Investigation of New Psychoactive Substances (NPS), Other Illicit Drugs, and Drug-Related Compounds in a Taiwanese Wastewater Sample Using High-Resolution Mass-Spectrometry-Based Targeted and Suspect Screening.

The proliferation of new psychoactive substances (NPSs) in recent years has posed a significant challenge to public health. Traditional monitoring methods have proven insufficient in tracking these constantly evolving substances, leading to the development of alternative approaches such as wastewater-based epidemiology (WBE). The present study aims to utilize high-resolution mass spectrometry (HRMS)-based targeted and suspect screening to profile NPS, other illicit drugs, and drug-related compounds in a Taiwanese wastewater sample. For the targeted analysis, 8 out 18 standards of illicit drugs have been identified. The suspect screening approach based on approximately 3600 substances in the SWGDRUG library can further identify 92 compounds, including opiate analgesics, synthetic cathinones, phenylalkylamines derivatives, phenethylamine derivatives, tryptamine derivatives, steroids, and ephedrine-related compounds. Additionally, the presence of 5-methoxy-2-aminoindane (MEAI) in the wastewater indicates that drug dealers have recently sold this potential NPS to evade drug regulations. This study firstly reports the HRMS-based comprehensive profile of NPS, other illicit drugs, and drug-related compounds in Taiwan, which could be applied as biomarkers for estimating the consumption of drugs.

Down-regulation of Notch-1 expression decreases PU.1-mediated myeloid differentiation signaling in acute myeloid leukemia.

The Notch receptor-mediated signaling pathways control cell fate in many types of organisms including neurogenesis, myogenesis and hematopoiesis in mammalian species. During normal hematopoiesis, Notch-1 promotes myeloid differentiation through up-regulation of the transcriptional factor PU.1. We therefore speculated that down-regulation of Notch-1 expression might be involved in the leukemogenesis of acute myeloid leukemia (AML). Here we investigated Notch-1 expression and its association with PU.1-mediated differentiation signaling in AML. The transcriptional level of Notch-1 and PU.1 was evaluated in 6 AML cell lines and 54 AML patient samples using real-time PCR analysis, and Western blot analysis of Notch-1, PU.1 and one of its downstream targets, the M-CSF receptor (MCSFR), was performed to test for confirmation. A significant decrease in the transcription levels of Notch-1 was noted in AML cell lines and patient samples, and decreased Notch-1 protein expression in AML was confirmed by Western blotting. Down-regulation of Notch-1 expression was associated with a decrease in PU.1/MCSFR expression in AML. Co-immunoprecipitation experiments showed that partial disruption of the Notch-1/PU.1 complex was noted in AML cells. No detectable mutation of Notch-1 (ANK, PEST) and PU.1 (PEST, DBD) was noted by PCR-single-strand conformation polymorphism (SSCP) assay. These results suggest that down-regulation of Notch-1 expression decreases PU.1/MCSFR expression and disrupts the Notch-1/PU.1 complex, which may impede the PU.1-mediated myeloid signaling and contribute to the leukemogenesis of AML.

Identification of critical residues in nervous necrosis virus B2 for dsRNA-binding and RNAi-inhibiting activity through by bioinformatic analysis and mutagenesis.

It is known that the non-structural B2 protein of nervous necrosis virus (NNV) plays an important role in viral replication and can inhibit the RNA interference system of the host cell. Moreover, the mechanism of NNV B2 protein to inhibit RNAi is by sequestration and protection of double strand (ds) RNA. In the flock house virus (FHV), a model alphanodavirus, the structural and mutational analysis of B2 identified that the positively charged Arg54 of the alpha2 helix mediated the dsRNA-binding activity. According to the betanodavirus B2 protein alignment and modeling results, the amino acid sequences and the predicted structure of betanodavirus B2 are different from alphanodaviruses. It was suggested that the four Arg residues of alpha3 helix between amino residues 52-60 of B2 may be involved in dsRNA-binding activity. Thus, this study replaced these four Arg residues with Gln at position 52 (R52Q), 53 (R53Q), 59 (R59Q), and 60 (R60Q) by site-directed mutagenesis method. The dsRNA-binding assays of these B2 mutants demonstrated that mB2(R53Q) and mB2(R60Q) mutants are dsRNA-binding defective. Moreover, we have found mB2(R53Q) and mB2(R60Q) could not antagonize RNAi by using HeLa cell as an RNAi inhibition model. These results suggested that Arg53 and Arg60 of betanodavirus B2 protein may be similar to Arg54 of alphanodavirus FHV B2 protein and are critical for dsRNA binding and RNAi-inhibiting. This study may serve as an example where bioinformatic analysis of related viral genomes may lead to meaningful structural and functional clues for certain viral proteins.

High prevalence of SV40 infection in patients with nodal non-Hodgkin's lymphoma but not acute leukemia independent of contaminated polio vaccines in Taiwan.

Recent studies have linked simian virus 40 (SV40) to non-Hodgkin's lymphoma (NHL), especially in countries in which people were exposed to contaminated polio vaccines prior to 1963. In Taiwan, nearly all children were not exposed to contaminated polio vaccine during this period; the relationship between SV40 infection and hematological malignancies is unclear and deserves to be studied. Using PCR amplification of SV40 large T antigen DNA, confirmed by Southern blot hybridization and sequence analysis, 91 frozen lymph nodes from NHL patients were examined. Thirteen (14.3 percent) showed positive for SV40. All other test samples, including diagnostic bone marrow from patients with acute leukemia, peripheral blood from 10 relatives of SV40 positive-patients and 91 age-matched normal volunteers, and 5 reactive hyperplastic lymphoid tissues, showed negative. These results may reflect that human-to-human transmission of SV40 is independent of contaminated polio vaccines; and SV40 is possibly associated with the development of NHL in Taiwan (p = 0.0001). Prospective studies are needed to determine the prevalence of SV40 infections in our and other human populations and to explore the means of transmission of the virus.

Extended lamivudine therapy against hepatitis B virus infection in hematopoietic stem cell transplant recipients.

Lamivudine has demonstrated efficacy in the treatment and prevention of hepatitis B virus (HBV) reactivation after hematopoietic stem cell transplantation (HSCT). However, most of these studies involved short durations of prophylaxis, so there is significant concern regarding lamivudine resistance in these patients. Between March 1984 and November 2002, 71 HBV surface antigen-positive HSCT recipients, including a subgroup of 16 who received pretransplantation lamivudine therapy, which was continued into the posttransplantation period to prevent reactivation hepatitis, were enrolled onto our study. The efficacy of lamivudine therapy was first evaluated for the subgroup of 16 patients in terms of treatment response, lamivudine resistance, and viral recurrence after discontinuation by using virologic assays. Efficacy was then evaluated for all patients in terms of the hazards of lamivudine therapy for reactivation hepatitis after transplantation. During a median lamivudine therapy period of 73 weeks (range, 19-153 weeks), the initial response showed a median reduction of 2.54 log10 in serum HBV DNA (-0.28 to 6.72 range). Lamivudine-resistant mutations were detected in 10 (63%) of 16 patients during therapy, and 1 (12%) of 16 patients finally developed a viral breakthrough. At a median follow-up of 30 months after discontinuation, 3 (27%) of 11 cases had recurrence of HBV infection. Despite the emergence of the mutations, no deaths were due to HBV reactivation or severe cases of hepatitis. In the Cox proportion regression model regarding reactivation hepatitis after transplantation of all enrolled patients, lamivudine therapy was found to be the only favorable factor for the event, with a hazard ratio of 0.122 (95% confidence interval, 0.016-0.908; P = .040). In conclusion, extended lamivudine therapy is safe and effective for the prevention of HBV reactivation in an HSCT setting and significantly decreases reactivation hepatitis after transplantation.

Decreased FHIT protein expression correlates with a worse prognosis in patients with diffuse large B-cell lymphoma.

This study was conducted to evaluate the expression of the presumptive tumor suppressor gene, FHIT (fragile histidine triad) and its clinical significance in non-Hodgkin's lymphoma (NHL). Nine lymphoma cell lines, 62 NHL samples including 31 diffuse large B-cell lymphomas (DLBCLs), and 10 benign hyperplastic lymph nodes were analyzed for FHIT transcription using RT-PCR, with DNA sequencing performed where aberrant transcripts were found. Immunohistochemistry (IHC) studies were conducted for evaluation of FHIT protein expression with clinical data collected for assessment of the clinical relevance of the FHIT expression in DLBCLs. Eight of nine (88.9%) cell lines showed abnormal FHIT mRNA transcription, including 4 with co-existence of normal and aberrant transcripts, and 4 with absence of mRNA expression. Abnormal FHIT mRNA expression was noted in 10 (32.3%) DLBCL patients, including 3 with loss of FHIT mRNA expression and 7 with aberrant transcripts. In all NHLs, abnormal FHIT transcripts were determined for 17 of 62 patients (27.4%). Regarding the FHIT protein expression in DLBCLs, 41.9% showed strong FHIT IHC expression (2+), where 58.1% patients revealed reduced (1+) or absence (0) of FHIT protein. Decreased FHIT protein expression was associated with a worse survival after univariate and multivariate analysis. Our result indicates that abnormal FHIT mRNA expression is noted frequently in NHL cell lines and a certain proportion of NHL patients, and decreased FHIT protein expression indicates a significantly worse prognosis in DLBCLs.