The N545S and K717N substitution at the N-glycosylation sites of the S2 subunit of avian infectious bronchitis virus can significantly enhance viral pathogenicity.

The S2 subunit of infectious bronchitis virus (IBV) is a heavily glycosylated protein that can impact various characteristics of the virus. It is currently known that N-glycosylation modifications are predominantly located on the S2 subunit. However, the exact role of their N-glycosylation modification remains undisclosed. To elucidate the function of these N-glycosylation sites, we identified 14 common sites distributed on the S2 subunit of the 5 genotypes of IBV in present study. Subsequently, we selected 7 sites to generate mutants and assessed their impact on viral virulence, replication ability, and antigenicity. Our finding revealed that only 2 substitutions, N545S and K717N, increased the viral replication titer and antigenicity, and ultimately the pathogenicity in chicks. To delve into the mechanisms underlying this increased pathogenicity, we discovered that K717N can change the structure of antigenic epitopes. The N545S substitution not only influenced antigenic epitope structure, but also enhanced the ability of the virus to enter CEKs during the early stages of viral replication. These results suggest that the enhanced viral pathogenicity associated with N545S and K717N substitutions is multifaceted, with acceleration of the viral membrane fusion process and alterations in epitope structure representing crucial factors in the capability of N-glycosylation modifications to boost viral virulence. These insights provide valuable guidance for the efficient development of live attenuated vaccines.

Sex Differences in the Antidepressant and Neurocognitive Effects of Nonconvulsive Electrotherapy in Patients with Treatment-Refractory Depression.

Objective: The objective of this study was to examine sex differences in the antidepressant and neurocognitive effects of adjunctive nonconvulsive electrotherapy (NET) in patients with treatment-refractory depression (TRD), which has not yet been thoroughly investigated. Methods: The study enrolled 20 patients with TRD, comprising 11 males and 9 females, who underwent a series of 6 NET sessions. The 17-item Hamilton Depression Rating Scale (HAMD-17) was used to assess depressive symptoms, response, and remission at baseline and after the first, third, and sixth NET sessions. The Wisconsin Card Sorting Test (WCST) was used to assess neurocognitive function at baseline and after the sixth NET session. Results: After completing 6 NET sessions, female patients experiencing TRD exhibited a higher inclination toward achieving an antidepressant response (77.8% vs. 45.5%, P = .197) and antidepressant remission (22.2% vs. 0%, P = .189) when compared to their male counterparts. No significant differences were observed in changes in the HAMD-17 and WCST subscale scores (all P > .05), including completing classification number, total error number, persistent error number, and random error number between males and females. Additionally, no significant correlations were observed between baseline WCST subscale scores and changes in HAMD-17 scores or endpoint scores, irrespective of sex (all P > .05). Conclusion: These pilot findings suggest that female patients with TRD exhibited increased rates of achieving antidepressant response and remission after undergoing NET. However, further studies should be conducted to confirm these findings.

Implantation and Decidualization in PCOS: Unraveling the Complexities of Pregnancy.

Polycystic Ovary Syndrome (PCOS) is a prevalent endocrine disorder in women of reproductive age, affecting 5-15% globally with a large proportion undiagnosed. This review explores the multifaceted nature of PCOS and its impact on pregnancy, including challenges in fertility due to hormonal imbalances and insulin resistance. Despite restoring ovulation pharmacologically, women with PCOS face lower pregnancy rates and higher risks of implantation failure and miscarriage. Our review focuses on the complexities of hormonal and metabolic imbalances that impair endometrial receptivity and decidualization in PCOS. Disrupted estrogen signaling, reduced integrity of endometrial epithelial tight junctions, and insulin resistance impair the window of endometrial receptivity. Furthermore, progesterone resistance adversely affects decidualization. Our review also examines the roles of various immune cells and inflammatory processes in the endometrium, contributing to the condition's reproductive challenges. Lastly, we discuss the use of rodent models in understanding PCOS, particularly those induced by hormonal interventions, offering insights into the syndrome's impact on pregnancy and potential treatments. This comprehensive review underscores the need for advanced understanding and treatment strategies to address the reproductive complications associated with PCOS, emphasizing its intricate interplay of hormonal, metabolic, and immune factors.

Testicular macrophages are recruited during a narrow fetal time window and promote organ-specific developmental functions.

A growing body of evidence demonstrates that fetal-derived tissue-resident macrophages have developmental functions. It has been proposed that macrophages promote testicular functions, but which macrophage populations are involved is unclear. Previous studies showed that macrophages play critical roles in fetal testis morphogenesis and described two adult testicular macrophage populations, interstitial and peritubular. There has been debate regarding the hematopoietic origins of testicular macrophages and whether distinct macrophage populations promote specific testicular functions. Here our hematopoietic lineage-tracing studies in mice show that yolk-sac-derived macrophages comprise the earliest testicular macrophages, while fetal hematopoietic stem cells (HSCs) generate monocytes that colonize the gonad during a narrow time window in a Sertoli-cell-dependent manner and differentiate into adult testicular macrophages. Finally, we show that yolk-sac-derived versus HSC-derived macrophages have distinct functions during testis morphogenesis, while interstitial macrophages specifically promote adult Leydig cell steroidogenesis. Our findings provide insight into testicular macrophage origins and their tissue-specific roles.

The conserved L1089 in the S2 subunit of avian infectious bronchitis virus determines viral kidney tropism by disrupting virus-cell fusion.

The virulence of avian gamma-coronavirus infectious bronchitis viruses (IBV) for the kidney has led to high mortality in dominant-genotype isolations, but the key sites of viral protein that determine kidney tropism are still not fully clear. In this study, the amino acid sequences of the S2 subunit of IBVs with opposing adaptivity to chicken embryonic kidney cells (CEKs) were aligned to identify putative sites associated with differences in viral adaptability. The S2 gene and the putative sites of the non-adapted CN strain were introduced into the CEKs-adapted SczyC30 strain to rescue seven mutants. Analysis of growth characteristics showed that the replacement of the entire S2 subunit and the L1089I substitution in the S2 subunit entirely abolished the proliferation of recombinant IBV in CEKs as well as in primary chicken oviduct epithelial cells. Pathogenicity assays also support the decisive role of this L1089 for viral nephrotropism, and this non-nephrotropic L1089I substitution significantly attenuates pathogenicity. Analysis of the putative cause of proliferation inhibition in CEKs suggests that the L1089I substitution affects neither virus attachment nor endocytosis, but instead fails to form double-membrane vesicles to initiate the viral replication and translation. Position 1089 of the IBV S2 subunit is conservative and predicted to lie in heptad repeat 2 domains. It is therefore reasonable to conclude that the L1089I substitution alters the nephrotropism of parent strain by affecting virus-cell fusion. These findings provide crucial insights into the adaptive mechanisms of IBV and have applications in the development of vaccines and drugs against IB.

Viral contamination on the surfaces of the personal protective equipment among health care professionals working in COVID-19 wards: A single-center prospective, observational study.

OBJECTIVE: To evaluate potential viral contamination on the surfaces of personal protective equipment (PPE) in COVID-19 wards. METHODS: Face shields, gloves, the chest area of PPE and shoe soles were sampled at different time points. The samples were tested for the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by PCR, and the cycle threshold (CT) values were recorded. RESULTS: The positive rate was 74.7% (239/320) for all PPE specimens. The CT values of the samples were ranked in the following order: face shields > chests > gloves > shoe soles (37.08±1.38, 35.48±2.02, 34.17±1.91 and 33.52±3.16, respectively; P for trend < .001). After disinfection, the CT values of shoe soles decreased compared with before disinfection (32.78±3.47 vs. 34.3±2.61, P = .037), whereas no significant effect of disinfection on the CT values of face shields, chests and gloves was observed. After disinfection, the CT values of specimens collected from shoe soles gradually increased; before disinfection, the CT values of shoe sole specimens were all less than 35. CONCLUSIONS: SARS-CoV-2 can attach to the surfaces of the PPE of healthcare professionals in COVID-19 wards, especially the shoe soles and undisinfected gloves. Shoe soles had the highest SARS-CoV-2 loads among all tested PPE items.

Perivascular cells support folliculogenesis in the developing ovary.

Supporting cells of the ovary, termed granulosa cells, are essential for ovarian differentiation and oogenesis by providing a nurturing environment for oocyte maintenance and maturation. Granulosa cells are specified in the fetal and perinatal ovary, and sufficient numbers of granulosa cells are critical for the establishment of follicles and the oocyte reserve. Identifying the cellular source from which granulosa cells and their progenitors are derived is an integral part of efforts to understand basic ovarian biology and the etiology of female infertility. In particular, the contribution of mesenchymal cells, especially perivascular cells, to ovarian development is poorly understood but is likely to be a source of new information regarding ovarian function. Here we have identified a cell population in the fetal ovary, which is a Nestin-expressing perivascular cell type. Using lineage tracing and ex vivo organ culture methods, we determined that perivascular cells are multipotent progenitors that contribute to granulosa, thecal, and pericyte cell lineages in the ovary. Maintenance of these progenitors is dependent on ovarian vasculature, likely reliant on endothelial-mesenchymal Notch signaling interactions. Depletion of Nestin+ progenitors resulted in a disruption of granulosa cell specification and in an increased number of germ cell cysts that fail to break down, leading to polyovular ovarian follicles. These findings highlight a cell population in the ovary and uncover a key role for vasculature in ovarian differentiation, which may lead to insights into the origins of female gonad dysgenesis and infertility.

Mutation of D201G near the receptor binding site significantly drives antigenic drift of circulating H9N2 subtype avian influenza virus.

The H9N2 subtype of avian influenza virus (H9N2 AIV) has caused significant losses in chicken flocks throughout China. Our previous research has shown that field isolates of H9N2 underwent antigenic drift to evolve into distinct groups with significant antigenic divergence from the commercially available vaccines. The present study sought to identify which single mutations that have naturally appeared in isolates from the past 5 years have driven antigenic drift. Six high-frequency mutation sites in/near the receptor binding site region were screened by comparing amino acid alignments of the H9N2 AIVs isolated from China between 2014 and 2019. Two substitutions (A168N and D201G) were demonstrated to have a significant impact on the antigenicity but did not change the growth kinetics of the virus. It is worth noting that the D201G substitution not only significantly changed the antigenicity but also caused immune escape against the parental virus. In conclusion, A168N and D201G substitution are newly discovered determinants that can significantly change the antigenicity of H9N2 AIV, which should be tracked during outbreaks.

Immune Cells as Critical Regulators of Steroidogenesis in the Testis and Beyond.

Steroidogenesis is an essential biological process for embryonic development, reproduction, and adult health. While specific glandular cells, such as Leydig cells in the testis, are traditionally known to be the principal players in steroid hormone production, there are other cell types that contribute to the process of steroidogenesis. In particular, immune cells are often an important component of the cellular niche that is required for the production of steroid hormones. For several decades, studies have reported that testicular macrophages and Leydig cells are intimately associated and exhibit a dependency on the other cell type for their proper development; however, the mechanisms that underlie the functional relationship between macrophages and Leydig cells are unclear. Beyond the testis, in certain instances immune cells themselves, such as certain types of lymphocytes, are capable of steroid hormone production, thus highlighting the complexity and diversity that underlie steroidogenesis. In this review we will describe how immune cells are critical regulators of steroidogenesis in the testis and in extra-glandular locations, as well as discuss how this area of research offers opportunities to uncover new insights into steroid hormone production.

Immune and vascular contributions to organogenesis of the testis and ovary.

Gonad development is a highly regulated process that coordinates cell specification and morphogenesis to produce sex-specific organ structures that are required for fertility, such as testicular seminiferous tubules and ovarian follicles. While sex determination occurs within specialized gonadal supporting cells, sexual differentiation is evident throughout the entire organ, including within the interstitial compartment, which contains immune cells and vasculature. While immune and vascular cells have been traditionally appreciated for their supporting roles during tissue growth and homeostasis, an increasing body of evidence supports the idea that these cell types are critical drivers of sexually dimorphic morphogenesis of the gonad. Myeloid immune cells, such as macrophages, are essential for multiple aspects of gonadogenesis and fertility, including for forming and maintaining gonadal vasculature in both sexes at varying stages of life. While vasculature is long known for supporting organ growth and serving as an export mechanism for gonadal sex steroids in utero, it is also an important component of fetal testicular morphogenesis and differentiation; additionally, it is vital for ovarian corpus luteal function and maintenance of pregnancy. These findings point toward a new paradigm in which immune cells and blood vessels are integral components of sexual differentiation and organogenesis. In this review, we discuss the state of the field regarding the diverse roles of immune and vascular cells during organogenesis of the testis and ovary and highlight outstanding questions in the field that could stimulate new research into these previously underappreciated constituents of the gonad.

Research Note: Effects of Escherichia coli co-infection on the protective efficacy assessment of two common infectious bronchitis vaccines.

Avian infectious bronchitis (IB), a highly contagious disease hazardous to the poultry industry, is caused by an etiological agent called the infectious bronchitis virus (IBV). Some IBV strains (IBVs) alone usually do not cause high mortality in field conditions if not with secondary pathogens including Escherichia coli (E. coli). Herein, we established an IBV and E. coli co-infection model to evaluate the protective efficacy of two IBV vaccine strains against a new emerging genotype GVI-1 with mild virulence in experimental conditions. Chickens were inoculated with IBV field isolate ZQX (genotype GVI-1) and challenged 4 dlater with the E. coli strain MS160427 (serotype O8). Subsequently, these chickens were euthanized at seven days postchallenge (d.p.c.) with E. coli. An autopsy revealed that lesions in the IBV plus E. coli co-infection group were more severe than those in the IBV-infected group. This pathological model was used to assess the protective effect of two commonly used vaccine strains (H120 and 4/91) against the IBV ZQX strain, and a significantly better protective efficacy was observed for 4/91 compared with H120. Thus, IBV and E. coli co-infection could be employed in assessing the protective efficacy of IBV vaccines.

In situ Synthesized Monosodium Urate Crystal Enhances Endometrium Decidualization via Sterile Inflammation During Pregnancy.

High level of uric acid (UA) is the major origin of gout, and is highly associated with various pregnant complications, such as preeclampsia and gestational diabetes. However, UA's level and role in the very early stage of pregnancy has not been uncovered. This study aims to investigate the relevance of serum UA and decidualization, an essential process for the establishment and maintenance of pregnancy in women and mice during the early stage of pregnancy. In this study, we first proved that expression level of UA synthase xanthine dehydrogenase (XDH) is highly increased along with decidualization of endometrial stromal cells in both in vitro and in vivo models. Furthermore, serum and endometrial levels of UA are higher in mice with decidualized uterin horn and in vitro decidualized stromal cells. The existence of monosodium urate (MSU) crystal was also confirmed by immunostaining. Next, the roles of MSU on decidualization were explored by both in vitro and in vivo models. Our data shows MSU crystal but not UA enhances the decidualization response of endometrial stromal cells, via the upregulation of inflammatory genes such Ptgs2 and Il11. inhibiting of Cox-2 activity abolishes MSU crystal induced higher expression of decidualization marker Prl8a2. At last, in women, we observed enriched expression of XDH in decidua compare to non-decidualized endometrium, the serum level of UA is significantly increased in women in very early stage of pregnancy, and drop down after elective abortion. In summary, we observed an increased serum UA level in the early stage of women's pregnancy, and proved that the increased level of UA results from the expressed XDH in decidualizing endometrium of both human and mouse, leading to the formation of MSU crystal. MSU crystal can enhance the decidualization response via inflammatory pathways. Our study has uncovered the association between UA, MSU, and decidualization during the early stage of pregnancy.

Loss of Mafb and Maf distorts myeloid cell ratios and disrupts fetal mouse testis vascularization and organogenesis†.

Testis differentiation is initiated when Sry in pre-Sertoli cells directs the gonad toward a male-specific fate. Sertoli cells are essential for testis development, but cell types within the interstitial compartment, such as immune and endothelial cells, are also critical for organ formation. Our previous work implicated macrophages in fetal testis morphogenesis, but little is known about genes underlying immune cell development during organogenesis. Here, we examine the role of the immune-associated genes Mafb and Maf in mouse fetal gonad development, and we demonstrate that deletion of these genes leads to aberrant hematopoiesis manifested by supernumerary gonadal monocytes. Mafb; Maf double knockout embryos underwent initial gonadal sex determination normally, but exhibited testicular hypervascularization, testis cord formation defects, Leydig cell deficit, and a reduced number of germ cells. In general, Mafb and Maf alone were dispensable for gonad development; however, when both genes were deleted, we observed significant defects in testicular morphogenesis, indicating that Mafb and Maf work redundantly during testis differentiation. These results demonstrate previously unappreciated roles for Mafb and Maf in immune and vascular development and highlight the importance of interstitial cells in gonadal differentiation.

Tryptophan and kynurenine stimulate human decidualization via activating Aryl hydrocarbon receptor: Short title: Kynurenine action on human decidualization.

Decidualization is essential for successful pregnancy in rodents and primates. Although L-Tryptophan and its metabolites are essential for mammalian pregnancy, the underlying mechanism is poorly defined. We explored effects of tryptophan and kynurenine on human in vitro decidualization in human endometrial stromal cell line and primary endometrial stromal cells. Tryptophan significantly stimulates the expression of prolactin and insulin growth factor binding protein 1, reliable markers for human decidualization. When stromal cells are treated with tryptophan, tryptophan hydroxylase-1 remains unchanged, but indoleamine 2,3-dioxygenase 1 is significantly increased, suggesting tryptophan is mainly metabolized through kynurenine pathway. Kynurenine significantly stimulates insulin growth factor binding protein 1 expression. Aryl hydrocarbon receptor and its target genes (P450 1A1 and P450 1B1) are significantly increased by tryptophan and kynurenine. The induction of tryptophan and kynurenine on insulin growth factor binding protein 1 is abrogated by CH223191, an aryl hydrocarbon receptor inhibitor. Cytochrome P450 1A1 and P450 1B1 catalyze the oxidative metabolism of estradiol to catechol estrogens (2-hydroxy estradiol and 4-hydroxy estradiol), respectively. Insulin growth factor binding protein 1 is up-regulated by 2-hydroxy estradiol and 4-hydroxy estradiol. Interferon-γ significantly induces the expression of indoleamine 2,3-dioxygenase 1, aryl hydrocarbon receptor and insulin growth factor binding protein 1. All the data are also verified in primary human stromal cells. Our data indicate that Interferon-γ-induced kynurenine pathway promotes human decidualization via aryl hydrocarbon receptor signaling.

Aldosterone from endometrial glands is benefit for human decidualization.

Local renin-angiotensin system (RAS) in female reproductive system is involved in many physiological and pathological processes, such as follicular development, ovarian angiogenesis, ovarian, and endometrial cancer progress. However, studies on the functional relevance of RAS in human endometrium are limited, especially for renin-angiotensin-aldosterone system (RAAS). In this study, we defined the location of RAS components in human endometrium. We found that angiotensin II type-1 receptor (AT1R) and aldosterone synthase (CYP11B2), major components of RAAS, are specifically expressed in endometrial gland during mid-secretory phase. Aldosterone receptor, mineralocorticoid receptor (MR), is elevated in stroma in mid-secretory endometrium. In vitro, MR is also activated by aldosterone during decidualization. Activated MR initiates LKB1 expression, followed by phosphorylating of AMPK that stimulates PDK4 expression. The impact of PDK4 on decidualization is independent on PDHE1α inactivation. Based on co-immunoprecipitation, PDK4 interacts with p-CREB to prevent its ubiquitination for facilitating decidualization via FOXO1. Restrain of MR activation interrupts LKB1/p-AMPK/PDK4/p-CREB/FOXO1 pathway induced by aldosterone, indicating that aldosterone action on decidualization is mainly dependent on MR stimulation. Aldosterone biosynthesized in endometrial gland during mid-secretory phase promotes decidualization via activating MR/LKB1/p-AMPK/PDK4/p-CREB/FOXO1 signaling pathway. This study provides the valuable information for understanding the underlying mechanism during decidualization.

A class â lentogenic newcastle disease virus strain confers effective protection against the prevalent strains.

The traditional vaccine strains, such as LaSota, do not completely prevent the shedding of NDV. An ideal vaccine which could not only prevent the clinical signs, but significantly reduce the shedding of NDV is urgently needed for the eradication of ND. In this study, an NDV isolate APMV-1/Chicken/China (SC)/PT3/2016 (hereafter referred as PT3) was identified as a class Ⅰ NDV and a lentogenic strain. The antigenic relationship between PT3 and 3 other NDV strains, including vaccine strain LaSota and 2 prevalent genotype Ⅶd and Ⅵb strains were analyzed. The protective efficacy of PT3 and LaSota against challenge with genotype Ⅶd and Ⅵb strains were assessed. The antigenic analysis result showed that 4 strains belong to the single serotype and the PT3 antiserum exhibited the highest HI titer against 3 other NDV strains. The results of protective efficacy showed that both of LaSota and PT3 could provide 100% survivability for infected chickens. However, PT3 performed better in inducing higher humoral responses and reducing virus shedding than the LaSota strain. Lentogenic strains from Class I NDV appear to be promising vaccine candidates for the control of ND, and allows for the easy discrimination of field NDV and vaccine strains.

Complete Genome Sequence of a Class I Newcastle Disease Virus Strain Isolate from a Breeding Chicken Flock in Sichuan, China.

A Newcastle disease virus (NDV) strain, APMV-1/Chicken/China(SC)/PT3/2016, was isolated from asymptomatic chickens at a breeding farm in China. The PT3 strain has a genome length of 15,198 nucleotides and is classified as subgenotype 1b of class I. Pathogenicity tests demonstrated that PT3 is a lentogenic strain.

Preparation and protective efficacy of a chicken embryo kidney cell-attenuation GI-19/QX-like avian infectious bronchitis virus vaccine.

Avian infectious bronchitis (IB) is a highly contagious disease, and hazardous to the poultry industry. Immune failure often occurs due to the emergence of new serotypes or field strains antigenically different from the vaccine strains. To prepare a candidate vaccine against the prevalent avian infectious bronchitis virus (IBV) in China, the GI-19/QX-like field isolate Sczy3 was selected as the progenitor strain and attenuated via passaging in chicken embryo kidney (CEK) cells for 100 times. The 100th generation of CEK-adapted strain, designated SczyC100, was safe to use on one-day old specific pathogen-free (SPF) chicken as determined by pathogenicity and virulence reversion test. The efficacies of SczyC100 and two commonly used commercial vaccines (H120 and 4/91) against prevalent GI-19/QX and GI-7/TWI type virulent strains were evaluated. Sczy3C100 effectively reduced the morbidity, mortality, mean lesion scores (MLSs), and viral load of trachea of chickens challenged by GI-19/QX and GI-7/TWI strains. CEK-adapted SczyC100 is therefore a potential vaccine candidate for the control of IB in China.

Molecular characterization of lysyl oxidase-mediated extracellular matrix remodeling during mouse decidualization.

The establishment of decidualization is a prerequisite of successful pregnancy. Lysyl oxidase (Lox) is a copper-containing amine oxidase which catalyzes cross-linking of collagen and elastin in the ECM. Lox is expressed in the subluminal stroma surrounding the implanting blastocyst on day 5 of pregnancy. From days 6 to 8, the signals for Lox mRNA and protein are strongly detected in the decidual cells. The expression of Lox is under the control of estrogen via the GSK-3ß/ß-catenin/c-myc pathway. Dtprp is decreased by the inhibition of Lox activity. Furthermore, the inhibition of Lox activity decreases stromal cell migration and embryo adhesion. Our findings highlight the crucial role of Lox in endometrial stromal cells and deepen our understanding of decidualization.

Alcohol Dehydrogenases and Acetaldehyde Dehydrogenases are Beneficial for Decidual Stromal Cells to Resist the Damage from Alcohol.

Aims: The aim of this study was to examine the effect of alcohol on the decidualization of human endometrial stromal cells during early pregnancy. Methods: During in vitro decidualization, human endometrial stromal cells were treated with alcohol, 4-methylpyrazole hydrochloride (FPZ), the inhibitor of alcohol dehydrogenases (ADHs), and tetraethylthiuram disulfide (DSF), the inhibitor of acetaldehyde dehydrogenases (ALDHs), respectively. Cell viability and decidualization were examined. Apoptosis and proliferation were also evaluated. Results: The findings showed that ADHs and ALDHs were up-regulated during decidualization. After alcohol treatment, the cell viability of decidual stromal cells was significantly higher than control, which was abrogated by FPZ or DSF. When cells were treated with alcohol, proliferation-related signal pathways were up-regulated in decidualized cells. Additionally, FOXO1 transcriptionally up-regulates ADH1B. Conclusion: Our study provided an evidence that highly expressed ADHs and ALDHs endow decidual stromal cells an ability to alleviate the harm from alcohol.