Inhibition of dengue viruses by N-methylcytisine thio derivatives through targeting viral envelope protein and NS2B-NS3 protease.

Dengue virus (DENV) is a significant global health threat, causing millions of cases worldwide each year. Developing antiviral drugs for DENV has been a challenging endeavor. Our previous study identified anti-DENV properties of two (-)-cytisine derivatives contained substitutions within the 2-pyridone core from a pool of 19 (-)-cytisine derivatives. This study aimed to expand on the previous research by investigating the antiviral potential of N-methylcytisine thio (mCy thio) derivatives against DENV, understanding the molecular mechanisms of antiviral activity for the active thio derivatives. The inhibitory assays on DENV-2-induced cytopathic effect and infectivity revealed that mCy thio derivatives 3 ((1R,5S)-3-methyl-1,2,3,4,5,6-hexahydro-8H-1,5-methanopyrido[1,2-a][1,5]diazocine-8-thione) and 6 ((1S,5R)-3-methyl-2-thioxo-1,2,3,4,5,6-hexahydro-8H-1,5-methanopyrido[1,2-a][1,5]diazocin-8-one) were identified as the active compounds against both DENV-1 and DENV-2. Derivative 6 displayed robust antiviral activity against DENV-2, with EC50 values ranging from 0.002 to 0.005 µM in different cell lines. Derivative 3 also exhibited significant antiviral activity against DENV-2. The study found that these compounds are effective at inhibiting DENV-2 at both the entry stage (including virus attachment) and post-entry stages of the viral life cycle. The study also investigated the inhibition of the DENV-2 NS2B-NS3 protease activity by these compounds. Derivative 6 demonstrated notably stronger inhibition compared to mCy thio 3, revealing its dual antiviral action at both the entry and post-entry stages. Molecular docking simulations indicated that mCy thio derivatives 3 and 6 bind to the domain I and III of the DENV E protein, as well as the active of NS2B-NS3 protease, suggesting their molecular interactions with the virus. The study demonstrates the antiviral efficacy of N-methylcytisine thio derivatives against DENV. It provides valuable insights into the potential interactions between these compounds and viral target proteins, which could be useful in the development of antiviral drugs for DENV.

Performance evaluation of the cobas SARS-CoV-2 Duo, a novel qualitative and quantitative assay, for the detection of SARS-CoV-2 RNA.

IMPORTANCE: Quantitative SARS-CoV-2 tests for viral load are necessary to guide patient treatment, as well as to determine infection control measures and policies. Although the real-time RT-PCR assays can report the Ct value to estimate the viral load, there are several serious concerns regarding the use of Ct values. Importantly, Ct values can vary significantly among between- and within-run methods. The diagnostic performance of the cobas SARS-CoV-2 Duo is appropriate. It is a precise, accurate, and sensitive method for the detection of SARS-CoV-2 RNA and is comparable to two qualitative assays (the cobas SARS-CoV-2 and the Liat cobas SARS-CoV-2 and Inf A/B). In contrast, using the Ct value to estimate viral load is not reliable, and utilization of a quantitative detection test, such as the cobas SARS-CoV-2 Duo, to accurately measure the viral load is needed.

Comparison between the analytical sensitivity and clinical performance of two cobas SARS-CoV-2 tests based on high-throughput and point-of-care systems.

Objectives: This study examined analytical sensitivity, specificity, and the clinical performance in detecting SARS-CoV-2 of the Cobas SARS-CoV-2 Test based on the high-throughput Cobas 6800 system and the Cobas SARS-CoV-2 & Flu A/B Test based on the point-of-care cobas Liat system. Methods: The commercial reagents containing SARS-CoV-2 RNA subgenomes were diluted for assessing the sensitivity of the RT-qPCR assay. 385 nasopharyngeal swab specimens taken from contacts of COVID-19 cases were tested for the SARS-CoV-2 detection with both Cobas SARS-CoV-2 Tests. Results: In analytical sensitivity assays, the Cobas SARS-CoV-2 & Flu A/B Test on the Liat system had a lower limit of detection (12.5-25 copies/mL) than the cobas SARS-CoV-2 Test on the cobas 6800 system (25-50 copies/mL). In clinical performance assays, the cobas SARS-CoV-2 Test demonstrated 89.36% (42 out of 47) PPA (positive percent agreement) and 98.82% (334 out of 338) NPA (negative percent agreement) compared to the results of the Cobas SARS-CoV-2 & Flu A/B test. Among five discordant specimens, four had the positive result of the cobas SARS-CoV-2 test, but the negative result of the cobas SARS-CoV-2 & Flu A/B Test. Moreover, these discordant specimens had the Ct values of greater than 33 for the cobas SARS-CoV-2 Test, implying a very small number of virions in the samples. Remarkably, four specimens with a presumptive positive result of the cobas SARS-CoV-2 test had been confirmed by the Cobas SARS-CoV-2 & Flu A/B Test. Next, the scatter plots of the Ct values showed a highly positive correlation between cobas SARS-CoV-2 & Flu A/B Test and the cobas SARS-CoV-2 Test (R-squared value = 0.954-0.962). Conclusions: Both SARS-CoV2 tests of the cobas 6800 and Liat systems produce reliable high throughput and point-of-care assays respectively for the early virus detection and the personal care decision-making during COVID-19 pandemic.

Tremella fuciformis Inhibits Melanogenesis in B16F10 Cells and Promotes Migration of Human Fibroblasts and Keratinocytes.

BACKGROUND/AIM: Natural skin whiteners have been investigated for centuries. The development of preparations that safely achieve whitening of hyper-pigmented skin lesions is a challenge for the cosmetics industry. Furthermore, promoting rapid wound healing and minimizing inflammation in injured skin are key to prevent from abnormal pigmentation in scar tissue. Natural products, including the fungus Tremella fuciformis (TF), are attracting attention as potential sources of lead compounds for these applications. MATERIALS AND METHODS: We investigated the in vitro effects of TF on melanogenesis in murine B16F10 cells. Melanin and tyrosinase levels were measured after treatment with TF. Wound healing in human keratinocytes (HaCaT) and fibroblasts (Detroit 551) was also determined via cell migration assay prior to TF exposure. RESULTS: TF significantly decreased melanin content and tyrosinase expression in a concentration-dependent manner in B16F10 cells. Furthermore, TF promoted wound healing in human HaCaT keratinocytes and Detroit 551 fibroblasts. CONCLUSION: TF proved effectively on inhibiting melanogenesis and promoting wound healing in vitro, demonstrating its potential as a novel skin-whitening agent. However, further clinical studies of safety and efficacy are required.

Comparison of the detection rates of RT-PCR and virus culture using a combination of specimens from multiple sites for enterovirus-associated encephalomyelitis during enterovirus 71 epidemic.

Enterovirus (EV) 71 may cause severe neurological illness in the pediatric population. The present study aimed to compare the detection rates of reverse transcription-polymerase chain reaction (RT-PCR) with pan-EV/EV71 type- specific primers and virus culture (VC) for the identification of EV and EV71 using specimens from multiple sites. In total, specimens from throat/rectal swabs, cerebrospinal fluid (CSF), and blood from 66 patients diagnosed with EV encephalomyelitis were subjected to both RT-PCR and VC for detecting the presence of pan-EV and EV71. The results revealed that the positive RT-PCR rate was higher in throat swabs (60.6%) and rectum swabs (50.0%) than in CSF (16.7%) and blood (15.6%). The same trend was also observed in case of VC: throat swabs (22.7%), rectum swabs (10.6%), and blood (3.0%). The detection rate of EV encephalomyelitis by RT-PCR was 90.9% for all samples, 63.6% of which were subtyped as EV71. The detection rates of RT-PCR were superior to those of VC, and identification using specimens from throat/rectal swabs yielded higher positive results. These findings may help physicians to identify the etiologies at an early stage during EV71 epidemics and to make emergent medical decisions for minimizing patient morbidity and mortality.

RAS, BRAF, and TP53 gene mutations in Taiwanese colorectal cancer patients.

BACKGROUND: Colorectal cancer (CRC) plays an important role in cancer mortality and morbidity. This study examined colorectal tissues for RAS, BRAF, and TP53 gene mutations to assess their value as indicators of outcomes of CRC therapy. MATERIAL AND METHODS: DNA was extracted from tissues taken from 165 patients with CRC. RAS gene mutations (exons 2 and 3) were detected by primer extension analysis. BRAF gene mutations (V600E) were detected by high resolution melting (HRM) analysis. TP53 gene mutations (exons 5-8) were detected by direct sequencing. RESULTS: RAS, BRAF, and TP53 mutations occurred in 36.97% (61/165), 4.24% (7/165), and 37.58% (62/165), respectively. The KRAS mutation is a predictor for poor 5-year survival (p = 0.05), and the co-presence of KRAS and TP53 mutations correlates with lymph node involvement (p = 0.029), tumor stage (p = 0.029), and poor survival (p = 0.021). Multivariate analysis adjusted for tumor size, histologic grade, lymph node metastasis, sex, and age also indicated that KRAS mutations correlate significantly with overall survival (p = 0.036). CONCLUSION: The KRAS mutation is not present in about one-third of CRC patients, and therefore other gene mutations need to be investigated to better understand the molecular mechanisms of CRC and its treatment.

Distribution of thalassemias and associated hemoglobinopathies identified by prenatal diagnosis in Taiwan.

BACKGROUND: Hemoglobin (Hb) gene disorders are common hereditary disorders in Taiwan, and α- and ß-thalassemias are among the well-known Hb disorders here. Our study provides a primary reference for designing a locally relevant antenatal diagnostic test to control the spread of thalassemia. METHODS: Between 1998 and 2011, prenatal diagnoses for identifying thalassemia and hemoglobinopathies were performed on 1240 fetuses at risk for α-hydrops and ß-thalassemia major. RESULTS: Of 1240 specimens analyzed, 1082 (87%) were obtained by amniocentesis; 125 (10%), by chorionic villus sampling; and 33 (3%), by cordocentesis. Prenatal diagnoses revealed that 21.5% of these fetuses as thalassemia major (including α-thalassemia hydrops, ß-thalassemia major, and Hb E/ß-thalassemia); 50.2%, for thalassemia minor (include α-thalassemia carrier, ß-thalassemia carrier, and α-thalassemia combined ß-thalassemia carrier); and 28.3% for normal type (include non-α, ß-thalassemia). The most common α-hydrops were SEA (Southeast Asian) and Philippine type (frequencies of 74.91 and 5.24%, respectively). The frequency of the IVS-II-654 combined codons 41/42 mutation, the most common ß-thalassemia major mutation in this region, was 5.24%. Two fetuses were found with E/ß-thalassemia (HbE/IVS-II-654 and HbE/codons 41/42, respectively). Since 1993, Taiwan's Department of Health adopted a national program for screening pregnancies to control spread of thalassemia. In the last 10years, less than 3 such cases have occurred per year. After 2003, this number was 0 for a total of 4years (2003, 2004, 2007, and 2008). CONCLUSION: In Taiwan, incidence and frequency of thalassemia genotypes were similar to those previously reported. The national program for screening pregnancies to control spread of thalassemia that resulted in a marked decline in the number of newborns with thalassemia major. Interestingly, prenatal diagnoses revealed 21.5% for thalassemia major, 50.2% for thalassemia minor, 28.3% normal comparison of thalassemia type distribution showed normal type increasing by 13.2% and major type decreasing by 14%. This unique and significant finding needs further clinical studies and discussion to explain such a phenomenon.

Molecular typing of Mycobacterium tuberculosis isolated from adult patients with tubercular spondylitis.

BACKGROUND/PURPOSE: Tuberculosis (TB) is endemic in Taiwan and usually affects the lung, spinal TB accounting for 1-3% of all TB infections. The manifestations of spinal TB are different from those of pulmonary TB. The purpose of this study was to define the epidemiological molecular types of mycobacterial strains causing spinal TB. METHODS: We retrospectively reviewed the medical charts of adult patients diagnosed with spinal TB from January 1998 to December 2007. Patients with positive culture results and/or pathological findings characteristic of TB were enrolled in this study. Spoligotyping was performed to type the Mycobacterium tuberculosis isolates. RESULTS: A total of 38 patients with spinal TB were identified. Their mean age was 68 years, and their median duration of symptoms was 60 days (range 3-720 days). The lumbar and thoracic spine accounted for 76% of the sites involved. Thirteen specimens (from seven male and six female patients) were available for typing. Spoligotyping of these 13 specimens revealed three Beijing (23%) and 10 non-Beijing types (77%). The non-Beijing types included two EAI2 Manilla (15%), two H3 (15%), two unclassified (15%), and one each of BOVIS1, U, T2, and orphan type. No significant predominant strain was found in this study, and no drug-resistant Beijing strains were identified. CONCLUSION: TB spondylitis was found to occur in older patients. Spoligotyping results showed that most of the TB spondylitis cases were caused by non-Beijing type Mycobacterium tuberculosis.

Evaluation of breast cancer susceptibility loci on 2q35, 3p24, 17q23 and FGFR2 genes in Taiwanese women with breast cancer.

AIM: Breast cancer is the most common cancer in women. In recent years, mounting evidence has identified the possibility that 2q35, 3p24, 17q23 and fibroblast growth factor receptor 2 (FGFR2) may be genetic susceptibility loci for breast cancer. This study aimed to evaluate the association of four polymorphic genotypes in these loci with breast cancer in Taiwanese women. PATIENTS AND METHODS: Eighty-eight patients with breast cancer and 70 controls without breast cancer were selected. Polymorphic variants of 2q35-rs13387042, 3p24-rs4973768, 17q23-rs650490 and FGFR2-rs2981578 were analyzed to test for their association with breast cancer susceptibility. The 2q35, 17q23 and FGFR2 polymorphisms were detected using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and the 3p24 polymorphism was detected using an amplification-created restriction site method. RESULTS: The distribution of genotypes of 2q35 were significantly different between the breast cancer group and the control group (p=0.035), while the distributions for 3p24, 17q23, and FGFR2 did not produce statistically significant differences (p>0.05). In addition, allele A of 2q35 conferred a higher risk for breast cancer risk than allele G (odds ratio, OR=2.95, 95% confidence interval, CI=1.29-6.71, p=0.008). Furthermore, the genotypic distribution of 2q35 was not significantly different among patients with different tumor stages, or from different specimen type. CONCLUSION: The 2q35 allele A may be a potential biomarker for breast cancer risk, but further confirmation is required to determine its role in breast carcinogenesis. Blood samples can be used for determining the genotypes for 2q35-rs13387042 in patients for risk of breast cancer.

Proteomic approaches to study epigallocatechin gallate-provoked apoptosis of TSGH-8301 human urinary bladder carcinoma cells: roles of AKT and heat shock protein 27-modulated intrinsic apoptotic pathways.

Epigallocatechin-3-gallate (EGCG), a polyphenol constituent present in green tea, has been shown to inhibit the growth of cancer cells in vitro and in vivo. However, studies regarding human bladder carcinoma cells are limited and not well investigated. Hence, our study focused on the evaluation of EGCG-triggered apoptosis in TSGH-8301 human urinary bladder carcinoma cells in vivo and in vitro as well as its related molecular mechanisms. In an in vivo study, EGCG inhibited xenograft tumor size of TSGH-8301 cells in a nude mouse model. Based on an in vitro study, EGCG resulted in morphological changes and increased growth inhibition in a dose- and time-dependent manner in TSGH-8301 cells. Furthermore, sub-G1 populations were shown and caspase-9 and -3 activities were stimulated in EGCG-treated TSGH-8301 cells. Moreover, a caspase-9 inhibitor (Z-LEHD-FMK) and a caspase-3 inhibitor (Z-DEVD-FMK) were able to reduce EGCG-stimulated caspase-9 and -3 activities, respectively. Loss of mitochondrial membrane potential (∆Ψm) resulted in an increase of protein levels of cytochrome c, Apaf-1, caspase-9 and -3 in TSGH-8301 cells following exposure to EGCG. Proteomic analysis revealed that EGCG affected the expression levels of various proteins, including HSP27, porin, tropomyosin 3 isoform 2, prohibitin and keratin 5, 14, 17 in TSGH-8301 cells. EGCG also suppressed AKT kinase activity and protein levels and also altered the expression levels of Bcl-2 family-related proteins such as Bcl-2, Bax, BAD and p-BAD. Based on the above findings, this study suggests that EGCG-provoked apoptotic death in TSGH-8301 cells is mediated through targeting AKT and HSP27 and modulating p-BAD, leading to activation of the intrinsic apoptotic pathway.

Molecular lesion frequency of hemoglobin gene disorders in Taiwan.

Hemoglobin (Hb) gene disorders are common inherited diseases in Taiwan. The α- and ß-thalassemias are among the well-known Hb diseases in this area. We reviewed abnormal hematological data in 3578 cases, identified between 1998 and 2009, as being at-risk for α-thalassemia (α-thal) (n = 1909; 53.3%), ß-thal (n = 743; 20.8%), non-α, ß-thal (n = 872; 24.4%), and α-thal combined with ß-thal (n = 54; 1.5%), and collected fetal blood samples for prenatal testing. The most common types of α(0)- and α(+)-thal were the SEA (Southeast Asian) deletion and the -α(3.7) rightward deletion, with frequencies of 87.79 and 4.85%, respectively. The frequency of the IVS-II-654 (C>T) mutation, the most common ß-thal mutation in this region, was 38.6%. Hb E [ß26(B8)Glu→Lys, GAG>AAG] was found to be the most common Hb variant, and it was concluded that Hb Tak [ß147 (+AC)], Hb G -Taichung (also known as Hb Q-Thailand) [α74(EF3)Asp→His, GAC>CAC (α1)], Hb Owari [α121(H4)Val→Met (GTG>ATG)], and Hb Phnom Penh [α117(GH5)Phe-Ile-α118(H1)Thr (α1)] were very rare. The results of this study provide a primary reference for designing a locally relevant antenatal diagnostic test for controlling the spread of thalassemia.

Molecular epidemiology of the 2005 enterovirus 71 outbreak in central Taiwan.

BACKGROUND: Since 1998, Taiwan has experienced annual outbreaks of enterovirus 71 (EV71) nationwide. The area around Taichung City experienced a particularly large outbreak in 2005, after which EV71 disappeared for 2 y before re-emerging in 2008. Here we present the clinical, genotypic, and epidemiological baseline data for the 2005 Taichung outbreak. METHODS: Throat swab, stool and cerebrospinal fluid samples were collected and stored in viral transport medium. Samples were tested by reverse-transcriptase polymerase chain reaction and viral culture. Epidemiological, laboratory, and clinical data were extracted from medical record reviews. A total of 27 virus isolates were selected for phylogenetic analysis. RESULTS: Confirmed phylogenetic results of the viruses were separated into 5 groups. The 5'-UTR regions served as a focus for investigating genetic relationships among the 27 EV71 isolates, all of which belonged to a distinct clade in the C4 genotype. Most of the strains belonged to 5 observed epidemic groups. CONCLUSION: In conclusion, the 2005 outbreak in central Taiwan was caused by divergent EV71 strains belonging to the C4 genotype.

Atypical presentation of cytomegalovirus endotheliitis: a case report.

PURPOSE: To describe an atypical case of cytomegalovirus (CMV) endotheliitis in a 74-year-old man who presented with chronic corneal edema without keratic precipitates (KPs) and intraocular pressure (IOP) elevation. DESIGN: Case report. METHODS: A complete ophthalmologic examination was performed. Polymerase chain reaction was used to test for herpes simplex virus, varicella zoster virus, and CMV DNA in aqueous humor samples to rule out viral endotheliitis. RESULTS: Severe bullous keratopathy was found in the temporal part of the cornea without KPs or elevated IOP. CMV DNA was detected. Corneal edema subsided with oral valganciclovior. CONCLUSIONS: CMV endotheliitis may present as corneal edema that lacks typical features, such as KPs or elevated IOP.