Identification of a novel neurovirulence factor encoded by the cryptic orphan gene UL31.6 of herpes simplex virus 1.

Although the herpes simplex virus type 1 (HSV-1) genome was thought to contain approximately 80 different protein coding sequences (CDSs), recent multi-omics analyses reported HSV-1 encodes more than 200 potential CDSs. However, few of the newly identified CDSs were confirmed to be expressed at the peptide or protein level in HSV-1-infected cells. Furthermore, the impact of the proteins they encode on HSV-1 infection is largely unknown. This study focused on a newly identified CDS, UL31.6. Re-analyzation of our previous chemical proteomics data verified that UL31.6 was expressed at the peptide level in HSV-1-infected cells. Antisera raised against a viral protein encoded by UL31.6 (pUL31.6) reacted with a protein with an approximate molecular mass of 37 kDa in lysates of Vero cells infected with each of three HSV-1 strains. pUL31.6 was efficiently dissociated from virions in high-salt solution. A UL31.6-null mutation had a minimal effect on HSV-1 gene expression, replication, cell-to-cell spread, and morphogenesis in Vero cells; in contrast, it significantly reduced HSV-1 cell-to-cell spread in three neural cells but not in four non-neural cells including Vero cells. The UL31.6-null mutation also significantly reduced the mortality and viral replication in the brains of mice after intracranial infection, but had minimal effects on pathogenic manifestations in and around the eyes, and viral replication detected in the tear films of mice after ocular infection. These results indicated that pUL31.6 was a tegument protein and specifically acted as a neurovirulence factor by potentially promoting viral transmission between neuronal cells in the central nervous system.IMPORTANCERecent multi-omics analyses reported the herpes simplex virus type 1 (HSV-1) genome encodes an additional number of potential coding sequences (CDSs). However, the expressions of these CDSs at the peptide or protein levels and the biological effects of these CDSs on HSV-1 infection remain largely unknown. This study annotated a cryptic orphan CDS, termed UL31.6, an HSV-1 gene that encodes a tegument protein with an approximate molecular mass of 37 kDa, which specifically acts as a neurovirulence factor. Our study indicates that HSV-1 proteins important for viral pathogenesis remain to be identified and a comprehensive understanding of the pathogenesis of HSV-1 will require not only the identification of cryptic orphan CDSs using emerging technologies but also step-by-step and in-depth analyses of each of the cryptic orphan CDSs.

MYBBP1A is required for efficient replication and gene expression of herpes simplex virus 1.

More than 100 different herpes simplex virus 1 (HSV-1) genes belong to three major classes, and their expression is coordinately regulated and sequentially ordered in a cascade. This complex HSV-1 gene expression is thought to be regulated by various viral and host cellular proteins. A host cellular protein, Myb-binding protein 1A (MYBBP1A), has been reported to be associated with HSV-1 viral genomes in conjunction with viral and cellular proteins critical for DNA replication, repair, and transcription within infected cells. However, the role(s) of MYBBP1A in HSV-1 infections remains unclear. In this study, we examined the effects of MYBBP1A depletion on HSV-1 infection and found that MYBBP1A depletion significantly reduced HSV-1 replication, as well as the accumulation of several viral proteins. These results suggest that MYBBP1A is an important host cellular factor that contributes to HSV-1 replication, plausibly by promoting viral gene expression.

3-Fr microcatheter-assisted endoscopic ultrasound-guided rendezvous technique with a 22-gauge needle and a 0.018-inch guidewire for difficult biliary cannulation.

Endoscopic ultrasound-guided rendezvous with a 22-gauge needle and a 0.018-inch guidewire, assisted by a 3-Fr microcatheter, effectively addresses challenges in biliary cannulation, improving guidewire manipulation and reducing risks of injury and leakage. Natsume and colleagues describe the successful extraction of common bile duct stones to demonstrate the efficacy of this technique.

Incidence of pancreatic injury and pancreatitis in patients treated with immune checkpoint inhibitors.

OBJECTIVES: Immune checkpoint inhibitors (ICIs) are being increasingly used to treat advanced malignancies. ICI-induced pancreatic injury (ICI-PI), which is an immune-related adverse event that may be a risk factor for ICI-associated pancreatitis, is not well documented in the literature. METHODS: Consecutive patients who received ICIs for advanced malignancies from August 2015 through October 2022 were analyzed with regard to the incidence of ICI-PI based on the Common Terminology Criteria for Adverse Events and ICI-associated pancreatitis. The imaging, clinical, and pathological findings of ICI-associated pancreatitis were also assessed. RESULTS: This study enrolled 843 patients. In multivariable analyses, dual or simultaneous immunotherapy and ≥10 cycles of ICI administration were significant predictive factors for all grades of pancreatic injury, including grade ≥ 3. Notably, patients who received simultaneous immunotherapy exhibited a higher incidence of grade ≥ 3 pancreatic injuries compared to those receiving asynchronous immunotherapy in univariable analysis (p = 0.032). One-fifth of the patients (16/70) with grade ≥ 3 pancreatic injuries had imaging evidence of pancreatitis similar to mild acute pancreatitis. ICI-associated pancreatitis was observed in 5.7% (48/843) of patients, including 1.8% (15/843) with moderate-to-severe pancreatitis (grade ≥ 2). Symptomatic cases (0.36%, 3/843) were treated with steroids with favorable outcomes. Immunohistochemistry for CD4 and CD8 revealed greater infiltration of CD8+ than CD4+ lymphocytes. CONCLUSION: Simultaneous immunotherapy and dual immunotherapy are risk factors for ICI-PI. Although most patients diagnosed with ICI-PI and ICI-associated pancreatitis were asymptomatic and had a low mortality likelihood, long-term outcomes, including endocrine and exocrine function should be carefully monitored.

Dual role of autotaxin as novel biomarker and therapeutic target in pancreatic neuroendocrine neoplasms.

Pancreatic neuroendocrine neoplasms (panNENs) are rare pancreatic neoplasms, and descriptions of treatment remain limited. Autotaxin (ATX) is a secreted autocrine motility factor involved in the production of lysophosphatidic acid (LPA), a lipid mediator that promotes the progression of various cancers. The aim of this study was to clarify the importance of the ATX-LPA axis in panNENs and to confirm its contribution to panNEN progression using clinical data, cell lines, and a mouse model. Serum ATX level was higher in patients with panNEN than in patients with other pancreatic diseases (chronic pancreatitis, pancreatic ductal adenocarcinoma [PDAC], intraductal papillary mucinous neoplasm, autoimmune pancreatitis) and healthy controls, and 61% of clinical specimens stained strongly for ATX. In a case we encountered, serum ATX level fluctuated with disease progression. An in vitro study showed higher ATX mRNA expression in panNEN cell lines than in PDAC cell lines. Cell proliferation and migration in panNEN cell lines were stimulated via the ATX-LPA axis and suppressed by RNA interference or inhibitors. An in vivo study showed that intraperitoneal injection of GLPG1690, an ATX inhibitor, suppressed tumor progression in a xenograft model. These findings revealed that ATX expression is significantly elevated in panNEN and is related to the progression of panNEN. We showed the potential of ATX as a novel biomarker and therapeutic target.

Dual impacts of a glycan shield on the envelope glycoprotein B of HSV-1: evasion from human antibodies in vivo and neurovirulence.

Identification of the mechanisms of viral evasion from human antibodies is crucial both for understanding viral pathogenesis and for designing effective vaccines. Here we show in cell cultures that an N-glycan shield on the herpes simplex virus 1 (HSV-1) envelope glycoprotein B (gB) mediated evasion from neutralization and antibody-dependent cellular cytotoxicity due to pooled γ-globulins derived from human blood. We also demonstrated that the presence of human γ-globulins in mice and immunity to HSV-1 induced by viral infection in mice significantly reduced replication in their eyes of a mutant virus lacking the glycosylation site but had little effect on the replication of its repaired virus. These results suggest that an N-glycan shield on a specific site of HSV-1 envelope gB mediated evasion from human antibodies in vivo and from HSV-1 immunity induced by viral infection in vivo. Notably, we also found that an N-glycan shield on a specific site of HSV-1 gB was significant for HSV-1 neurovirulence and replication in the central nervous system of naïve mice. Thus, we have identified a critical N-glycan shield on HSV-1 gB that has dual impacts, namely evasion from human antibodies in vivo and viral neurovirulence. IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes lifelong latent and recurrent infections in humans. To produce recurrent infections that contribute to transmission of the virus to new human host(s), the virus must be able to evade the antibodies persisting in latently infected individuals. Here, we show that an N-glycan shield on the specific site of the envelope glycoprotein B (gB) of HSV-1 mediates evasion from pooled γ-globulins derived from human blood both in cell cultures and mice. Notably, the N-glycan shield on the specific site of gB was also significant for HSV-1 neurovirulence in naïve mice. Considering the clinical features of HSV-1 infection, these results suggest that the glycan shield not only facilitates recurrent HSV-1 infections in latently infected humans by evading antibodies but is also important for HSV-1 pathogenesis during the initial infection.

STING signalling is terminated through ESCRT-dependent microautophagy of vesicles originating from recycling endosomes.

Stimulator of interferon genes (STING) is essential for the type I interferon response against a variety of DNA pathogens. Upon emergence of cytosolic DNA, STING translocates from the endoplasmic reticulum to the Golgi where STING activates the downstream kinase TBK1, then to lysosome through recycling endosomes (REs) for its degradation. Although the molecular machinery of STING activation is extensively studied and defined, the one underlying STING degradation and inactivation has not yet been fully elucidated. Here we show that STING is degraded by the endosomal sorting complexes required for transport (ESCRT)-driven microautophagy. Airyscan super-resolution microscopy and correlative light/electron microscopy suggest that STING-positive vesicles of an RE origin are directly encapsulated into Lamp1-positive compartments. Screening of mammalian Vps genes, the yeast homologues of which regulate Golgi-to-vacuole transport, shows that ESCRT proteins are essential for the STING encapsulation into Lamp1-positive compartments. Knockdown of Tsg101 and Vps4, components of ESCRT, results in the accumulation of STING vesicles in the cytosol, leading to the sustained type I interferon response. Knockdown of Tsg101 in human primary T cells leads to an increase the expression of interferon-stimulated genes. STING undergoes K63-linked ubiquitination at lysine 288 during its transit through the Golgi/REs, and this ubiquitination is required for STING degradation. Our results reveal a molecular mechanism that prevents hyperactivation of innate immune signalling, which operates at REs.

Theoretical step approach with 'Three-pillar' device assistance for successful endoscopic transpapillary gallbladder drainage.

BACKGROUND: Endoscopic transpapillary gallbladder drainage (ETGBD) has been reported as an alternative procedure for acute cholecystitis but remains a challenging procedure. AIMS: To elucidate the efficacy of a strategic approach for ETGBD that utilizes a four-step classification system and the optional use of 'Three-pillar' assistance with the following devices: cholangioscopy (SpyGlass DS, SG), a flex-type guidewire (Flex-GW), and a 3-Fr microcatheter (3-Fr Micro). METHODS: A total of 115 patients undergoing ETGBD were studied retrospectively. Characteristics and technical outcomes were compared between conventional ETGBD technique (Classical ETGBD, N = 50) and strategic ETGBD with optional Three-pillar assistance (Strategic ETGBD, N = 65). RESULTS: SG-assistance (15/65, 23.1%) was as an excellent troubleshooter in Category 1 (failure to identify the cystic duct [CD] orifice) and Category 2 (failure to advance the GW across the CD takeoff due to unfavorable angle). Flex-GW (19/65, 29.2%) worked for Category 3b (failure of GW access to the GB due to multiple tortuosities). 3-Fr Micro (11/65, 16.9%) was effective for Category 3a (failure of GW access to the GB due to CD obstruction) and Category 4 (failure of drainage stent insertion to the GB). The overall technical success rate was significantly higher for Strategic ETGBD (63/65, 96.9%) compared with Classical ETGBD (36/50, 72.0%) (p = 0.0001). CONCLUSIONS: Strategic ETGBD, which includes the Three-pillar assistance options of SG in the initial steps, Flex-GW for tortuous CD, and 3-Fr Micro for stenotic CD, achieved a significantly higher success rate than for Classical ETGBD.

Establishment of a system to quantify wild-type herpes simplex virus-induced cell-cell fusion reveals a role of N-glycosylation of HSV-1 envelope glycoprotein B in cell-cell fusion.

Wild-type herpes simplex virus (HSV) strains infrequently mediate cell-cell fusion in cell cultures and barely induce large multinucleated cells. In this study, we established a system to quantify infrequent cell-cell fusion induced by wild-type HSV strains. The established system clarified that the HSV-1 envelope glycoprotein B and its N-glycosylation at asparagine at position 141 were required for efficient cell-cell fusion. This study provides a link between cell-cell fusion induced by wild-type HSV-1 and viral pathogenesis in vivo.

Concomitant Pancreatic Ductal Adenocarcinoma and Type 1 Autoimmune Pancreatitis: A Potential Issue in the Diagnosis of Carcinoma by Endoscopic Ultrasound-guided Fine-needle Biopsy.

We herein report a 64-year-old man with concomitant pancreatic ductal adenocarcinoma (PDAC) and type 1 autoimmune pancreatitis (AIP). An endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) from the pancreatic head mass revealed level 2 histology of AIP and atypical glands. We diagnosed definitive focal AIP using the clinical diagnostic criteria. Computed tomography revealed that the pancreatic mass had not been reduced by steroid therapy. Surgery was performed after a histological PDAC diagnosis was made via a transpapillary biliary biopsy. The resected specimen revealed PDAC associated with AIP. It is important to consider the cooccurrence of PDAC and AIP even if the histological diagnosis via an EUS-FNB is AIP.

Clinical impact of bile-derived exosomal microRNAs as novel diagnostic and prognostic biomarkers for biliary tract cancers.

Sampling of bile juice during endoscopic retrograde cholangiopancreatography (ERCP) has potential benefit of being amenable to the identification of novel biomarkers in liquid biopsy. This study reports the results of a global investigation of exosomal microRNAs (miRNAs) in bile to identify potential biomarkers for biliary tract cancers (BTCs). Eighty-eight bile samples collected during ERCP (45 BTC and 43 noncancer control samples) were enrolled in this study. Eleven BTC samples and nine control samples were assigned as the discovery set. Exosomes in bile and serum samples were collected using a glass membrane column with size-controlled macroporous glass (MPG), and exosomal miRNA expression profiles were evaluated using comprehensive miRNA microarray analysis (3D-Gene). For validation, exosomal miRNA in the bile samples of 34 BTCs and 34 controls were comprehensively evaluated using 3D-Gene. In the discovery set, eight exosomal miRNAs in bile were identified as significant aberrant expression markers, while no miRNA with aberrant expression in serum was identified. In a comparison of the discovery and validation sets, miR-451a and miR-3619-3p were identified as reproducible upregulated markers, and the combination of the two bile miRNAs showed an excellent area under the curve (0.819) value for diagnosing BTCs. In addition, high miR-3619-3p expression in bile reflects poorer prognosis of BTCs (hazard ratio = 2.89). The MPG-extracted exosomal miRNAs in bile aspirated during ERCP provide a convenient new approach for diagnosing biliary diseases. Bile-derived miRNA analysis with miR-451a and miR-3619-3p represents a potentially valuable diagnostic strategy for identifying BTCs as well as a predictive indicator of BTC prognosis.

Redundant and Specific Roles of A-Type Lamins and Lamin B Receptor in Herpes Simplex Virus 1 Infection.

We investigated whether A-type lamins (lamin A/C) and lamin B receptor (LBR) are redundant during herpes simplex virus 1 (HSV-1) infection in HeLa cells expressing lamin A/C and LBR. Lamin A/C and LBR double knockout (KO) in HSV-1-infected HeLa cells significantly impaired expressions of HSV-1 early and late genes, maturation of replication compartments, marginalization of host chromatin to the nuclear periphery, enlargement of host cell nuclei, and viral DNA replication. Phenotypes of HSV-1-infected HeLa cells were restored by the ectopic expression of lamin A/C or LBR in lamin A/C and LBR double KO cells. Of note, lamin A/C single KO, but not LBR single KO, promoted the aberrant accumulation of virus particles outside the inner nuclear membrane (INM) and viral replication, as well as decreasing the frequency of virus particles inside the INM without affecting viral gene expression and DNA replication, time-spatial organization of replication compartments and host chromatin, and nuclear enlargement. These results indicated that lamin A/C and LBR had redundant and specific roles during HSV-1 infection. Thus, lamin A/C and LBR redundantly regulated the dynamics of the nuclear architecture, including the time-spatial organization of replication compartments and host chromatin, as well as promoting nuclear enlargement for efficient HSV-1 gene expression and DNA replication. In contrast, lamin A/C inhibited HSV-1 nuclear export through the INM during viral nuclear egress, which is a unique property of lamin A/C. IMPORTANCE This study demonstrated that lamin A/C and LBR had redundant functions associated with HSV-1 gene expression and DNA replication by regulating the dynamics of the nuclear architecture during HSV-1 infection. This is the first report to demonstrate the redundant roles of lamin A/C and LBR as well as the involvement of LBR in the regulation of these viral and cellular features in HSV-1-infected cells. These findings provide evidence for the specific property of lamin A/C to inhibit HSV-1 nuclear egress, which has long been considered but without direct proof.

Urolithin A targets the AKT/WNK1 axis to induce autophagy and exert anti-tumor effects in cholangiocarcinoma.

Urolithin A (UA; 3,8-dihydroxybenzo[c]chromen-6-one), a metabolite generated by intestinal bacteria during the biotransformation of ellagitannins, has gained considerable attention in treating several cancers. Cholangiocarcinoma (CCA) remains one of the most lethal cancers; it grows in a special environment constantly exposed to both blood and bile. Since UA is known to undergo enterohepatic recirculation, we hypothesized that UA might have significant antitumor effects in CCA. Here, we investigated the therapeutic potential of UA in CCA and aimed to elucidate its mechanisms, including autophagy. UA treatment inhibited cell proliferation and induced G2/M phase cell cycle arrest in CCA cells. UA also suppressed cell migration and invasion, but did not cause apoptosis. Furthermore, Western blotting and immunocytochemistry demonstrated increased LC3-II accumulation, while electron microscopy demonstrated induced autophagosomes after UA treatment, suggesting that UA upregulated autophagy in CCA cells. In xenograft mice treated with UA, tumor growth was inhibited with increased LC3-II levels. On the other hand, phospho-kinase array demonstrated downregulation of the AKT/WNK1 pathway. LC3-II expression was elevated in WNK1 knocked down cells, indicating that WNK1 is the key signal for regulating autophagy. Thus, UA exerted antitumor effects by suppressing the AKT/WNK1 signaling pathway and inducing autophagy. In conclusion, UA, a natural, well-tolerated compound, may be a promising therapeutic candidate for advanced CCA.

Use of Endoscopic Scraper and Cell Block Technique as a Replacement for Conventional Brush for Diagnosing Malignant Biliary Strictures.

Histological evidence is essential for diagnosing malignant biliary strictures. However, conventional brush cytology remains the primary method used worldwide, despite its low diagnostic sensitivity and accuracy, as it is technically easy, rapid, and cost-effective. The aim of this study was to evaluate the diagnostic performance of a recently introduced endoscopic scraper, the simplicity of which is comparable to that of a conventional brush, by comparing diagnostic yields and the number of collected cells. The sensitivity of the endoscopic scraper when using the cell block technique was significantly higher than when using brush cytology or a brush with the cell block technique (53.6% vs. 30.9%, p < 0.001; 53.6% vs. 31.6%, p = 0.024, respectively). Quantitative digital image analysis of cell block sections revealed that the median number of cells obtained with the endoscopic scraper was significantly higher than when using the brush (1917 vs. 1014 cells, p = 0.042). Furthermore, seven cases (8.3%; 7/84) were diagnosed by immunohistochemical analysis of the cell block section obtained from the endoscopic scraper. Given its simplicity and greater capacity for sample acquisition, use of the endoscopic scraper in conjunction with the cell block technique could replace brush cytology for the histological diagnosis of malignant biliary strictures.

Class III ß-Tubulin Expression Is of Value in Selecting nab -Paclitaxel and Gemcitabine as First-Line Therapy in Unresectable Pancreatic Cancer.

OBJECTIVES: Nab -paclitaxel and gemcitabine (GnP) or FOLFIRINOX (a combination of leucovorin, fluorouracil, irinotecan, and oxaliplatin [FFX]) is currently recognized as the standard first-line regimen for unresectable pancreatic ductal adenocarcinoma (PDAC). Class III ß-tubulin (TUBB3) has the potential to predict resistance to taxane in various tumors; therefore, this study aimed to clarify whether TUBB3 is a predictive marker for GnP response. METHODS: We retrospectively reviewed 113 patients with PDAC who received GnP or FFX as first-line chemotherapy and examined immunohistochemically the TUBB3 expression in specimens obtained by endoscopic ultrasound-guided fine-needle aspiration. RESULTS: High TUBB3 expression was associated with a significantly lower disease control rate ( P = 0.017) and shorter progression-free survival (PFS) ( P = 0.019), and multivariate analysis revealed that TUBB3 expression was an independent variable for PFS in the GnP first-line group ( P = 0.045). In addition, in the FFX first-line group, TUBB3 expression was not correlated with PFS or overall survival (OS). In all 113 patients, TUBB3 expression was not also associated with OS. CONCLUSIONS: Class III ß-tubulin might be a predictive factor for the response of GnP, but not a prognostic factor for OS, helping the selection of an optimized first-line chemotherapy regimen for unresectable PDAC.