Characterization of Human Norovirus Nonstructural Protein NS1.2 Involved in the Induction of the Filamentous Endoplasmic Reticulum, Enlarged Lipid Droplets, LC3 Recruitment, and Interaction with NTPase and NS4.

Human noroviruses (HuNVs) are the leading cause of gastroenteritis worldwide. NS1.2 is critical for HuNV pathogenesis, but the function is still unclear. The GII NS1.2 of HuNVs, unlike GI NS1.2, was localized to the endoplasmic reticulum (ER) and lipid droplets (LDs) and is accompanied by a distorted-filamentous ER morphology and aggregated-enlarged LDs. LC3 was recruited to the NS1.2-localized membrane through an autophagy-independent pathway. NS1.2, expressed from a cDNA clone of GII.4 norovirus, formed complexes with NTPase and NS4, which exhibited aggregated vesicle-like structures that were also colocalized with LC3 and LDs. NS1.2 is structurally divided into three domains from the N terminus: an inherently disordered region (IDR), a region that contains a putative hydrolase with the H-box/NC catalytic center (H-box/NC), and a C-terminal 251-330 a.a. region containing membrane-targeting domain. All three functional domains of NS1.2 were required for the induction of the filamentous ER. The IDR was essential for LC3 recruitment by NS1.2. Both the H-Box/NC and membrane-targeting domains are required for the induction of aggregated-enlarged LDs, NS1.2 self-assembly, and interaction with NTPase. The membrane-targeting domain was sufficient to interact with NS4. The study characterized the NS1.2 domain required for membrane targeting and protein-protein interactions, which are crucial for forming a viral replication complex.

Maternal risk factors associated with offspring biliary atresia: population-based study.

BACKGROUND: Biliary atresia (BA) is a progressive, idiopathic, fibro-obliterative disease of the intra and extrahepatic biliary tree. If untreated, it results in severe liver injury and death. The etiology and pathogenesis of BA remain unclear. Few studies have investigated the association between maternal illness/drug use and the occurrence of BA in offspring. METHODS: We used the data from the Birth Certificate Application of Taiwan and linked to National Health Insurance Research Database and Taiwan Maternal and Child Health Database for the years 2004 to 2017 (N = 1,647,231) on 2022/03, and identified BA cases according to diagnosis and procedure code. A total of 285 BA cases were identified. RESULTS: Mothers with type 2 diabetes mellitus and non-dependent drug abuse had higher rates having BA children than non-BA children, with an odds ratio of 2.17 (95% confidence interval [CI] = 1.04-4.53) and OR: 3.02 (95% CI = 1.34-6.78), respectively. CONCLUSION: These results support the notion that BA occurrence is related to maternal reasons. Further studies should be designed to identify additional maternal and pregnancy risk factors and to understand the underlying pathophysiology. IMPACT: 1. The occurrence of offspring biliary atresia may be related to maternal illness/drug use. 2. Maternal drug abuse and type 2 diabetes mellitus pose a high risk for offspring biliary atresia. 3. If maternal etiology is found, biliary atresia might be a preventable disease.

Identification and Characterization of Human Norovirus NTPase Regions Required for Lipid Droplet Localization, Cellular Apoptosis, and Interaction with the Viral P22 Protein.

The human norovirus (HuNV)-encoded nucleoside-triphosphatase (NTPase) is a multifunctional protein critically involved in viral replication and pathogenesis. Previously, we have shown that the viral NTPase is capable of forming vesicle clusters in cells, interacting with other viral proteins such as P22, and promoting cellular apoptosis. Herein, we demonstrate that NTPase-associated vesicle clusters correspond to lipid droplets (LDs) wrapped by the viral protein and show that NTPase-induced apoptosis is mediated through both caspase-8- and caspase-9-dependent pathways. Deletion analysis revealed that the N-terminal 179-amino-acid (aa) region of NTPase encompasses two LD-targeting motifs (designated LTM-1 and LTM-2), two apoptosis-inducing motifs, and multiple regulatory regions. Interestingly, the identified LTM-1 and LTM-2, which are located from aa 1 to 50 and from aa 51 to 90, respectively, overlap with the two apoptosis-inducing motifs. Although there was no positive correlation between the extent of LD localization and the degree of cellular apoptosis for NTPase mutants, we noticed that mutant proteins defective in LD-targeting ability could not induce cellular apoptosis. In addition to LD targeting, the amphipathic LTM-1 and LTM-2 motifs could have the potential to direct fusion proteins to the endoplasmic reticulum (ER). Furthermore, we found that the LTM-1 motif is a P22-interacting motif. However, P22 functionally augmented the proapoptotic activity of the LTM-2 fusion protein but not the LTM-1 fusion protein. Overall, our findings propose that NTPase may participate in multiple cellular processes through binding to LDs or to the ER via its N-terminal amphipathic helix motifs. IMPORTANCE Human noroviruses (HuNVs) are the major agent of global gastroenteritis outbreaks. However, due to the lack of an efficient cell culture system for HuNV propagation, functions of the viral-encoded proteins in host cells are still poorly understood. In the current study, we present that the viral NTPase is a lipid droplet (LD)-associated protein, and we identify two LD-targeting motifs, LTM-1 and LTM-2, in its N-terminal domain. In particular, the identified LTM-1 and LTM-2 motifs, which contain a hydrophobic region and an amphipathic helix, are also capable of delivering the fusion protein to the endoplasmic reticulum (ER), promoting cellular apoptosis, and physically or functionally associating with another viral protein P22. Since LDs and the ER have been linked to several biological functions in cells, our study therefore proposes that the norovirus NTPase may utilize LDs or the ER as replication platforms to benefit viral replication and pathogenesis.

Prediction of Nonalcoholic Fatty Liver Disease by Anthropometric Indices and Bioelectrical Impedance Analysis in Children.

Background: Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in children and is associated with obesity. Objectives: To test whether addition of bioelectrical impedance analysis (BIA) parameters to BMI and anthropometric indices improves the prediction performance of NAFLD than BMI z score (BAZ) alone. Methods: This cross-sectional study recruited 933 children 6-12 years of age for anthropometric measure, BIA, and liver ultrasound. Prediction models of the BAZ, anthropometric, and BIA sets were built in children with obesity using machine learning algorithms. Results: Prevalences of NAFLD were 44.4% (59/133) and 20% (12/60) in boys and girls with obesity, respectively. In both sexes, BAZ set performed worst; adding anthropometric indices into the model improved the model performance, whereas BIA parameters were the best approach for predicting NAFLD. The best result in boys achieved had an accuracy of 75.9% and area under receiver operating characteristic curve of 0.854. In girls, the best result achieved had an F-measure score of 0.615, Matthews correlation coefficient of 0.512, and area under precision-recalled curve of 0.697. Conclusion: BIA is a simple and highly precise tool that yields better NAFLD prediction model than anthropometric indices, and much better performance than BAZ. This study suggests BIA as a potential predictor for pediatric NAFLD.

The application of single-port laparoscopic percutaneous internal ring suture for the management of indirect inguinal hernia in female adults.

As most of the female inguinal hernias are of indirect type, we conducted this retrospective study to evaluate whether the single port laparoscopic percutaneous internal ring suture is feasible for the management of indirect inguinal hernia in female adults. From April 2016 to August 2019, there were 31 female adults who were diagnosed with inguinal hernias and received laparoscopic inspection at our surgical department. One patient who was finally diagnosed as an encysted hydrocele was excluded from the statistic study. All the 30 cases were of indirect type with a total of 35 single port laparoscopic percutaneous internal ring sutures performed. The median age was 38 years (range 20-88 years). The number and percentage of patients with right, left and bilateral hernias were 17 (56%), 11 (37%) and 2 (7%) respectively. Three contralateral patent processi vaginalium and 1 occult femoral hernia were found during operation. The percentages of the respective classifications according to the European Hernia Society system for the 35 PIRSs were L1: 40%, L2: 49%, and L3: 11%. The average operation time was 18 min for unilateral and 30 min for bilateral hernias. There were 1 recurrence and 1 chronic postoperative inguinal pain. Both had their symptoms and signs resolved after reoperation. The mean follow-up period was 13.6 months. We concluded that the single-port laparoscopic percutaneous internal ring suture is feasible for the management of indirect inguinal hernia in female adults.

Expression and regulation of the BKRF2, BKRF3 and BKRF4 genes of Epstein-Barr virus.

The BKRF2, BKRF3 and BKRF4 genes of Epstein-Barr virus (EBV) are located close together in the viral genome, which encode glycoprotein L, uracil-DNA glycosylase and a tegument protein, respectively. Here, we demonstrate that the BKRF2 gene behaves as a true-late lytic gene, whereas the BKRF3 and BKRF4 genes belong to the early lytic gene family. Our results further reveal that both BKRF3 and BKRF4 promoters are new synergistic targets of Zta and Rta, two EBV latent-to-lytic switch transactivators. Multiple Rta- and Zta-responsive elements within the BKRF3 and BKRF4 promoters were identified and characterized experimentally. Importantly, we show that DNA methylation is absolutely required for activation of the BKRF4 promoter by Zta alone or in combination with Rta. Moreover, we find that sodium butyrate, an inducing agent of EBV reactivation, is capable of activating the BKRF4 promoter through a mechanism independent of Zta and Rta. Overall, our studies highlight the complexity of transcriptional regulation of lytic genes within the BKRF2-BKRF3-BKRF4 gene locus.

Subcellular Localization and Functional Characterization of GII.4 Norovirus-Encoded NTPase.

The genotype II.4 (GII.4) variants of human noroviruses (HuNVs) are recognized as the major agent of global gastroenteritis outbreaks. Due to the lack of an efficient cell culture system for HuNV propagation, the exact roles of HuNV-encoded nonstructural proteins (including Nterm, NTPase, P22, VPg, Pro, and RdRp) in viral replication or pathogenesis have not yet been fully understood. Here, we report the molecular characterization of the GII.4 HuNV-encoded NTPase (designated GII-NTPase). Results from our studies showed that GII-NTPase forms vesicular or nonvesicular textures in the cell cytoplasm, and the nonvesicular fraction of GII-NTPase significantly localizes to the endoplasmic reticulum (ER) or mitochondria. Deletion analysis revealed that the N-terminal 179-amino-acid (aa) region of GII-NTPase is required for vesicle formation and for ER colocalization, whereas the C-terminal region is involved in mitochondrial colocalization. In particular, two mitochondrion-targeting domains were identified in the C-terminal region of GII-NTPase which perfectly colocalized with mitochondria when the N-terminal region of GII-NTPase was deleted. However, the corresponding C-terminal portions of NTPase derived from the GI HuNV did not show mitochondrial colocalization. We also found that GII-NTPase physically interacts with itself as well as with Nterm and P22, but not VPg, Pro, and RdRp, in cells. The Nterm- and P22-interacting region was mapped to the N-terminal 179-aa region of GII-NTPase, whereas the self-assembly of GII-NTPase could be achieved via a head-to-head, tail-to-tail, or head-to-tail configuration. More importantly, we demonstrate that GII-NTPase possesses a proapoptotic activity, which can be further enhanced by coexpression with Nterm or P22.IMPORTANCE Despite the importance of human norovirus GII.4 variants in global gastroenteritis outbreaks, the basic biological functions of the viral nonstructural proteins in cells remain rarely investigated. In this report, we focus our studies on characteristics of the GII.4 norovirus-encoded NTPase (GII-NTPase). We unexpectedly find that GII-NTPase can perfectly colocalize with mitochondria after its N-terminal region is deleted. However, such a phenomenon is not observed for NTPase encoded by a GI strain. We further reveal that the N-terminal 179-aa region of GII-NTPase is sufficient to mediate (i) vesicle formation, (ii) ER colocalization, (iii) the interaction with two other nonstructural proteins, including Nterm and P22, (iv) the formation of homodimers or homo-oligomers, and (v) the induction of cell apoptosis. Taken together, our findings emphasize that the virus-encoded NTPase must have multiple activities during viral replication or pathogenesis; however, these activities may vary somewhat among different genogroups.

A computed tomography phantom study of foam earplugs: Uncommon but potentially hazardous foreign body ingestion in children.

Ingestion of a foreign body is common among children. However, ingestion of foam earplugs (FEPs) has not been reported previously. A 7-month-old female infant presented with small bowel obstruction, which was finally proved to be a case of FEP ingestion.Computed tomography (CT) phantom study was performed to examine the imaging features of FEPs. We studied the following dry and fully wet FEPs, FEPs squeezed in pure water to varying degrees, and FEPs with different degrees of compression in the dry and wet states from day 0 to 6 and all scanned with a CT scanner.The density of a dry FEP is -843.5 ±â€Š4.5 Hounsfield units (HU) and it increases to 0.76 ±â€Š9.3 HU when fully wet. The densities of FEPs ranged from -844.2 to 1.0 HU with different water/air ratios, and some showed a heterogeneous geographic pattern. The densities of FEPs increase due to compression and gradual water absorption.FEPs can be potentially hazardous objects to children. Owing to the special foam structure of the FEP, it can mimic a fatty lesion if the density ranges from -100 to -50 HU; moreover, it can hide in the water if fully wet. However, it should not be mistaken as air, as the density of a dry FEP is -843.5 HU, and the contour can be observed if the window level is set appropriately. Because of its soft texture, the surgeon should be careful not to miss an FEP during the operation. Moreover, radiologists should be familiar with the CT features of FEPs so that they can be identified before surgery.

Identification and characterization of two novel spliced genes located in the orf47-orf46-orf45 gene locus of Kaposi's sarcoma-associated herpesvirus.

UNLABELLED: The orf47-orf46-orf45 gene cluster of Kaposi's sarcoma-associated herpesvirus (KSHV) is known to serially encode glycoprotein L (gL), uracil DNA glycosylase, and a viral tegument protein. Here, we identify two novel mRNA variants, orf47/45-A and orf47/45-B, alternatively spliced from a tricistronic orf47-orf46-orf45 mRNA that is expressed in the orf47-orf46-orf45 gene locus during the early stages of viral reactivation. The spliced gene products, ORF47/45-A and ORF47/45-B, consist of only a partial region of gL (ORF47), a unique 7-amino-acid motif, and the complete tegument protein ORF45. Like the ORF45 protein, ORF47/45-A and ORF47/45-B expressed in cells sufficiently activate the phosphorylation of p90 ribosomal S6 kinase (RSK) and extracellular signal-regulated protein kinase (ERK). However, unlike ORF45, both ORF47/45-A and ORF47/45-B contain a signal peptide sequence and are localized at the endoplasmic reticulum (ER). Additionally, we found that ORF47/45-A and ORF47/45-B have an extra function that mediates the upregulation of GRP78, a master regulator of ER homeostasis. The important event regarding GRP78 upregulation can be observed in all tested KSHV-positive cell lines after viral reactivation, and knockdown of GRP78 in cells significantly impairs viral lytic cycle progression, especially at late lytic stages. Compared with some other viral glycoproteins synthesized through the ER, our results strongly implicate that the ORF47/45 proteins may serve as key effectors for controlling GRP78 expression and ER homeostasis in cells. Taken together, our findings provide evidence showing the reciprocal association between the modulation of ER homeostasis and the progression of the KSHV lytic cycle. IMPORTANCE: Emerging evidence has shown that several viruses appear to use different strategies to control ER homeostasis for supporting their productive infections. The two parts of this study identify two aspects of the association between the regulation of ER homeostasis and the progression of the KSHV lytic cycle. The first part characterizes the function of two early lytic cycle proteins, ORF47/45-A and ORF47/45-B, on the activation of a major ER chaperone protein, GRP78. In addition to the ability to promote GRP78 upregulation, the ORF47/45 proteins also activate the phosphorylation of RSK and ERK. The second part reveals that upregulation of GRP78 is essential for the progression of the KSHV lytic cycle, especially at late stages. We therefore propose that activation of GRP78 expression by viral proteins at the early lytic stage may aid with the protection of host cells from severe ER stress and may directly involve the assembly or release of virions.

ORF50-dependent and ORF50-independent activation of the ORF45 gene of Kaposi's sarcoma-associated herpesvirus.

The ORF45 gene of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a multifunctional tegument protein. Here, we characterize the transcriptional control of the ORF45 gene and show that its promoter can be activated by ORF50 protein, a latent-lytic switch transactivator. The ORF45 promoter can also be induced by sodium butyrate (SB), a histone deacetylase inhibitor, in the absence of ORF50 protein. Although SB induces the ORF45 gene independently of ORF50, its full activation may require the presence of ORF50. Deletion and point mutation analyses revealed that two RBP-Jκ-binding sites in the ORF45 promoter confer the ORF50 responsiveness, whereas NF-Y and Sp1-binding sites mediate the response to SB. Direct binding of NF-Y, Sp1, or RBP-Jκ protein to the ORF45 promoter is required for the promoter activation induced by SB or by ORF50. In conclusion, our study demonstrates both ORF50-dependent and ORF50-independent transcriptional mechanisms operated on the activation of the ORF45 gene.

Positive and negative regulation in the promoter of the ORF46 gene of Kaposi's sarcoma-associated herpesvirus.

The ORF46 gene of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes uracil DNA glycosylase, an enzyme involved in DNA repair. In this study, we show that the transcriptional start site of the ORF46 gene is located at nucleotide 69,425 of the viral genome and ORF50 protein, a latent-lytic switch transactivator, activates the ORF46 promoter via RBP-Jκ protein. Three consensus RBP-Jκ-binding sites found in the ORF46 promoter are critical for the binding of RBP-Jκ protein and conferring the ORF50 responsiveness. In addition, a negative regulatory region has been determined in the ORF46 promoter, which mediates the suppression of the ORF50 responsiveness. The functional negative region of the ORF46 promoter is mainly composed of the Sp1-binding sites. Like the negative region of the ORF46 promoter, addition of Sp1-binding sequences alone in an ORF50-responsive promoter efficiently confers the suppression of the ORF50 responsiveness. Furthermore, sodium butyrate, a pleiotropic inducing agent for the KSHV lytic cycle, is able to relieve the negative regulation of the ORF46 promoter in the latently KSHV-infected cells. The identification of multiple positive and negative cis-acting regulatory elements in the viral promoters emphasizes the elaborate controls in the KSHV lytic cycle, which ensure the adequate expression of each viral lytic gene.

Fibrolamellar hepatocellular carcinoma- report of a case.

Fibrolamellar hepatocellular carcinoma (FLH) is a variant of hepatocellular carcinoma (HCC) with distinct clinical, histologic and prognostic features different from conventional HCC. Herein, we present a 14-year-old girl with a palpable mass over the right upper -quadrant of the abdomen. A well-defined mass in the left liver with heterogeneous density and a central linear band was demonstrated by ultrasound and computed tomogram. FLH was proved by the histopathology study of a liver specimen taken from an echo-guided liver core needle biopsy before surgery and from a left hepatectomy. No tumor recurrence was detected by ultrasound in the 2-year follow-up.

Gastric outlet obstruction in pediatric patients.

BACKGROUND: This study reports the etiologies, management and outcome of children with gastric outlet obstruction (GOO) in a children's hospital. METHODS: The medical records of 11 children with GOO not associated with idiopathic hypertrophic pyloric stenosis (IHPS) were reviewed. They were categorized into one group of anatomic abnormality (AA group) and one group of peptic ulcer disease (PD group). One case underwent episodes of GOO caused by anatomic abnormality and peptic ulcer disease, respectively. RESULTS: Six cases belonged to the AA group. Mean age was 58 months with a male to female ratio of two to four. Underlying etiologies were prepyloric mass (2), web (2) and gastric volvulus (2). Four patients underwent surgery. One patient was lost to follow-up. GOO did not recur in the follow-up period (mean duration 24 months) in the remaining cases. One case in the AA group and the remaining five patients composed the PD Group. Mean age was 49 months and all were male. Underlying causes were gastric ulcers (4) and chronic duodenal ulcers (2). Two of the five patients had Helicobacter pylori infection found by rapid urease test. Four patients recovered after medical management and another two, with normal serum gastrin levels, underwent surgery because of poor response to medical treatment. One case was lost to follow-up. No recurrence of GOO was noted in the follow-up period (mean duration 27 months) in the remaining cases. CONCLUSIONS: In our study, peptic ulcer disease was as important as anatomic abnormalities as the etiology for GOO not associated with IHPS, and medical management could release GOO caused by it. Compared to adult patients, H. pylori infection played a less important etiologic role in pediatric patients with GOO.

Congenital short bowel syndrome with malrotation.

Congenital short bowel syndrome (SBS) associated with malrotation and malabsorption is a very rare condition. We report on an infant girl with congenital SBS associated with malrotation and malabsorption. No polyhydraminos was noted during the regular prenatal examination. Protracted postnatal postprandial vomiting with progressive failure to thrive was noted. A laparotomy showed the small bowel was only about 20 cm in length. She eventually survived with short-term parenteral nutrition and use of oral L-glutamine supplementation. To our knowledge, this might be the shortest length of bowel loop ever reported. Currently, she is 15 months of age with a body weight of about 7 kg and good development.

Shigella flexneri sepsis in an infant.

Shigellosis continues to be an important public health problem in developed countries, since communication in the world village has become more frequent. In addition, this disease is difficult to be prevented because only a small number of bacteria are required to cause infection, and it has exhibited steady trends towards multiple drug resistance. This report describes a 7-month-old female infant with Shigella flexneri sepsis presenting initially with a high fever, watery diarrhea, and dehydration. She was successfully treated with ceftriaxone for 7 days. This case illustrates that Shigella should be included in the differential diagnosis of sepsis associated with diarrhea particularly in young infants traveling to or living in an endemic area. The choice of antimicrobial therapy and the optimal duration for treatment should be carefully evaluated because of the emergence of multidrug-resistant Shigella.

The presentation of asymptomatic palpable movable mass in female inguinal hernia.

UNLABELLED: The surgical approach to the inguinal canal in girls is identical to that in boys. The sliding hernia which contains the ovary with or without the fallopian tube occurs occasionally in female patients. In our clinical experience, we found that a hydrocele in the labium, also presenting with an asymptomatic palpable movable mass, mimics a sliding hernia with ovary. In an attempt to differentiate between hydrocele and sliding inguinal hernia with ovary in female patients, we report our experience dealing with the two situations at a single institution within a 5-year period. Between July 1997 and June 2002, 1800 infants and children underwent surgery for inguinal hernia at Chang Gung Children's Hospital, of whom 580 were female infants and girls aged 1 month to 14 years (mean, 5.7 years). Some 32 patients (5.3%) presented with an asymptomatic palpable movable mass over the labium major. Pre-operative sonography was performed for all cases. Twenty-six female infants aged 1 month to 18 months (mean 5 months) had sliding hernia; both the ovary and fallopian tube were contained. Six girls aged 2 years to 6 years (mean 4.6 years), had hydrocele of the canal of Nuck. The accuracy of pre-operative diagnosis with sonography was 100%. CONCLUSION: Sonography is an easy and accurate pre-operative diagnostic procedure. We suggest that sonography be performed routinely in all female cases with an inguinal hernia containing a palpable movable mass.

Idiopathic hypertrophic pyloric stenosis in identical twins.

Idiopathic hypertrophic pyloric stenosis (IHPS) was thought to be a congenital disease traditionally, even though several published reports assumed IHPS was an acquired disease. The pathogenesis and inheritance patterns of IHPS are not fully understood. Except for the familial recurrence of IHPS, concordance of IHPS in monozygotic or dizygotic twins was also noted, but occurrence in female twins is rare. From July 1992 through June 2000, 130 patients were diagnosed with IHPS in our hospital including one pair of female twins. We present the finding in the twins and review the associated articles about the pathogenesis and inheritance patterns of IHPS.