A Qualitative Study on the Psychological Experience of Emergency Department Medical Staff Caring for Patients with Suicide Attempts in Northeastern Sichuan.

The purpose of this study was to explore the psychological experiences of emergency department staff in northeastern Sichuan when treating patients with suicide attempts and to provide a theoretical basis for developing appropriate clinical interventions and improving mental health services for suicidal patients. Sixteen emergency department staff members who met recruitment requirements at two hospitals in Nanchong, China, were interviewed using Colizzi descriptive phenomenological analysis. The interviews were in-depth and semi-structured. The qualitative analysis of this study revealed three main themes: (1) aspects of the emotional experience that may be detrimental to helping people in crisis (e.g., sympathy and regret, confusion and bewilderment, worry and stress); (2) aspects of the cognitive experience (e.g., inability to deal with patients' psychological issues and having new perspective on the medical profession); and (3) raising awareness of mental health services. Future reform efforts should consider training medical staff in suicide prevention knowledge and communication skills, using a compassion-centered approach to alleviate the suffering of patients who attempt suicide, using the Safety Screening Scale (PSS-3), providing counselors for patients, developing family-focused interventions, and involving family members in suicide risk prevention and treatment.

Substandard feeding in children undergoing mechanical ventilation in pediatric intensive care unit: A retrospective cohort study in China.

Background: Enteral nutrition is a common yet vital practice in the pediatric intensive care unit (PICU). However, the status of substandard feeding of enteral nutrition in PICU children undergoing mechanical ventilation remains unclear and can be detrimental to the children's prognosis. Objective: This study aimed to evaluate the incidence, nursing care status, and influencing factors of substandard feeding in children undergoing mechanical ventilation in the PICU. Methods: This study employed a retrospective cohort design. Children undergoing mechanical ventilation and enteral nutrition in the PICU of a public hospital in China from 1 June 2021 to 31 December 2022 were selected using convenience sampling, and their characteristics were collected and evaluated. Pearson correlation analysis and multivariate logistic regression analysis were conducted to assess the influencing factors of substandard feeding in PICU children with mechanical ventilation. Results: A total of 156 PICU children undergoing mechanical ventilation were included for analysis in this study. The rate of substandard feeding in PICU children was 65.38%. Statistically significant differences were observed in diarrhea, vomiting, the use of sedatives, and average infusion speed between the substandard feeding group and the standard group (p <0.05). Pearson correlation results indicated that diarrhea (r = 0.595), vomiting (r = 0.602), and average infusion speed (r = 0.562) were correlated with substandard feeding and characteristics of included ICU children undergoing mechanical ventilation (p <0.05). Logistic regression results found that diarrhea (OR = 2.183, 95%CI: 1.855~2.742), vomiting (OR = 3.021, 95%CI: 2.256~4.294), and average infusion speed ≤40 mL/h (OR = 2.605, 95%CI: 1.921~3.357) were independent risk factors for substandard feeding in mechanically ventilated children in the ICU (p <0.05). Conclusion: The rate of substandard feeding in children with mechanical ventilation in the PICU was high. Diarrhea, vomiting, and slow infusion speed are important influencing factors for substandard feeding. It is suggested that nurses and other healthcare professionals take targeted measures, including the prevention and care of diarrhea and vomiting, as well as monitoring and adjusting the infusion speed of enteral nutrition, to reduce the occurrence of substandard feeding.

Development and evolution of human glutaminyl cyclase inhibitors (QCIs): an alternative promising approach for disease-modifying treatment of Alzheimer's disease.

Human glutaminyl cyclase (hQC) is drawing considerable attention and emerging as a potential druggable target for Alzheimer's disease (AD) due to its close involvement in the pathology of AD via the post-translational pyroglutamate modification of amyloid-ß. A recent phase 2a study has shown promising early evidence of efficacy for AD with a competitive benzimidazole-based QC inhibitor, PQ912, which also demonstrated favorable safety profiles. This finding has sparked new hope for the treatment of AD. In this review, we briefly summarize the discovery and evolution of hQC inhibitors, with a particular interest in classic Zinc binding group (ZBG)-containing chemicals reported in recent years. Additionally, we highlight several high-potency inhibitors and discuss new trends and challenges in the development of QC inhibitors as an alternative and promising disease-modifying therapy for AD.

Comparative Study of the Gut Microbiota Community between the Farmed and Wild Mastacembelus armatus (Zig-Zag Eel).

Cultivated and wild fish of the same species may exhibit different characteristics, such as in their flavor, growth and development. In some wild fish species, reproductive functions may even be retarded when wild individuals are moved into cultivated conditions. The gut microbiota may be one of the reasons for these phenomena as they have been reported to play an important role in host growth and development, as well as in normal reproductive functioning. Here, we used Mastacembelus armatus (zig-zag eel), a freshwater fish which shows anormal reproductive function in cultivated conditions, as a model to comparatively study the diversity, structure and function of gut microbiota in cultivated and wild groups by analyzing the 16S rRNA sequence of each group's microbiota. The results showed that Proteobacteria and Firmicutes were the dominant phyla in the gut microbiota of wild (accounting for 45.8% and 20.3% of the total number of Proteobacteria and Firmicutes, respectively) and farmed (accounting for 21.4% and 75.6% of the total number of Proteobacteria and Firmicutes, respectively) zig-zag eel. Wild zig-zag eels (Shannon = 3.56; Chao = 583.08; Ace = 579.18) had significantly higher alpha diversity than those in cultivated populations (Shannon = 2.09; Chao = 85.45; Ace = 86.14). A significant difference in the community structure of the gut microbiota was found between wild and cultivated populations. The wild zig-zag eel showed a high abundance of functional pathways in metabolism, genetic information processing and organismal system function. These results suggested that the diversity and function of gut microbiota in zig-zag eel were correlated with their diet and habitat conditions, which indicated that the management of cultivated populations should mimic the wild diet and habitat to improve the productivity and quality of farmed zig-zag eel.

Functional Peroral Infectivity Complex of White Spot Syndrome Virus of Shrimp.

White spot syndrome virus (WSSV) is a major cause of disease in shrimp cultures worldwide. The infection process of this large circular double-stranded DNA virus has been well studied, but its entry mechanism remains controversial. The major virion envelope protein VP28 has been implicated in oral and systemic viral infection in shrimp. However, genetic analysis of viral DNA has shown the presence of a few genes related to proteins of per os infectivity factor (PIF) complex in baculoviruses. This complex is essential for the entry of baculoviruses, large terrestrial circular DNA viruses, into the midgut epithelial cells of insect larvae. In this study, we aimed to determine whether a PIF complex exists in WSSV, the components of this complex, whether it functions as an oral infectivity complex in shrimp, and the biochemical properties that contribute to its function in a marine environment. The results revealed a WSSV PIF complex (~720 kDa) comprising at least eight proteins, four of which were not identified as PIF homologs: WSV134, VP124 (WSV216), WSSV021, and WSV136. WSV134 is suggested to be a PIF4 homolog due to predicted structural similarity and amino acid sequence identity. The WSSV PIF complex is resistant to alkali, proteolysis, and high salt, properties that are important for maintaining infectivity in aquatic environments. Oral infection can be neutralized by PIF-specific antibodies but not by VP28-specific antibodies. These results indicate that the WSSV PIF complex is critical for WSSV entry into shrimp; the complex's evolutionary significance is also discussed. IMPORTANCE White spot disease, caused by the white spot syndrome virus (WSSV), is a major scourge in cultured shrimp production facilities worldwide. This disease is only effectively controlled by sanitation. Intervention strategies are urgently needed but are limited by a lack of appropriate targets. Our identification of a per os infectivity factor (PIF) complex, which is pivotal for the entry of WSSV into shrimp, could provide new targets for antibody- or dsRNA-based intervention strategies. In addition, the presence of a PIF complex with at least eight components in WSSV, which is ancestrally related to the PIF complex of invertebrate baculoviruses, suggests that this complex is structurally and functionally conserved in disparate virus taxa.

Rotor-Tuning Boron Dipyrromethenes for Dual-Functional Imaging of Aß Oligomers and Viscosity.

Alzheimer's disease (AD), known as a common incurable and elderly neurodegenerative disease, has been widely explored for accurate detection of its biomarker (Aß oligomers) for early diagnosis. Although great efforts have been made, it is still of great importance to develop fluorescence probes for Aß oligomers with good selectivity and low background. Herein, starting from BODIPY493/503 (a commercial dye for neutral lipid droplets), which exhibited a small Stokes shift and no response toward Aß peptides, two fluorescence probes 5MB-SZ and B-SZ with a benzothiazole rotor at the 2-position of the BODIPY core and a methyl or benzyl group at the meso position have been designed and synthesized, which exhibited excellent optical properties/stability and could successfully image ß-amyloid fibrils and viscosity. Upon exposure to Aß oligomers, the fluorescence intensity of 5MB-SZ was enhanced by 43.64-fold with the corresponding fluorescence quantum yields changing from 0.85% to 27.43%. Meanwhile, probe 5MB-SZ showed a highly sensitive viscosity response in both solutions and living cells. In vitro and in vivo experiments confirmed that probe 5MB-SZ exhibited an excellent capacity for imaging ß-amyloid fibrils. Therefore, 5MB-SZ, as a rotor-tuning BODIPY analogue, could possibly serve as a highly potential and powerful fluorescence probe for early diagnosis of AD.

Endosomes and Microtubles are Required for Productive Infection in Aquareovirus.

Grass carp reovirus (GCRV), the genus Aquareovirus in family Reoviridae, is viewed as the most pathogenic aquareovirus. To understand the molecular mechanism of how aquareovirus initiates productive infection, the roles of endosome and microtubule in cell entry of GCRV are investigated by using quantum dots (QDs)-tracking in combination with biochemical approaches. We found that GCRV infection and viral protein synthesis were significantly inhibited by pretreating host cells with endosome acidification inhibitors NH4Cl, chloroquine and bafilomycin A1 (Bafi). Confocal images indicated that GCRV particles could colocalize with Rab5, Rab7 and lysosomes in host cells. Further ultrastructural examination validated that viral particle was found in late endosomes. Moreover, disruption of microtubules with nocodazole clearly blocked GCRV entry, while no inhibitory effects were observed with cytochalasin D treated cells in viral infection, hinting that intracellular transportation of endocytic uptake in GCRV infected cells is via microtubules but not actin filament. Notably, viral particles were observed to transport along microtubules by using QD-labeled GCRV. Altogether, our results suggest that GCRV can use endosomes and microtubules to initiate productive infection.

Functional Characterization of the Group I Alphabaculovirus Specific Gene ac73.

Baculoviridae is a family of large DNA viruses that specifically infect insects. It contains four genera, Alpha-, Beta-, Gamma-, and Deltabaculovirus. Alphabaculovirus is further divided into Group I and II, and Group I appears to be emerged most recently among all baculoviruses. Interestingly, there are 12 Group I specific genes that are only found in this lineage. Studying these genes is helpful to understand how baculoviruses evolved. Here, we reported the functional analyzing results of ac73, a function unknown Group I specific gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) which is the type species of baculovirus. The AC73 protein encoded by ac73 was found to be expressed during the late stage of infection and incorporated into the nucleocapsids of budded virus (BV) and occlusion-derived virus (ODV). In infected cells, AC73 resided mainly in the ring zone region of the nucleus, and appeared to be assembled into occlusion bodies (OBs). The ac73 knockout and repaired viruses were constructed and studied by in vitro and in vivo infection. Although ac73 was not essential for BV and ODV or OB formation, the BV titer and viral infectivity in insect larvae of ac73 knockout AcMNPV decreased by about 5-8 and 3-4 fold compared to those of wild type virus, respectively, suggesting ac73 contributed to infectious BV production and viral infectivity in vivo. This research provides new insight into the function of this Group I specific gene.

NS38 is required for aquareovirus replication via interaction with viral core proteins and host eIF3A.

Aquareoviruses contain an 11-segmented double-stranded RNA genome. Previous studies indicated that NS38, a virus-encoded putative single-stranded RNA binding protein, interacts with NS80 in viral inclusion bodies (VIBs). However, the role of NS38 in aquareovirus infection remained unclear. Here, we found that NS38 interacts with inner-capsid proteins (VP1-VP4 and VP6) and the NS80-RNA complex in both transfected and infected cells. Knockdown of NS38 by siRNAs-115/219 clearly reduced viral infection, with decreased mRNA and protein yields. Moreover, NS38 can interact with host cellular eukaryotic translation initiation factor 3 subunit A (eIF3A) in transfected cells, while no association was detected between eIF3A and NS80. This study is the first to define that the NS38 is essential to viral replication. Together, our findings indicate that NS38 might function as a mediator by interacting with viral and host cellular components in VIBs during replication.

Structure of RNA polymerase complex and genome within a dsRNA virus provides insights into the mechanisms of transcription and assembly.

Most double-stranded RNA (dsRNA) viruses transcribe RNA plus strands within a common innermost capsid shell. This process requires coordinated efforts by RNA-dependent RNA polymerase (RdRp) together with other capsid proteins and genomic RNA. Here we report the near-atomic resolution structure of the RdRp protein VP2 in complex with its cofactor protein VP4 and genomic RNA within an aquareovirus capsid using 200-kV cryoelectron microscopy and symmetry-mismatch reconstruction. The structure of these capsid proteins enabled us to observe the elaborate nonicosahedral structure within the double-layered icosahedral capsid. Our structure shows that the RdRp complex is anchored at the inner surface of the capsid shell and interacts with genomic dsRNA and four of the five asymmetrically arranged N termini of the capsid shell proteins under the fivefold axis, implying roles for these N termini in virus assembly. The binding site of the RNA end at VP2 is different from the RNA cap binding site identified in the crystal structure of orthoreovirus RdRp λ3, although the structures of VP2 and λ3 are almost identical. A loop, which was thought to separate the RNA template and transcript, interacts with an apical domain of the capsid shell protein, suggesting a mechanism for regulating RdRp replication and transcription. A conserved nucleoside triphosphate binding site was localized in our RdRp cofactor protein VP4 structure, and interactions between the VP4 and the genomic RNA were identified.

Identification of the caveolae/raft-mediated endocytosis as the primary entry pathway for aquareovirus.

Grass carp reovirus (GCRV), a member of the Aquareovirus genus in the Reoviridae family, is considered the most pathogenic aquareovirus. However, its productive viral entry pathways remain largely unclear. Using a combination of quantum dot (QD)-based live-virus tracking and biochemical assays, we found that extraction of cellular membrane cholesterol with methyl-ß-cyclodextrin (MßCD) and nystatin strongly inhibited the internalization of GCRVs, and supplementation with cholesterol restored viral infection. In addition, the entry of the virus was restrained by genistein, an inhibitor known to block caveolar endocytosis. Subsequent real-time tracking experiments revealed that the QD-labeled GCRV particles were colocalized with caveolin-1, and transfection of cells with dominant-negative mutant (caveolin-1 Y14F) significantly reduced GCRV infection. In contrast, no effects on virus infection were detected when the clathrin-mediated endocytosis or the macropinocytosis inhibitors were used. Our results collectively suggest that aquareoviruses can use caveolae/raft-mediated endocytosis as the primary entry pathway to initiate productive infection.

Identification and characterization of two cleavage fragments from the Aquareovirus nonstructural protein NS80.

Aquareovirus species vary with respect to pathogenicity, and the nonstructural protein NS80 of aquareoviruses has been implicated in the regulation of viral replication and assembly, which can form viral inclusion bodies (VIBs) and recruit viral proteins to its VIBs in infected cells. NS80 consists of 742 amino acids with a molecular weight of approximately 80 kDa. Interestingly, a short specific fragment of NS80 has also been detected in infected cells. In this study, an approximately 58-kDa product of NS80 was confirmed in various infected and transfected cells by immunoblotting analyses using α-NS80C. Mutational analysis and time course expression assays indicated that the accumulation of the 58-kDa fragment was related to time and infection dose, suggesting that the fragment is not a transient intermediate of protein degradation. Moreover, another smaller fragment with a molecular mass of approximately 22 kDa was observed in transfected and infected cells by immunoblotting with a specific anti-FLAG monoclonal antibody or α-NS80N, indicating that the 58- kDa polypeptide is derived from a specific cleavage site near the amino terminus of NS80. Additionally, different subcellular localization patterns were observed for the 22-kDa and 58-kDa fragments in an immunofluorescence analysis, implying that the two cleavage fragments of NS80 function differently in the viral life cycle. These results provide a basis for additional studies of the role of NS80 played in replication and particle assembly of the Aquareovirus.

The N-Terminal of Aquareovirus NS80 Is Required for Interacting with Viral Proteins and Viral Replication.

Reovirus replication and assembly occurs within viral inclusion bodies that formed in specific intracellular compartments of cytoplasm in infected cells. Previous study indicated that aquareovirus NS80 is able to form inclusion bodies, and also can retain viral proteins within its inclusions. To better understand how NS80 performed in viral replication and assembly, the functional regions of NS80 associated with other viral proteins in aquareovirus replication were investigated in this study. Deletion mutational analysis and rotavirus NSP5-based protein association platform were used to detect association regions. Immunofluorescence images indicated that different N-terminal regions of NS80 could associate with viral proteins VP1, VP4, VP6 and NS38. Further co-immunoprecipitation analysis confirmed the interaction between VP1, VP4, VP6 or NS38 with different regions covering the N-terminal amino acid (aa, 1-471) of NS80, respectively. Moreover, removal of NS80 N-terminal sequences required for interaction with proteins VP1, VP4, VP6 or NS38 not only prevented the capacity of NS80 to support viral replication in NS80 shRNA-based replication complementation assays, but also inhibited the expression of aquareovirus proteins, suggesting that N-terminal regions of NS80 are necessary for viral replication. These results provided a foundational basis for further understanding the role of NS80 in viral replication and assembly during aquareovirus infection.

Aquareovirus NS80 Initiates Efficient Viral Replication by Retaining Core Proteins within Replication-Associated Viral Inclusion Bodies.

Viral inclusion bodies (VIBs) are specific intracellular compartments for reoviruses replication and assembly. Aquareovirus nonstructural protein NS80 has been identified to be the major constituent for forming globular VIBs in our previous study. In this study, we investigated the role of NS80 in viral structural proteins expression and viral replication. Immunofluorescence assays showed that NS80 could retain five core proteins or inner-capsid proteins (VP1-VP4 and VP6), but not outer-capsid proteins (VP5 and VP7), within VIBs in co-transfected or infected cells. Further co-immunoprecipitation analysis confirmed that NS80 could interact with each core protein respectively. In addition, we found that newly synthesized viral RNAs co-localized with VIBs. Furthermore, time-course analysis of viral structural proteins expression showed that the expression of NS80 was detected first, followed by the detection of inner shell protein VP3, and then of other inner-capsid proteins, suggesting that VIBs were essential for the formation of viral core frame or progeny virion. Moreover, knockdown of NS80 by shRNA not only inhibited the expression of aquareovirus structural proteins, but also inhibited viral infection. These results indicated that NS80-based VIBs were formed at earlier stage of infection, and NS80 was able to coordinate the expression of viral structural proteins and viral replication.

VP5 autocleavage is required for efficient infection by in vitro-recoated aquareovirus particles.

Grass carp reovirus (GCRV) is a member of the genus Aquareovirus in the family Reoviridae, and contains five core proteins (VP1-VP4 and VP6) and two outer-capsid proteins (VP5 and VP7) in its particle. Previous studies have revealed that the outer-capsid proteins of reovirus are responsible for initiating infection, but the mechanism is poorly understood. Using baculovirus-expressed VP5 and VP7 to recoat purified cores, in vitro assembly of GCRV was achieved in this study. Recoated GCRV (R-GCRV) closely resembled native GCRV (N-GCRV) in particle morphology, protein composition and infectivity. Similar to N-GCRV, the infectivity of R-GCRV could be inhibited by treating cells with the weak base NH4Cl. In addition, recoated particles carrying an Asn→Ala substitution at residue 42 of VP5 (VP5N42A/VP7 R-GCRV) were no longer infectious. These results provide strong evidence that autocleavage of VP5 is critical for aquareovirus to initiate efficient infection.

Identification of a functional motif in the AqRV NS26 protein required for enhancing the fusogenic activity of FAST protein NS16.

Aquareoviruses AqRVs have a close relationship with orthoreoviruses. However, they contain an additional genome segment, S11, which encodes nonstructural protein NS26. We previously showed that NS26 can enhance the fusogenic activity of the fusion-associated small transmembrane FAST protein NS16 from AqRV. In this study, a TLPK motif in NS26 was identified as being important for the enhancement. When the TLPK motif was deleted from NS26, the enhanced efficiency of the NS16-mediated cellcell fusion was significantly impaired. Further mutational analysis showed that the lysine K residue in the TLPK motif was critical for the enhancement. Additionally, deletion of the TLPK motif prevented NS26 from interacting with lysosomes. These findings suggested that the TLPK motif is important for NS26 to enhance the fusogenic activity of NS16, and NS26 may utilize the lysosome to benefit the fusion process.