in 7-day postfertilization larvae using video motion analysis of up to 96 animals simultaneously. Results Xenopus oocyte electrophysiology showed that the wild-type zebrafish β3 gene encodes ion channels activated by propofol and etomidate, while the β3-/- zebrafish transgene does not. Compared to wild-type, β3-/- zebrafish showed similar morphology and growth, but more rapid swimming. Hypnotic EC50s (mean [95% CI]) were significantly higher for β3-/-versus wild-type larvae with etomidate (1.3 [1.0 to 1.6] vs. 0.6 [0.5 to 0.7] µM; P < 0.0001), propofol (1.1 [1.0 to 1.4] vs. 0.7 [0.6 to 0.8] µM; P = 0.0005), and pentobarbital (220 [190 to 240] vs. 130 [94 to 179] μM; P = 0.0009), but lower with ethanol (150 [106 to 213] vs. 380 [340 to 420] mM; P < 0.0001) and equivalent with other tested drugs. Comparing β3-/-versus wild-type sedative EC50s revealed a pattern similar to hypnosis. Conclusions Global β3-/- zebrafish are selectively insensitive to the same few sedative-hypnotics previously reported in β3 transgenic mice, indicating phylogenetic conservation of β3-containing GABAA receptors as anesthetic targets. Transgenic zebrafish are potentially valuable models for sedative-hypnotic mechanisms research.

One “silver lining” of the COVID-19 pandemic has been the reduction in air pollution that followed lockdowns. Unfortunately, this unintended air pollution decline will likely be short-lived. As regions begin to recover their economies, travel and industrial activity will increase the ambient pollutants quickly offsetting the improvement in air quality. Therefore, it is urgent to clarify the causal impact of reopening an economy on air quality during COVID-19. Based on city-level daily air quality data in China, this paper is the first to empirically analyze the causal effect of reopening the economy in the provincial capital Lanzhou on concentrations of four air pollutants using the synthetic control method. The results show that the reopening caused a significant increase in the concentration of NO2 by as much as 30 μg/m3 (an increase of 75% from the lockdown level) and a significant increase in O3 concentrations by 60μg/m3 (a 60% increase) which peaked on the 6th day after the restart. The reopening also led to significant fluctuations in SO2 and CO concentrations. This study contains useful conclusions by providing timely and reliable causal evidence on the lasting impact of COVID-19 on air quality.

Isoflurane exposure adversely influences subsequent fear memory formation in mice. Calcineurin (CaN), a phosphatase, prevents the establishment of emotional memory by dephosphorylating substrates and inhibiting the expression of learning and memory related genes. We investigated whether isoflurane impairment of fear memory formation was associated with altered CaN activity and downstream phosphorylated-extracellular signal-regulated kinases (p-ERK) and early growth response gene-1 (Egr-1) expression in hippocampus and amygdala. We also tested whether memory performance can be rescued by the CaN inhibitor FK506. Adult C57BL/6 mice were injected FK506 or vehicle after being exposed to 1.3% isoflurane or air for 1 h. After a 1 h- recovery, mice underwent classical fear conditioning (FC) training. Fear memory were tested 30 min, 48 h and 7 days after training. The activity of CaN, and expression of p-ERK and Egr-1 in hippocampus and amygdala were analyzed. Isoflurane exposure reduced mice freezing time in contextual and tone FC tests 30 min and 48 h after training. Hippocampus and amygdala from isoflurane-exposed mice had enhanced CaN activity, reduced p-ERK/ERK and Egr-1 expression. All these changes in isoflurane-exposed mice were attenuated by FK506 treatment. These results indicate calcineurin/p-ERK/Egr-1 Pathway is involved in fear memory impairment after isoflurane exposure in mice.

BACKGROUND Sevoflurane is the most widely used inhalational anesthetic in pediatric medicine. Despite this, sevoflurane has been reported to exert potentially neurotoxic effects on the developing brain. Clinical interventions and treatments for these effects are limited. Tanshinone IIA (Tan IIA), extracted from Salvia miltiorrhiza (Danshen), has been documented to alleviate cognitive decline in traditional applications. Therefore, we hypothesized that preadministration of Tan IIA may attenuate sevoflurane-induced neurotoxicity, suggesting that Tan IIA is a new and promising drug capable of counteracting the effects of cognitive dysfunction produced by general anesthetics. METHODS To test this hypothesis, neonatal C57 mice (P6) were exposed to 3% sevoflurane for 2 hours with or without Tan IIA pretreatment at a dose of 10 mg/kg or 20 mg/kg for 3 consecutive days. Cognitive behavior tests such as open field tests and fear conditioning were performed to evaluate locomotor and cognitive function at P31 and P32. At P8, other separate tests, including TdT mediated dUTP Nick End Labeling (TUNEL) assay, immunohistochemistry, Western blotting, enzyme-linked immunosorbent assay, and electron microscopy, were performed. The mean differences among groups were compared using 1-way analysis of variance followed by Bonferroni post hoc multiple comparison tests. RESULTS Repeated exposure to sevoflurane leads to significant cognitive impairment in mice, which may be explained by increased apoptosis, overexpression of neuroinflammatory markers, and changes in synaptic ultrastructure. Interestingly, preadministration of Tan IIA ameliorated these neurocognitive deficits, as shown by increased freezing percentages on the fear conditioning test (sevoflurane+Tan IIA [20 mg/kg] versus sevoflurane, mean difference, 19, 99% confidence interval for difference, 6.4-31, P < .0001, n = 6). The treatment also reduced the percentage of TUNEL-positive nuclei (sevoflurane versus sevoflurane+Tan IIA [20 mg/kg], 2.6, 0.73-4.5, P = .0004, n = 6) and the normalized expression of cleaved caspase-3 (sevoflurane versus sevoflurane+Tan IIA [20 mg/kg], 0.27, 0.02-0.51, P = .0046, n = 5). Moreover, it attenuated the production of the neuroinflammatory mediators interleukin (IL)-1β and IL-6 (normalized sevoflurane versus sevoflurane+Tan IIA [20 mg/kg]: IL-1β: 0.75, 0.47-1.0; P < .0001; IL-6: 0.66, 0.35-0.97; P < .0001; n = 10 per group). Finally, based on measurements of postsynaptic density, the treatment preserved synaptic ultrastructure (sevoflurane+Tan IIA [20 mg/kg] versus sevoflurane, 42, 20-66; P < .0001; n = 12 per group). CONCLUSIONS These results indicate that Tan IIA can alleviate sevoflurane-induced neurobehavioral abnormalities and may decrease neuroapoptosis and neuroinflammation.

The fault location accuracy of traction electric lines protection device based on impedance characteristics is usually over thousands of meters. To find out the reasons that affect the accuracy of fault location, this paper studies some the key factors including rail potential, feedback current through ground and fault point impedance characteristics of traction electric lines. Then the calculation method of impedance increment and the reason of the location error are found. It is of great significance for the setting calculation and fault location error analysis of the protection system.

SummaryObjective The DBA/1 mouse is a relevant animal model of sudden unexpected death in epilepsy (SUDEP), as it exhibits seizure-induced respiratory arrest (S-IRA) evoked by acoustic stimulation, followed by cardiac arrhythmia and death. Defects in serotonergic neurotransmission may contribute to S-IRA. The tryptophan hydroxylase-2 (TPH2) enzyme converts L-tryptophan to 5-hydroxytryptophan (5-HTP), a precursor for central nervous system (CNS) serotonin (5-HT) synthesis; and DBA/1 mice have a polymorphism that decreases TPH2 activity. We, therefore, hypothesized that supplementation with 5-HTP may bypass TPH2 and suppress S-IRA in DBA/1 mice. Methods TPH2 expression was examined by Western blot in the brainstem of DBA/1 and C57BL/6J mice both with and without acoustic stimulation. Changes in breathing and cardiac electrical activity in DBA/1 and C57BL/6J mice that incurred sudden death during generalized seizures evoked by pentylenetetrazole (PTZ) were studied by plethysmography and electrocardiography. The effect of 5-HTP administration on seizure-induced mortality evoked by acoustic stimulation or by PTZ was investigated in DBA/1 mice. Results Repetitive acoustic stimulation resulted in reduced TPH2 protein in the brainstem of DBA/1 mice as compared with C57BL/6J mice. S-IRA evoked by acoustic stimulation in DBA/1 mice was significantly reduced by 5-HTP. Following S-IRA, cardiac electrical activity could be detected for minutes before terminal asystole and death in both DBA/1 and C57BL/6J mice after PTZ treatment. The incidence of S-IRA by PTZ administration was greater in DBA/1 than in C57BL/6J mice, and administration of 5-HTP also significantly reduced S-IRA by PTZ in DBA/1 mice. Significance Our data suggest that S-IRA is the primary event leading to death incurred in most DBA/1 and some C57BL/6J mice during PTZ-evoked seizures. Suppression of S-IRA by 5-HTP suggests that 5-HT transmission contributes to the pathophysiology of S-IRA, and that 5-HTP, an over-the-counter supplement available for human consumption, may be clinically useful in preventing SUDEP.

Purpose To explore the quantitative distributions of different lymphocyte subsets in the lacrimal sac mucosa and identify changes of Th1- and Th2-associated cytokines in the tears of patients with chronic dacryocystitis. Methods Lacrimal sac mucosal specimens from patients with chronic dacryocystitis were analyzed. Hematoxylin-eosin staining and Masson staining were performed for pathologic analysis, and immunohistochemical staining was performed for the detection of CD3+, CD4+, CD8+, CD20+, Th1, and Th2 lymphocytes. Quantitative real-time PCR was performed to analyze IFN-γ and IL-4 mRNA expression. In addition, tear samples from patients with chronic dacryocystitis and healthy volunteers were collected and analyzed with an antibody array system for Th1- and Th2-associated cytokines and chemokines. Results Different distribution patterns of lymphocyte subsets were observed in the lacrimal sac walls. Both CD20+ B lymphocytes and CD3+ T lymphocytes accumulated in organized lymphoid follicles, and CD3+ T cells were also distributed in a diffuse manner. Among the two subsets of T cells, CD4+ T cells were more abundant than CD8+ T cells. Both the immunohistochemical staining and real-time PCR results revealed significantly higher expression levels of IFN-γ than those of IL-4. The levels of Th1- and Th2-related cytokines and chemokines measured were significantly higher in the tears of patients than in those of controls. Conclusions The different distribution patterns of lymphocyte subsets provide insight into a potential immunologic mechanism for dacryocystitis. The cytokines secreted by Th1 or Th2 cells may play a major role in the pathogenesis of dacryocystitis and could be explored as therapeutic targets.

Compared to neutral memory, emotional memory is extremely strong and persistent immediately after acquisition, therefore it may recruit specific mechanisms during acquisition. The calcineurin-dependent mechanisms engaging early growth response 1 (Egr-1) have been proved to determine the strength of emotional memory during establishment. Isoflurane, a widely used inhalation anesthetic, can interfere with the acquisition of emotional memory. We hypothesized that isoflurane impairs the acquisition of conditioned taste aversion (CTA) memory in rats and the Egr-1 expression regulation via calcineurin (CaN) and ERK signaling pathway is involved in isoflurane-induced repression of CTA memory. To examine this, we investigated the influence of isoflurane on CTA memory and the expression and activity of CaN, the phosphorylation level of ERK and the expression of Egr-1 in amygdala in response to CTA training in rats. The results showed that isoflurane exposure (1.5%, 2h) before training impaired the acquisition of CTA memory in rats. Isoflurane exposure increased the CaN activity and decreased the p-ERK and Egr-1 in amygdala in rats. These findings suggest that isoflurane can disrupt the establishment of aversion memory, and CaN activation associating with p-ERK and Egr-1 down-regulation may contribute to the isoflurane induced impairment of aversion memory acquisition.

The temporal-spatial distribution of Neolithic sites shows a long period of absence of human settlements during the early Holocene on the Yangtze Delta, and the area has not been colonized until 7000 aBP, corresponding to the onset of the Majiabang culture. Besides the regions east to the sandbar of Maqiao Site in Shanghai, foraminifera are not found in Neolithic cultural layers since 7000 aBP, indicating a widespread transgression between 10000 and 7000 aBP. Evidence of radiocarbon-dated cultural relics, foraminifera and pollen records from the Hemudu Site suggests that the Hemudu culture emerged and developed during the mid-Holocene Climatic Optimum. Therefore, the hypothesis of a mid-Holocene sea-level highstand along the Yangtze Delta and Ningshao Plain from 7000 to 5000 aBP is an open debate. New evidence from archaeological excavations and bio-stratigraphy suggests that, prior to 7000 aBP, a high sea level occurred along the Ningshao Plain. Subsequently, the sea level was lowered between 7000 and 5000 aBP, and the area suffered from frequent floods from 5000 to 3900 aBP.