Viral tegument proteins restrict cGAS-DNA phase separation to mediate immune evasion.

DNA-induced liquid-liquid phase separation of cyclic GMP-AMP synthase (cGAS) triggers a potent response to detect pathogen infection and promote innate immune signaling. Whether and how pathogens manipulate cGAS-DNA condensation to mediate immune evasion is unknown. We report the identification of a structurally related viral tegument protein family, represented by ORF52 and VP22 from gamma- and alpha-herpesvirinae, respectively, that employs a conserved mechanism to restrict cGAS-DNA phase separation. ORF52/VP22 proteins accumulate into, and effectively disrupt, the pre-formed cGAS-DNA condensation both in vitro and in cells. The inhibition process is dependent on DNA-induced liquid-liquid phase separation of the viral protein rather than a direct interaction with cGAS. Moreover, highly abundant ORF52 proteins carried within viral particles are able to target cGAS-DNA phase separation in early infection stage. Our results define ORF52/VP22-type tegument proteins as a family of inhibitors targeting cGAS-DNA phase separation and demonstrate a mechanism for how viruses overcome innate immunity.

RNF213 modulates γ-herpesvirus infection and reactivation via targeting the viral Replication and Transcription Activator.

Interferons (IFNs) and the products of interferon-stimulated genes (ISGs) play crucial roles in host defense against virus infections. Although many ISGs have been characterized with respect to their antiviral activity, their target specificities and mechanisms of action remain largely unknown. Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that is linked to several human malignancies. Here, we used the genetically and biologically related virus, murine gammaherpesvirus 68 (MHV-68) and screened for ISGs with anti-gammaherpesvirus activities. We found that overexpression of RNF213 dramatically inhibited MHV-68 infection, whereas knockdown of endogenous RNF213 significantly promoted MHV-68 proliferation. Importantly, RNF213 also inhibited KSHV de novo infection, and depletion of RNF213 in the latently KSHV-infected iSLK-219 cell line significantly enhanced lytic reactivation. Mechanistically, we demonstrated that RNF213 targeted the Replication and Transcription Activator (RTA) of both KSHV and MHV-68, and promoted the degradation of RTA protein through the proteasome-dependent pathway. RNF213 directly interacted with RTA and functioned as an E3 ligase to ubiquitinate RTA via K48 linkage. Taken together, we conclude that RNF213 serves as an E3 ligase and inhibits the de novo infection and lytic reactivation of gammaherpesviruses by degrading RTA through the ubiquitin-proteasome pathway.

Meta-learning shows great potential in plant disease recognition under few available samples.

Plant diseases worsen the threat of food shortage with the growing global population, and disease recognition is the basis for the effective prevention and control of plant diseases. Deep learning has made significant breakthroughs in the field of plant disease recognition. Compared with traditional deep learning, meta-learning can still maintain more than 90% accuracy in disease recognition with small samples. However, there is no comprehensive review on the application of meta-learning in plant disease recognition. Here, we mainly summarize the functions, advantages, and limitations of meta-learning research methods and their applications for plant disease recognition with a few data scenarios. Finally, we outline several research avenues for utilizing current and future meta-learning in plant science. This review may help plant science researchers obtain faster, more accurate, and more credible solutions through deep learning with fewer labeled samples.

CircBRD7 attenuates tumor growth and metastasis in nasopharyngeal carcinoma via epigenetic activation of its host gene.

BRD7 was identified as a tumor suppressor in nasopharyngeal carcinoma (NPC). Circular RNAs (CircRNAs) are involved in the occurrence and development of NPC as oncogenes or tumor suppressors. However, the function and mechanism of the circular RNA forms derived from BRD7 in NPC are not well understood. In this study, we first identified that circBRD7 was a novel circRNA derived from BRD7 that inhibited cell proliferation, migration, invasion of NPC cells, as well as the xenograft tumor growth and metastasis in vivo. Mechanistically, circBRD7 promoted the transcriptional activation and expression of BRD7 by enhancing the enrichment of histone 3 lysine 27 acetylation (H3K27ac) in the promoter region of its host gene BRD7, and BRD7 promoted the formation of circBRD7. Therefore, circBRD7 formed a positive feedback loop with BRD7 to inhibit NPC development and progression. Moreover, restoration of BRD7 expression rescued the inhibitory effect of circBRD7 on the malignant progression of NPC. In addition, circBRD7 demonstrated low expression in NPC tissues, which was positively correlated with BRD7 expression and negatively correlated with the clinical stage of NPC patients. Taken together, circBRD7 attenuates the tumor growth and metastasis of NPC by forming a positive feedback loop with its host gene BRD7, and targeting the circBRD7/BRD7 axis is a promising strategy for the clinical diagnosis and treatment of NPC.

Concurrent attention to hetero-depth surfaces in 3-D visual space is governed by theta rhythm.

When simultaneously confronted with multiple attentional targets, visual system employs a time-multiplexing approach in which each target alternates for prioritized access, a mechanism broadly known as rhythmic attentional sampling. For the past decade, rhythmic attentional sampling has received mounting support from converging behavioral and neural findings. However, so compelling are these findings that a critical test ground has been long overshadowed, namely the 3-D visual space where attention is complicated by extraction of the spatial layout of surfaces extending beyond 2-D planes. It remains unknown how attentional deployment to multiple targets is accomplished in the 3-D space. Here, we provided a time-resolved portrait of the behavioral and neural dynamics when participants concurrently attended to two surfaces defined by motion-depth conjunctions. To characterize the moment-to-moment attentional modulation effects, we measured perceptual sensitivity to the hetero-depth surface motions on a fine temporal scale and reconstructed their neural representations using a time-resolved multivariate inverted encoding model. We found that the perceptual sensitivity to the two surface motions rhythmically fluctuated over time at ~4 Hz, with one's enhancement closely tracked by the other's diminishment. Moreover, the behavioral pattern was coupled with an ongoing periodic alternation in strength between the two surface motion representations in the same frequency. Together, our findings provide the first converging evidence of an attentional "pendulum" that rhythmically traverses different stereoscopic depth planes and are indicative of a ubiquitous attentional time multiplexor based on theta rhythm in the 3-D visual space.

A specific super-enhancer actuated by berberine regulates EGFR-mediated RAS-RAF1-MEK1/2-ERK1/2 pathway to induce nasopharyngeal carcinoma autophagy.

Nasopharyngeal carcinoma (NPC), primarily found in the southern region of China, is a malignant tumor known for its highly metastatic characteristics. The high mortality rates caused by the distant metastasis and disease recurrence remain unsolved clinical problems. In clinic, the berberine (BBR) compound has widely been in NPC therapy to decrease metastasis and disease recurrence, and BBR was documented as a main component with multiple anti-NPC effects. However, the mechanism by which BBR inhibits the growth and metastasis of nasopharyngeal carcinoma remains elusive. Herein, we show that BBR effectively inhibits the growth, metastasis, and invasion of NPC via inducing a specific super enhancer (SE). From a mechanistic perspective, the RNA sequencing (RNA-seq) results suggest that the RAS-RAF1-MEK1/2-ERK1/2 signaling pathway, activated by the epidermal growth factor receptor (EGFR), plays a significant role in BBR-induced autophagy in NPC. Blockading of autophagy markedly attenuated the effect of BBR-mediated NPC cell growth and metastasis inhibition. Notably, BBR increased the expression of EGFR by transcription, and knockout of EGFR significantly inhibited BBR-induced microtubule associated protein 1 light chain 3 (LC3)-II increase and p62 inhibition, proposing that EGFR plays a pivotal role in BBR-induced autophagy in NPC. Chromatin immunoprecipitation sequencing (ChIP-seq) results found that a specific SE existed only in NPC cells treated with BBR. This SE knockdown markedly repressed the expression of EGFR and phosphorylated EGFR (EGFR-p) and reversed the inhibition of BBR on NPC proliferation, metastasis, and invasion. Furthermore, BBR-specific SE may trigger autophagy by enhancing EGFR gene transcription, thereby upregulating the RAS-RAF1-MEK1/2-ERK1/2 signaling pathway. In addition, in vivo BBR effectively inhibited NPC cells growth and metastasis, following an increase LC3 and EGFR and a decrease p62. Collectively, this study identifies a novel BBR-special SE and established a new epigenetic paradigm, by which BBR regulates autophagy, inhibits proliferation, metastasis, and invasion. It provides a rationale for BBR application as the treatment regime in NPC therapy in future.

Risk factors for amputation in diabetic foot ulcers: A retrospective analysis.

Diabetic foot ulcers (DFUs) are chronic, difficult-to-heal wounds with a very high incidence of amputation. For patients with DFUs, prevention of amputation is crucial. However, the risk factors associated with DFU amputation and the extent to which different risk factors increase the risk of amputation are still uncertain. This study intends to provide a clinical basis for early intervention in DFU by retrospectively analysing the risk factors for DFU amputation. A retrospective analysis of 200 patients with DFUs admitted between October 2019 and October 2023 was conducted. Sixty-eight of the 200 underwent amputations. The overall amputation rate was 34%. Multiple logistic regression model showed that neutrophil/lymphocyte ratio (OR = 1.943; 95% CI:1.826-2.139), white blood cell (OR = 1.143; 95% CI:1.034-1.267), C-reactive protein (OR = 1.307; 95% CI:1.113-2.194) and Wagner grading (OR = 2.783; 95% CI: 1.751-4.302) were independent risk factors for amputation, while haemoglobin (OR = 0.742; 95% CI:0.638-0.965) and high density lipoprotein were independent protective factors for amputation (OR = 0.168; 95% CI:0.037-0.716), and further Receiver Operating Characteristic Curve curves showed that they showed high accuracy and were good predictors of amputation of DFUs.

Correlation of iron deficiency indexes with Eradication and Recurrence of Helicobacter Pylori Infection in Children.

Objective: To explore the correlation of iron deficiency (ID) indexes with eradication and recurrence of Helicobacter pylori (Hp) infection in children. Methods: This is a clinical comparative study. One hundred and twenty-six children who were first diagnosed as Hp infection in Baoding Children's Hospital (Hp infection group); and the control group included 200 children without Helicobacter Pylori infection (negative stool Hp antigen test and/or 13C-urea breath test) in local region at the same time from January 2020 to January 2022. Enrolled children were subjected to routine blood test, serum ferritin (SF), serum iron (SI) and total iron binding capacity (TIBC) detection. Meanwhile, children with Hp infection were given triple therapy for eradication and followed up for one year. Results: The levels of SI, SF and Hb in non-eradication group were lower than those in eradication group (P<0.05); while TIBC level in the former group was higher than that in the latter group (P<0.05). Furthermore, SF level in the recurrence group was lower than that in the non-recurrence group (P<0.05). While there was no significant difference in Hb, SI and TIBC levels between the recurrence group and the non-recurrence group (P>0.05). Conclusion: Low level of SF may be a risk factor for difficulty in eradication and recurrence after eradication in children with Hp infection. Meanwhile, low levels of Hb and SI are influential factors for difficulty in eradication in children with Hp infection.

The Interaction between Tegument Proteins ORF33 and ORF45 Plays an Essential Role in Cytoplasmic Virion Maturation of a Gammaherpesvirus.

Tegument, which occupies the space between the nucleocapsid and the envelope, is a unique structure of a herpesvirion. Tegument proteins are major components of tegument and play critical roles in virus life cycle. Murine gammaherpesvirus 68 (MHV-68), a member of the gammaherpesvirus subfamily, is closely related to two human herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). We have previously shown that MHV-68 ORF33, conserved among all herpesviruses, encodes a tegument protein that is associated with intranuclear capsids and is essential for virion morphogenesis and egress. Another tegument protein ORF45, which is conserved only among gammaherpesviruses, also plays an essential role in virion morphogenesis of MHV-68. In this study, we investigated the underlying mechanism and showed that these two proteins colocalize and interact with each other during virus infection. We mapped the ORF33-interacting domain to the conserved carboxyl-terminal 23 amino acids (C23) of ORF45. Deletion of the C23 coding sequence in the context of viral genome abolished the production of infectious virions. Transmission electron microscopy results demonstrated that C23 of ORF45 are essential for virion tegumentation in the cytoplasm. We further mapped the ORF45-interacting domain to the N-terminal 17 amino acids (N17) of ORF33. Deletion of the N17 coding sequence in the context of viral genome also abolished production of infectious virions, and N17 of ORF33 are also essential for virion tegumentation in the cytoplasm. Taken together, our data strongly indicate that the interaction between ORF45 and ORF33 plays an essential role in cytoplasmic maturation of MHV-68 virions. IMPORTANCE A critical step in viral lytic replication is the assembly of progeny viral particles. Herpesviruses are important pathogens. A herpesvirus particle comprises, from inside to outside, four layers: DNA core, capsid, tegument, and envelope. The tegument layer contains dozens of virally encoded tegument proteins, which play critical roles in virus assembly. Murine gammaherpesvirus 68 (MHV-68) is a tumor-associated herpesvirus and is closely related to Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. We previously found that the absence of either tegument protein ORF33 or ORF45 inhibits the translocation of nucleocapsids to the cytoplasm and blocks virion maturation, but the underlying mechanism remained unclear. Here, we showed that ORF33 interacts with ORF45. We mapped their interaction domains and constructed viral mutants with defects in ORF33-ORF45 interaction. Transmission electron microscopy data demonstrated that the assembly of these viral mutants in the cytoplasm is blocked. Our results indicate that ORF33-ORF45 interaction is essential for gammaherpesvirus replication.

Chiral-Boron-Complex-Catalyzed Asymmetric [3 + 2] Cycloaddition of ß-Trifluoromethyl α,ß-Unsaturated Ketones with N,N'-Cyclic Azomethine Imines.

The (R)-3,3'-(3,5-(CF3)2-C6H3)2-BINOL-boron-complex-catalyzed asymmetric 1,3-dipolar cycloaddition of ß-trifluoromethyl α,ß-unsaturated ketone with N,N'-cyclic azomethine imines was developed to afford N,N'-bicyclic pyrazolidine derivatives bearing a stereogenic carbon center containing CF3 motifs in high yields with excellent diastereo- and enantioselectivities (up to >20:1 dr, and >99% ee). This catalytic system features mild reaction conditions, high efficiency, and a broad substrate scope.

Molecular mechanism underlying selective inhibition of mRNA nuclear export by herpesvirus protein ORF10.

Viruses employ multiple strategies to inhibit host mRNA nuclear export. Distinct to the generally nonselective inhibition mechanisms, ORF10 from gammaherpesviruses inhibits mRNA export in a transcript-selective manner by interacting with Rae1 (RNA export 1) and Nup98 (nucleoporin 98). We now report the structure of ORF10 from MHV-68 (murine gammaherpesvirus 68) bound to the Rae1-Nup98 heterodimer, thereby revealing detailed intermolecular interactions. Structural and functional assays highlight that two highly conserved residues of ORF10, L60 and M413, play critical roles in both complex assembly and mRNA export inhibition. Interestingly, although ORF10 occupies the RNA-binding groove of Rae1-Nup98, the ORF10-Rae1-Nup98 ternary complex still maintains a comparable RNA-binding ability due to the ORF10-RNA direct interaction. Moreover, mutations on the RNA-binding surface of ORF10 disrupt its function of mRNA export inhibition. Our work demonstrates the molecular mechanism of ORF10-mediated selective inhibition and provides insights into the functions of Rae1-Nup98 in regulating host mRNA export.

The emerging roles of the interaction between m6A modification and c-Myc in driving tumorigenesis and development.

N6-methyladenosine (m6A) is an extremely common and conservative posttranscriptional modification, that can specifically target and regulate the expression or stability of a series of tumor-related genes, thus playing critical roles in the occurrence and development of tumors. c-Myc is an important tumorigenic transcription factor that promotes tumorigenesis and development by mainly regulating the expression of downstream target genes. Increasing evidence shows that m6A modification, as well as abnormal expression and regulation of c-Myc, is critical molecular mechanisms driving tumorigenesis and development. Although more evidence has been uncovered about the individual roles of m6A modification or c-Myc in tumors, the interaction between m6A modification and c-Myc in tumorigenesis and development has not been systematically summarized. Therefore, this review is focused on the mutual regulation between m6A modification and c-Myc expression and stability as well as its roles in tumorigenesis and development. We also summarized the potential value of the interaction between m6A modification and m6A expression and stability in tumor diagnosis and treatment, which provides a specific reference for revealing the mechanism of tumor occurrence and development as well as clinical diagnosis and treatment.

Correlation between helicobacter pylori infection and iron deficiency in children.

Objectives: To investigate the correlation between Helicobacter pylori (Hp) infection and Iron deficiency (ID) in children. Methods: This cross-sectional study was conducted at Baoding Children's Hospital from January 2018 to December 2019. A total of one thousand children who came to our hospital for physical examination and met the inclusion criteria were continuously included in this study. All the children were given questionnaires (personal and family social and economic status), a stool Hp antigen test and/or a 13C-urea breath test, as well as measurements of hemoglobin (Hb), mean red blood cell volume (MCV), red blood cell distribution width (RDW), mean corpuscular hemoglobin concentration (MCHC), serum ferritin (SF), serum iron (SI), and total iron binding capacity (TIBC). Children who tested positive for Hp were divided into the Hp group and children who tested negative for Hp were divided into the control group. ID or IDA was diagnosed based on the child's blood test results. Results: A total of 902 children met the inclusion criteria, including 194 (21.5%) in the Hp group and 708 (78.5%) in the control group. The incidence of ID and IDA in Hp group was higher than that in control group (2=9.112, 2=4.478; All P < 0.05); The levels of MCV, SI, SF and Hb in Hp group were lower than those in control group (t=5.288; T = 3.864; T = 6.751; T =11.841, all P < 0.05), TIBC level was higher than that of control group (T =7.630, P < 0.05); The levels of MCHC and RDW in THE Hp group were not statistically significant compared with the control group. Logistic regression showed that Hp infection was not a combined risk factor for ID. Older age, higher educational background of the mother, living in the city, and higher family income were the combined protective factors to prevent the occurrence of ID in children. Conclusion: Hp infection is not a combined risk factor for the development of ID in children. The influence of family social and economic factors should be taken into consideration when analyzing the correlation between Hp infection and ID.

Correlation between the Level of Inflammatory Cytokines and Prognosis in Children with IVIG-sensitive Kawasaki Disease and IVIG-resistant Kawasaki Disease.

Objectives: To investigate whether the levels of interleukin 1ß (IL-1ß), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α) in children with Kawasaki disease (KD) are correlated with coronary artery lesion (CAL) and resistance to intravenous immunoglobulin (IVIG) treatment. Methods: A total of 216 children in line with KD diagnostic criteria were continuously included as subjects, and 50 healthy children at the same period were selected as the control group, and their levels of IL-1ß, IFN-γ, and TNF-α were detected. Results: Subjects were subdivided according to the presence or absence of CAL: 42 cases (19.4%) of 216 children with KD developed CAL and were subdivided into the CAL group, while 174 (80.6%) of those who did not develop CAL were subdivided into the NCAL group. The levels of IL-1ß, IFN-γ, and TNF-α in the CAL group and the NCAL group were higher than those in the control group (P<0.05), and the levels of those in the CAL group were higher than those in the NCAL group (P<0.05). Subjects were subdivided according to the effect of IVIG treatment: 194 cases (89.8%) of 216 children with KD had a good control of inflammation after the initial IVIG treatment, and were considered to have IVIG-sensitive KD and divided into the IVIG-sensitive group; 22 cases (10.2%) could not get good control of inflammation after the initial IVIG treatment, and were considered to have IVIG-resistant KD and divided into the IVIG-resistant group. The levels of IL-1ß, IFN-γ, and TNF-α in the IVIG-sensitive group and the IVIG-resistant group were higher than those in the control group; The levels of IL-1ß, IFN-γ, and TNF-α in the IVIG-resistant group were higher than those in the IVIG-sensitive group (P<0.05), while the fever time of the IVIG-sensitive group was lower than that of the IVIG-resistant group (P<0.05). Conclusion: Children with KD may experience changes in IL-1ß, IFN-γ, and TNF-α levels in the acute phase. Such a significant increase in levels may be a risk factor for CAL and resistance to IVIG treatment in children with KD, while the prolonged fever time is a risk factor for resistance to IVIG treatment in children with KD.

Changes in metabolomics and lipidomics in brain tissue and their correlations with the gut microbiome after chronic food-derived arsenic exposure in mice.

Arsenic can cause neurodegenerative diseases of the brain, but the definite mechanism is still unknown. In this study, to discuss the disturbances on brain metabolome and lipidome under subchronic arsenic exposure, we treated mice with the arsenic-containing feed (concentration of total arsenic = 30 mg/kg) prepared in accordance with the proportion of rice arsenicals for 16 weeks and performed metabolomics and lipidomics studies respectively using UHPLC-Triple-TOF-MS/MS and UHPLC-Q Exactive Focus MS/MS on mice brain. In addition, the distributions of arsenical metabolites along the feed-gut-blood-brain chain were analyzed by ICP-MS and HPLC-ICP-MS, and fecal microbial variations were investigated by 16 s sequencing. The data showed that although only a tiny amount of arsenic (DMA=0.101 mg/kg, uAs=0.071 mg/kg) enters the brain through the blood-brain barrier, there were significant changes in brain metabolism, including 118 metabolites and 17 lipids. These different metabolites were involved in 30 distinct pathways, including glycometabolism, and metabolisms of lipid, nucleic acid, and amino acid were previously reported to be correlated with neurodegenerative diseases. Additionally, these different metabolites were significantly correlated with 12 gut bacterial OTUs, among which Lachnospiraceae, Muribaculaceae, Ruminococcaceae, and Erysipelotrichaceae were also previously reported to be related to the distortion of metabolism, indicating that the disturbance of metabolism in the brain may be associated with the disturbance of gut microbes induced by arsenic. Thus, the current study demonstrated that the brain metabolome and lipidome were significantly disturbed under subchronic arsenic exposure, and the disturbances also significantly correlated with some gut microbiome and may be associated with neurodegenerative diseases. Although preliminary, the results shed some light on the pathophysiology of arsenic-caused neurodegenerative diseases.

The novel TP53 3'-end methylation pattern associated with its expression would be a potential biomarker for breast cancer detection.

BACKGROUND: Deficiency or silence of TP53 is an early event in breast tumorigenesis. Aberrant methylation and mutation in regulatory regions were considered as crucial regulators of gene expression. METHODS: Using multiplex-PCR and next-generation sequencing technology, we analyzed TP53 mutation spectrum in its promoter region. Using PCR target sequence enrichment and next-generation bisulfite sequencing technology, we analyzed the methylation profile of the promoter and 3'-end regions of TP53 gene in paired breast tumor and normal tissues from 120 breast cancer patients. The expression of TP53 and the flanking gene ATP1B2 was explored with qPCR method in the same cohort. RESULTS: No promoter mutation of TP53 gene was found in the cohort of the 120 breast cancer patients. The 3'-end of TP53 gene was hyper-methylated (average 78.71%) compared with the promoter region (average less than 1%) in breast tumor tissues. TP53 was significantly lower expressed (P = 1.68E-15) and hyper-methylated in 3'-end (P = 1.82E-18) in tumor. Negative cis correlation was found between the TP53 expression and its 3'-end methylation (P = 9.02E-8, R = 0.337). TP53 expression was significantly associated with PR status (P = 0.0128), Ki67 level (P = 0.0091), and breast cancer subtypes (P = 0.0109). TP53 3'-end methylation and expression showed a good performance in discriminating breast cancer and normal tissues with an AUC of 0.930. CONCLUSIONS: The 3'-end methylation of TP53 might be a crucial regulator for its expression in breast cancer, suggesting that TP53 3'-end hyper-methylation associated with its lower expression could be a potential biomarker for breast cancer diagnosis.

Functional Identification and Characterization of the Nuclear Egress Complex of a Gammaherpesvirus.

The herpesvirus nuclear egress complex (NEC) is composed of two viral proteins. They play key roles in mediating the translocation of capsids from the nucleus to the cytoplasm by facilitating the budding of capsids into the perinuclear space (PNS). The NEC of alphaherpesvirus can induce the formation of virion-like vesicles from the nuclear membrane in the absence of other viral proteins. However, whether the NEC of gammaherpesvirus harbors the ability to do so in mammalian cells remains to be determined. In this study, we first constructed open reading frame 67 (ORF67)-null and ORF69-null mutants of murine gammaherpesvirus 68 (MHV-68) and demonstrated that both ORF67 and ORF69 play critical roles in nuclear egress and hence viral lytic replication. Biochemical and bioimaging analyses showed that ORF67 and ORF69 interacted with each other and were sufficient to induce the formation of virion-like vesicles from the nuclear membrane in mammalian cells. Thus, we designated ORF67 and ORF69 components of MHV-68 NEC. Furthermore, we identified amino acids critical for mediating the interaction between ORF67 and ORF69 through homology modeling and verified their function in nuclear egress, providing insights into the molecular basis of NEC formation in gammaherpesviruses.IMPORTANCE Increasing amounts of knowledge indicate that the nuclear egress complex (NEC) is critical for the nuclear egress of herpesvirus capsids, which can be viewed as a vesicle-mediated transport pathway through the nuclear membrane. In this study, we identified open reading frame 67 (ORF67) and ORF69 as components of the NEC in murine gammaherpesvirus 68 (MHV-68) and demonstrated that they efficiently induce virion-like vesicles from the nuclear membrane in mammalian cells. This is the first time that the NEC of a gammaherpesvirus has been found to demonstrate such an essential characteristic. In addition, we identified amino acids critical for mediating the interaction between ORF67 and ORF69 as well as nuclear egress. Notably, these amino acids are conserved in Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), providing a structural basis to design antigammaherpesvirus drugs.

A Large-Scale Multicenter Study Validates Aldo-Keto Reductase Family 1 Member B10 as a Prevalent Serum Marker for Detection of Hepatocellular Carcinoma.

Aldo-keto reductase family 1 member B10 (AKR1B10) is a secretory protein overexpressed in hepatocellular carcinoma (HCC). We aimed to evaluate AKR1B10 as a serum marker for detection of HCC. Herein, we conducted a cohort study that consecutively enrolled 1,244 participants from three independent hospitals, including HCC, healthy controls (HCs), benign liver tumors (BLTs), chronic hepatitis B (CHB), and liver cirrhosis (LC). Serum AKR1B10 was tested by time-resolved fluorescent assays. Data were plotted for receiver operating characteristic (ROC) curve analyses. Alpha-fetoprotein (AFP) was analyzed for comparison. An exploratory discovery cohort demonstrated that serum AKR1B10 increased in patients with HCC (1,567.3 ± 292.6 pg/mL; n = 69) compared with HCs (85.7 ± 10.9 pg/mL; n = 66; P < 0.0001). A training cohort of 519 participants yielded an optimal diagnostic cutoff of serum AKR1B10 at 267.9 pg/mL. When ROC curve was plotted for HCC versus all controls (HC + BLT + CHB + LC), serum AKR1B10 had diagnostic parameters of the area under the curve (AUC) 0.896, sensitivity 72.7%, and specificity 95.7%, which were better than AFP with AUC 0.816, sensitivity 65.1%, and specificity 88.9%. Impressively, AKR1B10 showed promising diagnostic potential in early-stage HCC and AFP-negative HCC. Combination of AKR1B10 with AFP increased diagnostic accuracy for HCC compared with AKR1B10 or AFP alone. A validation cohort of 522 participants confirmed these findings. An independent cohort of 68 patients with HCC who were followed up showed that serum AKR1B10 dramatically decreased 1 day after operation and was nearly back to normal 3 days after operation. Conclusion: AKR1B10 is a potent serum marker for detection of HCC and early-stage HCC, with better diagnostic performance than AFP.

Virus-Like Vesicles of Kaposi's Sarcoma-Associated Herpesvirus Activate Lytic Replication by Triggering Differentiation Signaling.

Virus-like vesicles (VLVs) are membrane-enclosed vesicles that resemble native enveloped viruses in organization but lack the viral capsid and genome. During the productive infection of tumor-associated gammaherpesviruses, both virions and VLVs are produced and are released into the extracellular space. However, studies of gammaherpesvirus-associated VLVs have been largely restricted by the technical difficulty of separating VLVs from mature virions. Here we report a strategy of selectively isolating VLVs by using a Kaposi's sarcoma-associated herpesvirus (KSHV) mutant that is defective in small capsid protein and is unable to produce mature virions. Using mass spectrometry analysis, we found that VLVs contained viral glycoproteins required for cellular entry, as well as tegument proteins involved in regulating lytic replication, but lacked capsid proteins. Functional analysis showed that VLVs induced the expression of the viral lytic activator RTA, initiating KSHV lytic gene expression. Furthermore, employing RNA sequencing, we performed a genomewide analysis of cellular responses triggered by VLVs and found that PRDM1, a master regulator in cell differentiation, was significantly upregulated. In the context of KSHV replication, we demonstrated that VLV-induced upregulation of PRDM1 was necessary and sufficient to reactivate KSHV by activating its RTA promoter. In sum, our study systematically examined the composition of VLVs and demonstrated their biological roles in manipulating host cell responses and facilitating KSHV lytic replication.IMPORTANCE Cells lytically infected with tumor-associated herpesviruses produce a high proportion of virus-like vesicles (VLVs). The composition and function of VLVs have not been well defined, largely due to the inability to efficiently isolate VLVs that are free of virions. Using a cell system capable of establishing latent KSHV infection and robust reactivation, we successfully isolated VLVs from a KSHV mutant defective in the small capsid protein. We quantitatively analyzed proteins and microRNAs in VLVs and characterized the roles of VLVs in manipulating host cells and facilitating viral infection. More importantly, we demonstrated that by upregulating PRDM1 expression, VLVs triggered differentiation signaling in targeted cells and facilitated viral lytic infection via activation of the RTA promoter. Our study not only demonstrates a new strategy for isolating VLVs but also shows the important roles of KSHV-associated VLVs in intercellular communication and the viral life cycle.

Tegument Protein ORF45 Plays an Essential Role in Virion Morphogenesis of Murine Gammaherpesvirus 68.

Tegument proteins play critical roles in herpesvirus morphogenesis. ORF45 is a conserved tegument protein of gammaherpesviruses; however, its role in virion morphogenesis is largely unknown. In this work, we determined the ultrastructural localization of murine gammaherpesvirus 68 (MHV-68) ORF45 and found that this protein was incorporated into virions around the site of host-derived vesicles. Notably, the absence of ORF45 inhibited nucleocapsid egress and blocked cytoplasmic virion maturation, demonstrating that ORF45 is essential for MHV-68 virion morphogenesis.