A human pluripotent stem cell-based model of SARS-CoV-2 infection reveals an ACE2-independent inflammatory activation of vascular endothelial cells through TLR4.

To date, the direct causative mechanism of SARS-CoV-2-induced endotheliitis remains unclear. Here, we report that human ECs barely express surface ACE2, and ECs express less intracellular ACE2 than non-ECs of the lungs. We ectopically expressed ACE2 in hESC-ECs to model SARS-CoV-2 infection. ACE2-deficient ECs are resistant to the infection but are more activated than ACE2-expressing ones. The virus directly induces endothelial activation by increasing monocyte adhesion, NO production, and enhanced phosphorylation of p38 mitogen-associated protein kinase (MAPK), NF-κB, and eNOS in ACE2-expressing and -deficient ECs. ACE2-deficient ECs respond to SARS-CoV-2 through TLR4 as treatment with its antagonist inhibits p38 MAPK/NF-κB/ interleukin-1ß (IL-1ß) activation after viral exposure. Genome-wide, single-cell RNA-seq analyses further confirm activation of the TLR4/MAPK14/RELA/IL-1ß axis in circulating ECs of mild and severe COVID-19 patients. Circulating ECs could serve as biomarkers for indicating patients with endotheliitis. Together, our findings support a direct role for SARS-CoV-2 in mediating endothelial inflammation in an ACE2-dependent or -independent manner.

Broken even-odd symmetry in self-selection of distances between nanoclusters due to the presence or absence of topological solitons.

Depositing particles randomly on a 1D lattice is expected to result in an equal number of particle pairs separated by even or odd lattice units. Unexpectedly, the even-odd symmetry is broken in the self-selection of distances between indium magic-number clusters on a Si(100)-2×1 reconstructed surface. Cluster pairs separated by even units are less abundant because they are linked by silicon atomic chains carrying topological solitons, which induce local strain and create localized electronic states with higher energy. Our findings reveal a unique particle-particle interaction mediated by the presence or absence of topological solitons on alternate lattices.

Experimental Study on Plasmodium knowlesi Normocyte Binding Protein Xa Region II (PkNBPXaII) for Erythrocyte Binding.

Normocyte binding protein Xa (NBPXa) has been implied to play a significant role in parasite invasion of human erythrocytes. Previous phylogenetic studies have reported the existence of three types of NBPXa for Plasmodium knowlesi (PkNBPXa). PkNBPXa region II (PkNBPXaII) of type 1, type 2 and type 3 were expressed on mammalian cell surface and interacted with human and macaque (Macaca fascicularis) erythrocytes. The binding activities of PkNBPXaII towards human and macaque erythrocytes were evaluated using erythrocyte-binding assay (EBA). Three parameters were evaluated to achieve the optimal protein expression of PkNBPXaII and erythrocyte binding activity in EBA: types of mammalian cells, post transfection time and erythrocyte incubation time. COS-7, HEK-293, and CHO-K1 cells showed successful expression of PkNBPXaII, despite the protein expression is weak compared to the positive control. COS-7 was used in EBA. All three types of PkNBPXaII showed rosette formation with macaque erythrocytes but not with human erythrocytes. Future studies to enhance the PkNBPXaII expression on surface of mammalian cells is indeed needed in order to elucidate the specific role of PkNBPXaII in erythrocytes invasion.

Enteral myiasis causing acute dysentery: A case report.

Enteral myiasis or intestinal myiasis is acquired by ingesting food or water contaminated with dipteran fly eggs or larvae. Here, we describe a patient with intestinal myiasis presenting with acute dysentery caused by the larva of Hermetia illucens. The larva was identified morphologically, and its species confirmed through molecular analysis using polymerase chain reaction and sequencing based on mitochondrial cytochrome c oxidase subunit I gene (COI).

High incidence of Plasmodium knowlesi malaria compared to other human malaria species in several hospitals in Malaysia.

Through the regional control programme, Malaysia has been successfully reducing the incidence of Plasmodium falciparum and Plasmodium vivax infections. However, the incidence of zoonotic malaria Plasmodium knowlesi infection is increasing and now has been the major cause of malaria in Malaysia especially Malaysian Borneo. The emergence of knowlesi infection has threatened the malaria elimination programme which the government aims to reduce the overall malaria infections by 2020. Unlike other benign human Plasmodium spp., P. knowlesi can cause fatal infections. The aim of this study was to determine the incidence and distribution of five human malaria parasites including P. knowlesi in Peninsular Malaysia and Malaysian Borneo. A total of 112 blood samples were collected from seven states and district hospitals in Peninsular Malaysia and Malaysian Borneo from year 2015 to 2016. The samples were examined by microscopy and further confirmed by nested PCR assay targeting 18S rRNA gene of Plasmodium spp. Following the nested PCR assays, a total of 54 (48.2%) samples were positive for P. knowlesi infections, 12 (10.7%) cases were positive for P. vivax infections, followed by 7 (6.3%) cases of P. falciparum and 4 (3.5%) cases of P. malariae. There were 3 cases (2.7%) of mixed infections (P. knowlesi/P. vivax). However, no cases were identified as P. ovale. A total of 32 (28.6%) cases were found as negative infections. LoopMediated Isothermal Amplification Assay (LAMP) was performed to confirm inconclusive results produced by microscopy and nested PCR. P. knowlesi showed the highest prevalence in Sarawak (n= 30), Sabah (n=13), Pulau Pinang (n=5) and Pahang (n=6). PCR and LAMP was not able to detect a large number of microscopy positive samples due to DNA degradation during storage and shipping. Among all the states involved in this study, the highest prevalence of P. knowlesi infection was found in Sabah and Sarawak.

Vitamin D receptor gene polymorphisms and distinct clinical phenotypes of hepatitis B carriers in Taiwan.

Vitamin D exhibits immunomodulatory and antiproliferative effects through vitamin D receptor (VDR) in chronic infections and cancers. We genotyped the BsmI (rs1544410), ApaI (rs7975232) and TaqI (rs731236) polymorphisms of VDR gene in 250 Taiwanese chronic hepatitis B virus (HBV) carriers who were categorized into six phenotypes. After adjustment for age and sex, the frequencies of the VDR B/b, B/a, B/T, B/a/T in patients with hepatitis flare(s) were lower than those without (7 vs 20%, P=0.009; 1 vs 9%, P=0.004; 3 vs 10%, P=0.007; 1 vs 9%, P=0.005, respectively); in contrast, T/t, A/T, A/t, b/A/t were higher in flare(s) (8 vs 3%, P=0.003; 49 vs 34%, P=0.027; 2 vs 1%, P=0.004; 0.5 vs 0%, P=0.001, respectively). In addition, B/b, B/B, T/t, b/A, B/a, B/A, B/T, B/t, A/t, b/A/T, B/a/T, B/A/T, B/A/t, b/A/t were higher in patients positive for HBeAg. The distribution of VDR genotypes was comparable between patients with and without hepatocellular carcinoma (HCC). VDR gene polymorphisms are associated with distinct clinical phenotypes in Taiwanese HBV carriers but not with HCC development.

Elimination of contamination in loop-mediated isothermal amplification assay for detection of human malaria.

The LAMP assay, amplifies the target DNA rapidly, with 10-fold greater sensitivity than conventional PCR. The greater sensitivity also comes with greater risks of contamination. To overcome this issue, the current project includes either uracil DNA glycosylase (UDG) or a mineral oil overlay in the LAMP assay. Our results indicated that UDG or a mineral oil overlay can effectively prevent carryover contamination in the LAMP assay for the detection of human malaria. By incorporating these preventative methods, contamination can be eliminated and LAMP can potentially be used in the field; and point of care diagnosis for human malaria.

Pegylated interferon alpha-2a versus standard interferon alpha-2a for treatment-naive dialysis patients with chronic hepatitis C: a randomised study.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is prevalent in dialysis patients, and standard interferon monotherapy is the current standard of care for such patients. AIM: To investigate whether pegylated interferon has a better therapeutic efficacy and safety profile than standard interferon in dialysis patients with chronic hepatitis C. METHODS: 50 such patients were randomly assigned to receive either pegylated interferon alpha-2a 135 microg subcutaneously once per week or standard interferon alpha-2a 3 million units subcutaneously thrice per week for 24 weeks. The primary efficacy and safety end points were sustained virological response (SVR) by intention-to-treat analysis and treatment-related withdrawal rate during the study. RESULTS: In univariate analysis, patients receiving pegylated interferon alpha-2a tended to have a higher sustained virological response (SVR) than those receiving standard interferon alpha-2a (48% vs 20%, p = 0.07). By using multivariate analysis, treatment with pegylated interferon alpha-2a (p = 0.02) and pretreatment HCV RNA level <800 000 IU/ml (p = 0.007) were independently predictive of an SVR. All patients failing to achieve a rapid virological response (RVR) could not achieve an SVR. In addition, patients receiving pegylated interferon alpha-2a had a significantly lower treatment-related withdrawal rate than those receiving standard interferon alpha-2a (0% vs 20%, p = 0.04). CONCLUSIONS: Pegylated interferon alpha-2a once weekly provides more effective and safer therapy than standard interferon alpha-2a thrice weekly for treatment-naive dialysis patients with chronic hepatitis C.

P38-associated pathway involvement in apoptosis induced by photodynamic therapy with Lonicera japonica in human lung squamous carcinoma CH27 cells.

Photodynamic therapy (PDT) is an effective therapy for local malignant tumors. Lonicera japonica was found to have the anti-tumor effect. The aim of this study is to explore the mechanisms of apoptosis induced by PDT in lung CH27 carcinoma cells with alcohol extract from Lonicera japonica as photosensitizer. Our study indicated that Lonicera japonica extracts exhibited significant photocytotoxicity in CH27 cells at a concentration range of 50-150 microg/ml, with 0.4-1.2J/cm2 light dose. PDT with Lonicera japonica extracts-induced cell death is a typical apoptosis that was accompanied by DNA condensation, externalization of phosphatidylserine and formation of apoptotic bodies. PDT with Lonicera japonica extracts was shown to be caspase-3-independent apoptosis via activation of AIF in this study. P38-associated pathway may be involved in apoptosis induced by PDT with Lonicera japonica extracts in CH27 cells. We also have demonstrated that PDT with Lonicera japonica extracts-induced CH27 cells apoptosis was probably related to its ability to change the protein expression and distribution of heat shock protein 27.

Acute exacerbations of chronic type B hepatitis are accompanied by increased T cell responses to hepatitis B core and e antigens. Implications for hepatitis B e antigen seroconversion.

T cell proliferative responses to hepatitis B virus-encoded envelope antigen (S + preS2 + preS1), recombinant core antigen (HBcAg), and natural hepatitis B e antigen (HBeAg) were examined in 22 HBeAg-positive patients with chronic type B hepatitis and 17 healthy hepatitis B surface antigen (HBsAg) carriers. The results showed that HBeAg-positive patients had (a) higher levels of T cell responses to HBcAg/HBeAg than those of healthy HBsAg carriers (P less than 0.001 and P less than 0.01, respectively); (b) a further increase in these T cell responses during acute exacerbations (P less than 0.05 and P less than 0.05, respectively); (c) subsidence in the T cell responses to HBcAg/HBeAg after recovery from acute exacerbations and HBeAg seroconversion, whereas the responses would persist at high levels if the patients did not enter a clinical remission; and (d) low levels of T cell responses to S + preS2 + preS1 either before or after HBeAg seroconversion. The appearance of increasing T cell responses to HBcAg/HBeAg usually occurred in the early phase of acute exacerbations. These findings imply that HBcAg/HBeAg-specific T cells play an important role in the exacerbations of chronic hepatitis B and in HBeAg seroconversion. HBcAg/HBeAg-specific precursor T cell frequencies were serially studied in selected cases by limiting dilution assay. Elevation (two- to fourfold) of HBcAg/HBeAg-specific precursor T cell frequencies contributed to the increase of HBcAg/HBeAg-specific T cell proliferation during acute exacerbations.

Induction of liver alpha-1 acid glycoprotein gene expression involves both positive and negative transcription factors.

Expression of the alpha-1 acid glycoprotein (AGP) gene is liver specific and acute phase responsive. Within the 180-bp region of the AGP promoter, at least five cis elements have been found to interact with trans-acting factors. Four of these elements (A, C, D, and E) interacted with AGP/EBP, a liver-enriched transcription factor, as shown by footprinting analysis and by an anti-AGP/EBP antibody-induced supershift in a gel retardation assay. Modification of these sites by site-directed mutagenesis coupled with transfection analysis indicated that AGP/EBP binding to all of these sites resulted in positive regulation of the promoter. Dose-response data suggest that AGP/EBP binding to these sites results in the cooperative activation of the promoter. In contrast, functional assays showed that element B is a negative regulatory element; this element is recognized by heat-stable DNA-binding factors which are found in many cells and tissues. The regulation of these binding proteins was studied in rat liver treated with lipopolysaccharide (LPS), which induced an acute-phase reaction. We found that LPS treatment resulted in a two- to threefold increase in AGP/EBP activity and a severalfold decrease in the activity of factors that bind to element B in the liver. These results indicate that expression of the AGP gene can be regulated by both positive and negative factors and that the modulation of these factors can account for the LPS induction of the AGP gene.

Purification and characterization of nucleolin and its identification as a transcription repressor.

Expression of the acute-phase response genes, such as that for alpha-1 acid glycoprotein (AGP), involves both positive and negative transcription factors. A positive transcription factor, AGP/EBP, and a negative transcription factor, factor B, have been identified as the two most important factors responsible for the induction of the AGP gene. In this paper we report the purification, characterization, and identification of a B-motif-binding factor from the mouse hepatoma cell line 129p. The purified factor has been identified as nucleolin by amino acid sequence analysis. Biochemical and functional studies further established that nucleolin is a transcription repressor for regulation of AGP and possibly other acute-phase response genes. Thus, in addition to the many known functions of nucleolin, such as rRNA transcription, processing, ribosome biogenesis, and the shuttling of proteins between the cytoplasmic and nuclear compartments, it may also function as a transcriptional repressor.

Inhibition of 12-lipoxygenase during baicalein-induced human lung nonsmall carcinoma H460 cell apoptosis.

Baicalein is known as a 12-lipoxygenase (12-LOX) inhibitor. The 12-LOX is found to be involved in the progression of human cancers and the inhibitor of 12-LOX offers a target for the prevention cancer. We demonstrated the inhibitory effect of baicalein on the gene and protein expression of 12-LOX in H460 human lung nonsmall carcinoma cell line. Treatment of baicalein inhibited the growth of H460 cells in a dose-dependent manner. Following 24h exposure to 50muM baicalein, cell cycle analysis revealed an increase in the cell population in S-phase. During the S-phase arrest, baicalein decreased the protein levels of cdk1 and cyclin B1, which are the regulating proteins of S-phase transition to G2/M-phase, in this study. Furthermore, baicalein induced the most of H460 cell apoptosis after treatment for 48h. H460 cells formed vesicles and apoptotic body, and then floated after treatment with baicalein. Baicalein-induced H460 cell apoptosis was confirmed by DNA condensation and fragmentation. Baicalein-induced apoptosis were also accompanied by decreasing in Bcl-2 and proform of caspase-3 and increasing p53 and Bax protein levels. Pretreatment with a specific caspase-3 inhibitor, Ac-DEVD-CHO, partially reduced baicalein-induced cell death, indicating baicalein induces apoptosis is partially dependent on caspase-3 pathway in H460 cells. These data suggest that baicalein, a 12-LOX inhibitor, inhibits the proliferation of H460 cells via S-phase arrest and induces apoptosis in association with the regulation of molecules in the cell cycle and apoptosis-related proteins.

Successful treatment of hepatitis B virus infection with Lamivudine after heart transplantation.

Patients with hepatitis B virus (HBV) infection have a higher morbidity and mortality after heart transplantation (HT). HBV infection is endemic in Taiwan. We studied the effect of lamivudine treatment of HBV infection after HT. From July 1987 to July 2005, 252 patients underwent HT. All recipients and donors underwent routine screening of hepatitis B surface antigen (HBsAg), hepatitis B e antigen, antibody to hepatitis B surface antigen, antibody to hepatitis B core antigen, antibody to hepatitis B e antigen, and an alanine aminotransferase (ALT) level before HT. When ALT was two times greater than the upper limit of normal or serum bilirubin was higher than 3 mg/dL in HBsAg-positive patients, HBV-DNA were checked by a branched DNA assay or polymerase chain reaction. When HVB-DNA was greater than 100,000 copies/mL, lamivudine (100 mg per day) was prescribed indefinitely. There were 14 patients under lamivudine treatment after HT, among whom, none suffered severe adverse reactions from lamivudine. Four patients died: one due to end-stage cirrhosis while awaiting liver transplantation at 14 months after HT. Two died of sudden death at 54 months and 138 months after HT. Another died of diffuse B cell lymphoma at 62 months after HT. All the survivors have normal ALT and undetectable HBV-DNA after lamivudine treatment. But the YMDD mutant was detected in two patients. With successful treatment of HBV infection in HT, it is not necessary to exclude HBV infection patients from HT.

Splicing mutations of the p53 gene in human hepatocellular carcinoma.

Point mutations in exons of the tumor suppressor p53 gene occur frequently in many human tumors including hepatocellular carcinoma and are extensively studied. However, intronic point mutations are rare and are totally unknown for hepatocellular carcinoma. By reverse transcription and polymerase chain reaction amplification of p53 RNA from hepatocellular carcinoma tissues of 45 Taiwanese patients, we found amplified complementary DNA fragments of abnormal size in 4 (9%) tumor samples. Sequence analysis of these complementary DNA products revealed aberrant retention of intron 7 in one sample, insertion of 49 base pairs of the 3' end of intron 6 in 2 samples, and deletion of exon 4 in the other sample. Direct sequencing of their genomic DNA revealed relevant point mutations at consensus sequence at either the 5' or 3' splice site of intron 7, 6, 6, and 3, respectively. The splicing mutations produced p53 mutants with truncation of COOH-terminus that are identical to those found in lung cancers. Three of the 4 patients with splicing mutations were younger and had huge tumors. The results suggest a possible role of these p53 mutants in the development of human cancers.

Frequent genetic alterations at the distal region of chromosome 1p in human hepatocellular carcinomas.

Cytogenetic analysis of hepatocellular carcinoma (HCC) cell lines and primary HCC tissues has demonstrated chromosome 1p to be the region most commonly affected. To refine the altered locus, genetic abnormalities of this region were surveyed systemically by microsatellite polymorphism analysis. Twelve sets of primers evenly distributed on chromosome 1p which can amplify di- or tetranucleotide repeat length polymorphism by polymerase chain reaction were selected. The results were then supplemented by the conventional restriction fragment length polymorphism study. A comparison of the allele patterns between 30 pairs of HCC and their corresponding nontumor DNAs discovered chromosome 1p aberrations in 15 of 30 tumors (50%). The abnormalities can be classified into three groups. The first aberration was typical loss of heterozygosity that was found in 9 HCCs (30%). The second aberration was a 2-3-fold increase of allelic dosage, which was detected in 6 HCCs (20%). The third aberration was the novel microsatellite polymorphism, which was detected in 3 cases (10%). These abnormalities seemed to cluster at the distal part of chromosome 1p, with a common region mapped to 1p35-36, which is also the region with frequent loss of heterozygosity in neuroblastoma and colorectal and breast cancers. Therefore, loss of putative tumor suppressor gene(s) in this locus may participate in the development of hepatocellular carcinoma and a wide range of human cancers.

Mutation of p53 gene in hepatocellular carcinoma in Taiwan.

To elucidate the role of p53 mutation in hepatocarcinogenesis in Taiwan, a hepatitis B viral infection hyperendemic area, exons 5 to 8 of the p53 gene in the tumor tissue of 61 hepatocellular carcinomas were amplified and sequenced. A total of 20 cases (32.8%) were found to have mutations; 36.6% (15 of 41) for the hepatitis B surface antigen positive group and 25.0% (5 of 20) for the hepatitis B surface antigen negative group. The corresponding normal liver showed no mutation. The mutation is widely distributed throughout exons 5 to 8. Only 4 cases (6.6%), all positive for hepatitis B surface antigen, had a specific hot spot mutation at codon 249 with G to T transversion. Our results show that scattered point mutations in p53 are not uncommon in hepatocellular carcinoma samples from Taiwan and may be important in the development of this cancer. However, the aflatoxin related specific mutation seems much less related to the genesis of hepatocellular carcinoma in Taiwan.

Evaluation of third-generation hepatitis C antibody assay in chronic hepatitis C and chronic non-B, non-C hepatitis.

Second-generation hepatitis C virus (HCV) antibody assays are more sensitive and specific than first-generation assays in the detection of HCV infection; such tests are widely used. However, there are still HCV-infected patients who test seronegative for anti-HCV even by second-generation assays. In this study we evaluated the performance of the new third-generation HCV assay (HCV 3.0) in 230 individuals with different second-generation anti-HCV (HCV 2.0) and HCV RNA patterns. Our results showed the followings: only one healthy adult had a discrepant result in 200 subjects negative (group I) or positive (group II) for HCV 2.0 and HCV RNA; 7 of 13 (54%) HCV 2.0-negative but HCV RNA-positive patients (group III) were HCV 3.0-positive; two of 17 (12%) so-called chronic non-B, non-C hepatitis patients (group IV) were positive for HCV 3.0. We conclude that third-generation anti-HCV assays are more sensitive and specific than second-generation assays in the detection of chronic HCV infection; however false-positive results may be observed among low-risk healthy persons.

Interferon-alpha reduces insulin resistance and beta-cell secretion in responders among patients with chronic hepatitis B and C.

This study aimed at elucidating the effects of interferon (IFN)-alpha on glucose metabolism in patients with chronic hepatitis B and C infections. Twenty-eight biopsy-proven patients with chronic hepatitis B (ten cases) and hepatitis C (18 cases) were given IFN-alpha for a total of 24 weeks. The patients received a 75 g oral glucose tolerance test (OGTT), glucagon stimulation test, tests for type 1 diabetes-related autoantibodies and an insulin suppression test before and after IFN-alpha therapy. Ten of the 28 patients responded to IFN-alpha therapy. Steady-state plasma glucose of the insulin suppression test decreased significantly in responders (13.32+/-1.48 (S.E.M.) vs 11.33+/-1.19 mmol/l, P=0.0501) but not in non-responders (12.29+/-1.24 vs 11.11+/-0.99 mmol/l, P=0.2110) immediately after completion of IFN-alpha treatment. In the oral glucose tolerance test, no significant difference was observed in plasma glucose in either responders (10.17+/-0.23 vs 10.03+/-0.22 mmol/l) or non-responders (10.11+/-0.22 vs 9.97+/-0.21 mmol/l) 3 Months after completion of IFN-alpha treatment. However, significant differences were noted in C-peptide in both responders (2.90+/-0.13 vs 2.20+/-0.09 nmol/l, P=0.0040) and non-responders (2.45+/-0.11 vs 2.22+/-0.08 nmol/l, P=0.0287) before vs after treatment. The changes of C-peptide in an OGTT between responders and non-responders were also significantly different (P=0.0028), with responders reporting a greater reduction in C-peptide. No case developed autoantibodies during the treatment. In patients who were successfully treated with IFN-alpha, insulin sensitivity improved and their plasma glucose stayed at the same level without secreting as much insulin from islet beta-cells.