African swine fever virus I73R is a critical virulence-related gene: A potential target for attenuation.

African swine fever virus (ASFV) is a large, double-stranded DNA virus that causes a fatal disease in pigs, posing a threat to the global pig industry. Whereas some ASFV proteins have been found to play important roles in ASFV-host interaction, the functional roles of many proteins are still largely unknown. In this study, we identified I73R, an early viral gene in the replication cycle of ASFV, as a key virulence factor. Our findings demonstrate that pI73R suppresses the host innate immune response by broadly inhibiting the synthesis of host proteins, including antiviral proteins. Crystallization and structural characterization results suggest that pI73R is a nucleic-acid-binding protein containing a Zα domain. It localizes in the nucleus and inhibits host protein synthesis by suppressing the nuclear export of cellular messenger RNA (mRNAs). While pI73R promotes viral replication, the deletion of the gene showed that it is a nonessential gene for virus replication. In vivo safety and immunogenicity evaluation results demonstrate that the deletion mutant ASFV-GZΔI73R is completely nonpathogenic and provides effective protection to pigs against wild-type ASFV. These results reveal I73R as a virulence-related gene critical for ASFV pathogenesis and suggest that it is a potential target for virus attenuation. Accordingly, the deletion mutant ASFV-GZΔI73R can be a potent live-attenuated vaccine candidate.

Novel mutation N588 residue in the NS1 protein of feline parvovirus greatly augments viral replication.

Feline parvovirus (FPV) infection is highly fatal in felines. NS1, which is a key nonstructural protein of FPV, can inhibit host innate immunity and promote viral replication, which is the main reason for the severe pathogenicity of FPV. However, the mechanism by which the NS1 protein disrupts host immunity and regulates viral replication is still unclear. Here, we identified an FPV M1 strain that is regulated by the NS1 protein and has more pronounced suppression of innate immunity, resulting in robust replication. We found that the neutralization titer of the FPV M1 strain was significantly lower than that of the other strains. Moreover, FPV M1 had powerful replication ability, and the FPV M1-NS1 protein had heightened efficacy in repressing interferon-stimulated genes (ISGs) expression. Subsequently, we constructed an FPV reverse genetic system, which confirmed that the N588 residue of FPV M1-NS1 protein is a key amino acid that bolsters viral proliferation. Recombinant virus containing N588 also had stronger ability to inhibit ISGs, and lower ISGs levels promoted viral replication and reduced the neutralization titer of the positive control serum. Finally, we confirmed that the difference in viral replication was abolished in type I IFN receptor knockout cell lines. In conclusion, our results demonstrate that the N588 residue of the NS1 protein is a critical amino acid that promotes viral proliferation by increasing the inhibition of ISGs expression. These insights provide a reference for studying the relationship between parvovirus-mediated inhibition of host innate immunity and viral replication while facilitating improved FPV vaccine production.IMPORTANCEFPV infection is a viral infectious disease with the highest mortality rate in felines. A universal feature of parvovirus is its ability to inhibit host innate immunity, and its ability to suppress innate immunity is mainly accomplished by the NS1 protein. In the present study, FPV was used as a viral model to explore the mechanism by which the NS1 protein inhibits innate immunity and regulates viral replication. Studies have shown that the FPV-NS1 protein containing the N588 residue strongly inhibits the expression of host ISGs, thereby increasing the viral proliferation titer. In addition, the presence of the N588 residue can increase the proliferation titer of the strain 5- to 10-fold without affecting its virulence and immunogenicity. In conclusion, our findings provide new insights and guidance for studying the mechanisms by which parvoviruses suppress innate immunity and for developing high-yielding FPV vaccines.

Drug screening identified that handelin inhibits feline calicivirus infection by inhibiting HSP70 expression in vitro.

Feline calicivirus (FCV) is considered one of the major pathogens of cats worldwide and causes upper respiratory tract disease in all cats. In some cats, infection is by a highly virulent strain of FCV (vs.-FCV), which can cause severe and fatal systemic disease symptoms. At present, few antiviral drugs are approved for clinical treatment against FCV. Therefore, there is an imminent need for effective FCV antiviral agents. Here, we used observed a cytopathic effect (CPE) assay to screen 1746 traditional Chinese medicine monomer compounds and found one that can effectively inhibit FCV replication, namely, handelin, with an effective concentration (EC50) value of approximately 2.5 µM. Further study showed that handelin inhibits FCV replication via interference with heat shock protein 70 (HSP70), which is a crucial host factor and plays a positive role in regulating viral replication. Moreover, handelin and HSP70 inhibitors have broad-spectrum antiviral activity. These findings indicate that handelin is a potential candidate for the treatment of FCV infection and that HSP70 may be an important drug target.

The CDE region of feline Calicivirus VP1 protein is a potential candidate subunit vaccine.

BACKGROUND: Feline calicivirus (FCV) infection causes severe upper respiratory disease in cats, but there are no effective vaccines available for preventing FCV infection. Subunit vaccines have the advantages of safety, low cost and excellent immunogenicity, but no FCV subunit vaccine is currently available. The CDE protein is the dominant neutralizing epitope region of the main antigenic structural protein of FCV, VP1. Therefore, this study evaluated the effectiveness of the CDE region as a truncated FCV VP1 protein in preventing FCV infection to provide a strategy for developing potential FCV subunit vaccines. RESULTS: Through the prediction of FCV VP1 epitopes, we found that the E region is the dominant neutralizing epitope region. By analysing the spatial structure of VP1 protein, 13 amino acid sites in the CD and E regions were found to form hydrogen bonding interactions. The results show the presence of these interaction forces supports the E region, helping improve the stability and expression level of the soluble E protein. Therefore, we selected the CDE protein as the immunogen for the immunization of felines. After immunization with the CDE protein, we found significant stimulation of IgG, IgA and neutralizing antibody production in serum and swab samples, and the cytokine TNF-α levels and the numbers of CD4+ T lymphocytes were increased. Moreover, a viral challenge trial indicated that the protection generated by the CDE subunit vaccine significantly reduced the incidence of disease in animals. CONCLUSIONS: For the first time, we studied the efficacy of the CDE protein, which is the dominant neutralizing epitope region of the FCV VP1 protein, in preventing FCV infection. We revealed that the CDE protein can significantly activate humoral, mucosal and cellular immunity, and the resulting protective effect can significantly reduce the incidence of animal disease. The CDE region of the FCV capsid is easy to produce and has high stability and excellent immunogenicity, which makes it a candidate for low-cost vaccines.

Thriving at work as a mediator of the relationship between psychological resilience and the work performance of clinical nurses.

OBJECTIVE: This study aims to investigate the relationship between psychological resilience, thriving at work, and work performance among nurses, as well as analyse the mediating role of thriving at work in the relationship between psychological resilience and the work performance of nurses. The findings are intended to serve as a reference for nursing managers to design tailored work performance intervention programs. METHOD: Using convenience sampling, 308 clinical nurses were selected from a tertiary hospital in Changsha City, Hunan Province, China, from February to April 2023. The Connor-Davidson Resilience Scale (CD-RISC), the Thriving at Work Scale, and the Work Performance Scale were employed for the questionnaire survey. Pearson correlation analysis was used to explore the relationship between psychological resilience, thriving at work and work performance. The SPSS 26.0 software's 'Process' plugin was utilised for mediation effect analysis. RESULTS: Significantly positive correlations were found between psychological resilience and thriving at work (r = 0.806, P < 0.01), thriving at work and work performance (r = 0.571, P < 0.01) as well as psychological resilience and work performance (r = 0.572, P < 0.01). Psychological resilience significantly predicted work performance positively (ß = 0.558, t = 11.165, P < 0.01), and this prediction remained significant when thriving at work (the mediating variable), was introduced (ß = 0.371, t = 4.772, P < 0.01). Psychological resilience significantly predicted thriving at work positively (ß = 0.731, t = 20.779, P < 0.01), and thriving at work significantly predicted work performance positively (ß = 0.256, t = 3.105, P < 0.05). The mediating effect size of thriving at work between psychological resilience and work performance was 33.49% (P < 0.05). CONCLUSION: Thriving at work plays a partial mediating role between psychological resilience and work performance. The level of work performance among clinical nurses was relatively high. Nursing managers can enhance thriving at work by fostering psychological resilience among clinical nurses, thereby further improving their work performance to ensure high-quality and efficient nursing care.

Identification of a Novel Neutralizing Epitope on the N-Terminal Domain of the Human Coronavirus 229E Spike Protein.

The receptor binding domain (RBD) of the coronavirus spike protein (S) has been verified to be the main target for potent neutralizing antibodies (nAbs) in most coronaviruses, and the N-terminal domain (NTD) of some betacoronaviruses has also been indicated to induce nAbs. For alphacoronavirus HCoV-229E, its RBD has been shown to have neutralizing epitopes, and these epitopes could change over time. However, whether neutralizing epitopes exist on the NTD and whether these epitopes change like those of the RBD are still unknown. Here, we verified that neutralizing epitopes exist on the NTD of HCoV-229E. Furthermore, we characterized an NTD targeting nAb 5H10, which could neutralize both pseudotyped and authentic HCoV-229E VR740 in vitro. Epitope mapping indicated that 5H10 targeted motif E1 (147-167 aa) and identified F159 as critical for 5H10 binding. More importantly, our results revealed that motif E1 was highly conserved among clinical isolates except for F159. Further data proved that mutations at position 159 gradually appeared over time and could completely abolish the neutralizing ability of 5H10, supporting the notion that position 159 may be under selective pressure during the human epidemic. In addition, we also found that contemporary clinical serum has a stronger binding capacity for the NTD of contemporary strains than historic strains, proving that the epitope on the NTD could change over time. In summary, these findings define a novel neutralizing epitope on the NTD of HCoV-229E S and provide a theoretical basis for the design of vaccines against HCoV-229E or related coronaviruses. IMPORTANCE Characterization of the neutralizing epitope of the spike (S) protein, the major invasion protein of coronaviruses, can help us better understand the evolutionary characteristics of these viruses and promote vaccine development. To date, the neutralizing epitope distribution of alphacoronaviruses is not well known. Here, we identified a neutralizing antibody that targeted the N-terminal domain (NTD) of the alphacoronavirus HCoV-229E S protein. Epitope mapping revealed a novel epitope that was not previously discovered in HCoV-229E. Further studies identified an important residue, F159. Mutations that gradually appeared over time at this site abolished the neutralizing ability of 5H10, indicating that selective pressure occurred at this position in the spread of HCoV-229E. Furthermore, we found that the epitopes within the NTD also changed over time. Taken together, our findings defined a novel neutralizing epitope and highlighted the role of the NTD in the future prevention and control of HCoV-229E or related coronaviruses.

Imaging via Correlation of X-Ray Fluorescence Photons.

We demonstrate that x-ray fluorescence emission, which cannot maintain a stationary interference pattern, can be used to obtain images of structures by recording photon-photon correlations in the manner of the stellar intensity interferometry of Hanbury Brown and Twiss. This is achieved utilizing femtosecond-duration pulses of a hard x-ray free-electron laser to generate the emission in exposures comparable to the coherence time of the fluorescence. Iterative phasing of the photon correlation map generated a model-free real-space image of the structure of the emitters. Since fluorescence can dominate coherent scattering, this may enable imaging uncrystallised macromolecules.

Evaluation of career planning group counseling and its effectiveness for intern male nursing students.

OBJECTIVE: To investigate the influence of group counselling on the career planning and career maturity of male nursing students. METHOD: Sixty male nursing students were randomly selected from a specific-level first-class hospital in Hunan Province from July to August 2020 by using the convenience sampling method and were subsequently divided into the control group and the experimental group using the random number table method. The control group received routine pre-job training, including aspects concerning the hospital profile, nurse etiquette, nursing core systems, professional ethics, nursing emergency treatment and career prospects and planning. In the experimental group, career planning group counselling was added after the regular pre-service training (once a week) with each session lasting 2 h for a total of six training sessions. At six weeks and three months after the intervention, the career status evaluation scale and the college students' career maturity scale were used to compare the career planning and career maturity status of the two groups of male nursing students. RESULTS: After six weeks and three months of intervention, all the dimensions and total scores of both the career status evaluation scale and the career maturity scale in the experimental group were superior to those in the control group with statistically significant differences (all P < 0.05). The repeated measures of variance analysis indicated that the differences in the total score for career planning and the four dimensions in terms of intergroup effect, time effect and interaction effect between the two groups were statistically significant (P < 0.05). The intergroup effect, time effect and interaction effect of the total score for vocational maturity, career goal, career confidence, career value, career freedom and career reference of the two groups were statistically significant (P < 0.05), while the time effect of the relative dependency dimension was also statistically significant (P < 0.05). CONCLUSION: Group counselling can significantly improve the career planning and career maturity status of male nursing students and has a certain long-term effect.

A novel function of African Swine Fever Virus pE66L in inhibition of host translation by the PKR/eIF2α pathway.

African swine fever virus (ASFV) is one of the most contagious and lethal viruses infecting pigs. This virus is endemic in many countries and has very recently spread to China, but no licensed vaccines or treatments are currently available. Despite extensive research, the basic question of how ASFV-encoded proteins inhibit host translation remains. Here, we examined how ASFV interfered with host translation and optimized viral gene expression. We found that 14 ASFV proteins inhibited Renilla luciferase (Rluc) activity greater than 5-fold, and the protein with the strongest inhibitory effect was pE66L, which was not previously reported. Combined with bioinformatical analysis and biochemical experiment, we determined that the transmembrane (TM) domain (amino acids 13-34) of pE66L was required for the inhibition of host gene expression. Notably, we constructed a recombinant plasmid with the TM domain linked to enhanced green fluorescent protein (EGFP) and further demonstrated that this domain broadly inhibited protein synthesis. Confocal and biochemical analyses indicated that the TM domain might help proteins locate to the endoplasmic reticulum (ER) to suppress translation though the PKR/eIF2α pathway. Deletion of the E66L gene had little effect on virus replication in macrophages, but significantly recovered host gene expression. Taken together, our findings complement studies on the host translation of ASFV proteins and suggest that ASFV pE66L induces host translation shutoff, which is dependent on activation of the PKR/eIF2α pathway.Importance African swine fever virus (ASFV) is a member of the nucleocytoplasmic large DNA virus superfamily that predominantly replicates in the cytoplasm of infected cells. The ASFV double-stranded DNA genome varies in length from approximately 170 to 193 kbp depending on the isolate and contains between 150 and 167 open reading frames (ORFs), of which half the encoded proteins have not been explored. Our study showed that 14 proteins had an obvious inhibitory effect on Renilla luciferase (Rluc) gene synthesis, with pE66L showing the most significant effect. Furthermore, the transmembrane (TM) domain of pE66L broadly inhibited host protein synthesis in a PKR/eIF2a pathway-dependent manner. Loss of pE66L during ASFV infection had little effect on virus replication, but significantly recovered host protein synthetic. Based on the above results, our findings expand our view of ASFV in determining the fate of host-pathogen interactions.

Establishment of Full-Length cDNA Clones and an Efficient Oral Infection Model for Feline Coronavirus in Cats.

Feline infectious peritonitis virus (FIPV) is the etiologic agent of feline infectious peritonitis (FIP) and causes fatal disease in cats of almost all ages. Currently, there are no clinically approved drugs or effective vaccines for FIP. Furthermore, the pathogenesis of FIP is still not fully understood. There is an urgent need for an effective infection model of feline infectious peritonitis induced by FIPV. Here, we constructed a field type I FIPV full-length cDNA clone, pBAC-QS, corresponding to the isolated FIPV QS. By replacing the FIPV QS spike gene with the commercially available type II FIPV 79-1146 (79-1146_CA) spike gene, we established and rescued a recombinant virus, designated rQS-79. Moreover, we constructed 79-1146_CA infectious full-length cDNA pBAC-79-1146_CA, corresponding to recombinant feline coronavirus (FCoV) 79-1146_CA (r79-1146_CA). In animal experiments with 1- to 2-year-old adult cats orally infected with the recombinant virus, rQS-79 induced typical FIP signs and 100% mortality. In contrast to cats infected with rQS-79, cats infected with 79-1146_CA did not show obvious signs. Furthermore, by rechallenging rQS-79 in surviving cats previously infected with 79-1146_CA, we found that there was no protection against rQS-79 with different titers of neutralizing antibodies. However, high titers of neutralizing antibodies may help prolong the cat survival time. Overall, we report the first reverse genetics of virulent recombinant FCoV (causing 100% mortality in adult cats) and attenuated FCoV (causing no mortality in adult cats), which will be powerful tools to study pathogenesis, antiviral drugs, and vaccines for FCoV. IMPORTANCE Tissue- or cell culture-adapted feline infectious peritonitis virus (FIPV) usually loses pathogenicity. To develop a highly virulent FIPV, we constructed a field isolate type I FIPV full-length clone with the spike gene replaced by the 79-1146 spike gene, corresponding to a virus named rQS-79, which induces high mortality in adult cats. rQS-79 represents the first described reverse genetics system for highly pathogenic FCoV. By further constructing the cell culture-adapted FCoV 79-1146_CA, we obtained infectious clones of virulent and attenuated FCoV. By in vitro and in vivo experiments, we established a model that can serve to study the pathogenic mechanisms of FIPV. Importantly, the wild-type FIPV replicase skeleton of serotype I will greatly facilitate the screening of antiviral drugs, both in vivo and in vitro.

The structural basis of African swine fever virus core shell protein p15 binding to DNA.

African swine fever (ASF) is an acute, hemorrhagic, and highly contagious disease caused by African swine fever virus (ASFV). The mortality rate of acute infection up to 100% have posed an unprecedented challenge of the swine industry. Currently no commercial antiviral drug is available for the control and treatment of ASFV. The structural resolution of ASFV virions reveals the details of ASFV morphogenesis, providing a new perspective for the research and promotion of the development of ASFV vaccines. Although the architecture of ASFV have been solved via cryo-EM, the structural details of four of the five viral layers remain unclear (except the outer capsid). In this study, we resolved the crystal structure of the ASFV core shell protein p15. The secondary structural elements of a protomer include four α-helix structures and six antiparallel ß-strands. Further analysis revealed that ASFV p15 forms disulfide-linked trimers between the Cys9 from one protomer and Cys30 from other protomer. Additionally, the nucleic acid-binding property was characterized by electrophoretic mobility shift assay. Two critical amino acid Lys10 and Lys39 have been identified which is essential to the nucleic acid-binding affinity of ASFV p15. Together, these findings may provide new insight into antiviral drug development.

Bone marrow mesenchymal stem cells therapy on bilateral pelvic nerve crush-induced voiding dysfunction in rats.

INTRODUCTION AND HYPOTHESIS: Neurogenic voiding dysfunction can be induced after radical pelvic surgery and severely affects patients' quality of life. This study aims to investigate the effects of bone marrow mesenchymal stem cells (BMSCs) on neurogenic voiding dysfunction in male rats and explore the underlying mechanisms. METHODS: Thirty 4-week-old male Sprague-Dawley rats were randomly divided into three groups: (1) sham-operated (sham, n = 10), (2) intrabladder wall injection of phosphate buffer solution (PBS) after bilateral pelvic nerve crush (BPNC+PBS, n = 10), and (3) intrabladder wall injection of BMSCs after bilateral pelvic nerve crush (BPNC+BMSCs, n = 10). Four weeks postoperatively, functional and morphological examinations were performed. RESULTS: Compared to the sham group, BPNC rats manifested significant augmentation in the frequency of non-voiding contractions and postvoid residual and bladder capacity, and they had decreases in intravesical pressure and voiding efficiency. However, they were markedly improved after BMSC injection. Masson's trichrome staining showed that the ratio of collagen area in bladder wall tissue significantly increased in the BPNC+PBS group but was reduced following BMSC injection. BPNC increased the protein expression of TGF-ß1, Smad2/3, and collagen I/III but decreased the expression of α-SMA. BMSC injection stimulated higher expression levels of α-SMA and lower expression levels of the other target proteins. The expression levels of vesicular acetylcholine transporters were reduced at 4 weeks post-BPNC, whereas injection of BMSCs boosted the expression quantity. CONCLUSIONS: BMSC therapy suppressed detrusor fibrosis, improved intravesical pressure and voiding efficiency, and partially restored voiding function in male rats after BPNC.

Retracted: Anticancer Activity of Phloretin Against Human Gastric Cancer Cell Lines Involves Apoptosis, Cell Cycle Arrest, and Inhibition of Cell Invasion and JNK Signalling Pathway.

This publication has been retracted by the Editor due to concerns regarding the originality of the figure images. Reference: Min Xu, Weiguang Gu, Zhou Shen, Fang Wang. Anticancer Activity of Phloretin Against Human Gastric Cancer Cell Lines Involves Apoptosis, Cell Cycle Arrest, and Inhibition of Cell Invasion and JNK Signalling Pathway. Med Sci Monit, 2018; 24: 6551-6558. DOI: 10.12659/MSM.910542.

A cross-scale study on the relationship between urban expansion and ecosystem services in China.

Clarifying the relationship between urban expansion and ecosystem services (ESs) is critical for sustainable management of land resources and ecosystems. However, little is known about the relationship between the two at the cross-scale (particularly at the national-provincial scale). Therefore, we conducted a systematic assessment of the spatiotemporal dynamics and the relationship between urban expansion and ESs including food production (FP), soil conservation (SC), carbon sequestration (CS), and water yield (WY) in China from 1992 to 2020 on the national-provincial scale. The results show that China's urban expansion took up a large amount of cropland, accounting for 79.35% of the newly-added built-up land. Shandong had the largest expansion scale and the highest speed, Shanghai had the most pronounced expansion intensity, and more than 50% of the provinces were dominated by outlying expansion pattern. In terms of total change, the three ESs of FP, SC, and WY increased by 286.5 × 106 t, 1893.61 × 106 t, and 8337.20 × 106 mm, respectively, and CS decreased by 683.90 × 106 Mg C. However, in the urban expansion area, FP and CS net decreased by 1757.6 × 104 t and 19,640.19 × 104 Mg C, respectively, while SC and WY net increased by 347.52 × 104 t and 20,264.11 × 104 mm, respectively. Shandong contributed the most to changes in ESs in urban expansion areas. Urban expansion was significantly negatively correlated with FP and CS with the correlation coefficients > -0.8; it was significantly positively correlated with SC and WY, with coefficients of 0.714 and 0.413, respectively, and urban expansion had a lagged effect on ESs. The impact of urban expansion on ESs had a spatial spillover effect and showed prominent spatial clustering in Anhui, Henan, and Shandong. Based on these results, we proposed urban planning countermeasures grounded in the perspective of ES improvement, which would provide policy references for the sustainable management of the ecological environment and land resources.

Lysine 164 is critical for SARS-CoV-2 Nsp1 inhibition of host gene expression.

The emerging pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused social and economic disruption worldwide, infecting over 9.0 million people and killing over 469 000 by 24 June 2020. Unfortunately, no vaccine or antiviral drug that completely eliminates the transmissible disease coronavirus disease 2019 (COVID-19) has been developed to date. Given that coronavirus nonstructural protein 1 (nsp1) is a good target for attenuated vaccines, it is of great significance to explore the detailed characteristics of SARS-CoV-2 nsp1. Here, we first confirmed that SARS-CoV-2 nsp1 had a conserved function similar to that of SARS-CoV nsp1 in inhibiting host-protein synthesis and showed greater inhibition efficiency, as revealed by ribopuromycylation and Renilla luciferase (Rluc) reporter assays. Specifically, bioinformatics and biochemical experiments showed that by interacting with 40S ribosomal subunit, the lysine located at amino acid 164 (K164) was the key residue that enabled SARS-CoV-2 nsp1 to suppress host gene expression. Furthermore, as an inhibitor of host-protein expression, SARS-CoV-2 nsp1 contributed to cell-cycle arrest in G0/G1 phase, which might provide a favourable environment for virus production. Taken together, this research uncovered the detailed mechanism by which SARS-CoV-2 nsp1 K164 inhibited host gene expression, laying the foundation for the development of attenuated vaccines based on nsp1 modification.

Tubularized urethral reconstruction using everted saphenous vein graft in a beagle model.

BACKGROUND: A long segment stricture in the anterior urethra is a challenge in urology. We conducted a study to investigate the efficacy of anterior urethral reconstruction using an everted saphenous vein graft (SVG) in a tubular fashion. METHODS: Twelve male beagles were randomly divided into three groups: experimental group (n = 5), control group (n = 5) and normal group (n = 2). A 3 cm defect in the anterior urethra was created. Autologous SVG was harvested. In the experimental group, urethral defect was replaced by an everted SVG in a tubular fashion. In the control group, urethral reconstruction was performed using an uneverted SVG. Beagles in all groups received retrograde urethrography to evaluate urethral patency and were killed for histological examination 6 months after operation. RESULTS: Four beagles in the experimental group had no voiding difficulty and the other one could not void spontaneously. Retrograde urethrography showed the four beagles in experimental group had wide urethral lumens. Ether urethral stricture or fistula were detected in all animals in the control group. Histological analysis of the four beagles in the experimental group indicated the everted SVG completely integrated into the urethra. The reconstructed urethra contained a wide lumen and was completely covered by urothelium. The periurethral collagen and muscle fibers formed and were highly organized. Everted SVG showed a high ability of neovascularization. In the control group, the reconstructed segment showed a fibrotic urethral lumen where the urothelium was not intact. Only few new capillaries were formed. CONCLUSIONS: Everted SVG demonstrates for a promising strategy for potential urethral stricture repair.

Umbilical two-port laparoscopic percutaneous extraperitoneal closure for patent processus vaginalis in boys: incision-hiding and solo-like surgery.

BACKGROUND: Transumbilical two-port laparoscopic percutaneous extraperitoneal closure for the treatment of processus vaginalis patency in boys has been practising recent years. The applicable instruments and skills are still evolving. In this study, we used a self-made needle assisted by a disposable dissecting forceps to practise this minimal invasive method for patent processus vaginalis in boys. Its safety and effectiveness were studied. The methods for depth and orientation perceptions were analyzed. METHODS: From January 2020 to November 2020, boys characteristic of symtomatic patency of processus vaginalis were performed open surgery consecutively. From December 2020, the authors begun to propose transumbilical two-port laparoscopic percutaneous extraperitoneal closure for this kind of boy patients. The open group included fifteen boys and the laparoscopic group included ten ones. The data of the patients age, constituent ratios of unilateral and bilateral patency, operating time, postoperative stay in hospital, follow-up time, conversion, postoperative complications were assessed. Throughout the laparoscopic process, the parallel and synchronous movements of lens pole and dissecting forceps were maintained. Vas deferens protrude was imagined as one of the point to form the triangular manipulation plane. RESULTS: There were no statistically significant difference between the laparoscopic group and the open group for the following items: age, operating time, the constituent ratios of unilateral or bilateral patency of processus vaginalis (P > 0.05). Postoperative stay in hospital and follow-up time of the laparoscopic group was significantly shorter than that of the open group (P = 0.0000). No laparoscopic case was converted to open surgery. After 10 cases of laparoscopic practice, orientation perception was established. There were no postoperative complications for all the patients. CONCLUSION: Our preliminary experience suggested that umbilical two-port laparoscopic percutaneous extraperitoneal closure is safe and convenient for patent processus vaginalis treatment in boys. It has the advantage of incision-hiding and can be manipulated like a solo-like surgery.

A conserved region of nonstructural protein 1 from alphacoronaviruses inhibits host gene expression and is critical for viral virulence.

Coronaviruses are enveloped, single-stranded RNA viruses that are distributed worldwide. They include transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and the human coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), many of which seriously endanger human health and well-being. Only alphacoronaviruses and betacoronaviruses harbor nonstructural protein 1 (nsp1), which performs multiple functions in inhibiting antiviral host responses. The role of the C terminus of betacoronavirus nsp1 in virulence has been characterized, but the location of the alphacoronavirus nsp1 region that is important for virulence remains unclear. Here, using TGEV nsp1 as a model to explore the function of this protein in alphacoronaviruses, we demonstrate that alphacoronavirus nsp1 inhibits host gene expression. Solving the crystal structure of full-length TGEV at 1.85-Å resolution and conducting several biochemical analyses, we observed that a specific motif (amino acids 91-95) of alphacoronavirus nsp1 is a conserved region that inhibits host protein synthesis. Using a reverse-genetics system based on CRISPR/Cas9 technology to construct a recombinant TGEV in which this specific nsp1 motif was altered, we found that this mutation does not affect virus replication in cell culture but significantly reduces TGEV pathogenicity in pigs. Taken together, our findings suggest that alphacoronavirus nsp1 is an essential virulence determinant, providing a potential paradigm for the development of a new attenuated vaccine based on modified nsp1.

Crystal structures of Babesia microti lactate dehydrogenase BmLDH reveal a critical role for Arg99 in catalysis.

The tick- and transfusion-transmitted human pathogen Babesia microti infects host erythrocytes to cause the pathologic symptoms associated with human babesiosis, an emerging disease with worldwide distribution and potentially fatal clinical outcome. Drugs currently recommended for the treatment of babesiosis are associated with a high failure rate and significant adverse events, highlighting the urgent need for more-effective and safer babesiosis therapies. Unlike other apicomplexan parasites, B. microti lacks a canonical lactate dehydrogenase (LDH) but instead expresses a unique enzyme, B. microti LDH (BmLDH), acquired through evolution by horizontal transfer from a mammalian host. Here, we report the crystal structures of BmLDH in apo state and ternary complex (enzyme-NADH-oxamate) solved at 2.79 and 1.89 Å. Analysis of these structures reveals that upon binding to the coenzyme and substrate, the active pocket of BmLDH undergoes a major conformational change from an opened and disordered to a closed and stabilized state. Biochemical assays using wild-type and mutant B. microti and human LDHs identified Arg99 as a critical residue for the catalytic activity of BmLDH but not its human counterpart. Interestingly, mutation of Arg99 to Ala had no impact on the overall structure and affinity of BmLDH to NADH but dramatically altered the closure of the enzyme's active pocket. Together, these structural and biochemical data highlight significant differences between B. microti and human LDH enzymes and suggest that BmLDH could be a suitable target for the development of selective antibabesial inhibitors.-Yu, L., Shen, Z., Liu, Q., Zhan, X., Luo, X., An, X., Sun, Y., Li, M., Wang, S., Nie, Z., Ao, Y., Zhao, Y., Peng, G., Ben Mamoun, C., He, L., Zhao, J. Crystal structures of Babesia microti lactate dehydrogenase BmLDH reveal a critical role for Arg99 in catalysis.

Insight into the evolution of nidovirus endoribonuclease based on the finding that nsp15 from porcine Deltacoronavirus functions as a dimer.

Nidovirus endoribonucleases (NendoUs) include nonstructural protein 15 (nsp15) from coronaviruses and nsp11 from arteriviruses, both of which have been reported to participate in the viral replication process and in the evasion of the host immune system. Results from a previous study of coronaviruses SARS-CoV, HCoV-229E, and MHV nsp15 indicate that it mainly forms a functional hexamer, whereas nsp11 from the arterivirus PRRSV is a dimer. Here, we found that porcine Deltacoronavirus (PDCoV) nsp15 primarily exists as dimers and monomers in vitro Biological experiments reveal that a PDCoV nsp15 mutant lacking the first 27 amino acids of the N-terminal domain (Asn-1-Asn-27) forms more monomers and displays decreased enzymatic activity, indicating that this region is important for its dimerization. Moreover, multiple sequence alignments and three-dimensional structural analysis indicated that the C-terminal region (His-251-Val-261) of PDCoV nsp15 is 10 amino acids shorter and forms a shorter loop than that formed by the equivalent sequence (Gln-259-Phe-279) of SARS-CoV nsp15. This result may explain why PDCoV nsp15 failed to form hexamers. We speculate that NendoUs may have originated from XendoU endoribonucleases (XendoUs) forming monomers in eukaryotic cells, that NendoU from arterivirus gained the ability to form dimers, and that the coronavirus variants then evolved the capacity to assemble into hexamers. We further propose that PDCoV nsp15 may be an intermediate in this evolutionary process. Our findings provide a theoretical basis for improving our understanding of NendoU evolution and offer useful clues for designing drugs and vaccines against nidoviruses.