A method to synthesize specific active Cu sites of single-atom catalysts with high stability for acetylene hydration.

Single-atom catalysts (SACs) have received much attention in the realm of energy and catalytic conversion due to their maximum atomic efficiency. Herein, we report a cascade anchoring strategy for the preparation of a Cu-S1O2 species of single-atom catalyst attached to a carbon carrier containing P and S (Cu-S1O2 SA/CPS) with a content of 12.4 wt%. Over the Cu-S1O2 SA/CPS catalyst, the conversion of 95.8% and selectivity of 87.2% for acetylene hydration could still be achieved at 70 h (T = 200°C, GHSV(C2H2) = 90 h-1 and VH2O/VC2H2 = 4). X-ray absorption spectroscopy (XAS) and X-ray photoelectron spectroscopy (XPS) tests reveal that the Cu atoms of Cu-S1O2 SA/CPS are predominantly coordinated in a trinary manner (Cu-S1O2). Based on high-resolution aberration-corrected high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM), it is demonstrated that the Cu single-atom sites are highly dispersed in Cu-S1O2 SA/CPS. It is evident from the scanning electron microscopy (SEM) that Cu-S1O2 SA/CPS has a two-dimensional layered structure. The specific structure of the active site Cu is primarily attributed to the coordination of S and O atoms, resulting in its superior stability for acetylene hydration towards the synthesis of acetaldehyde. Density functional theory (DFT) calculations confirm that the formation of the Cu-S1O2 site facilitates the activation of acetylene, which is a pivotal step in the acetylene hydration process and considered as the rate-determining step. This article not only introduces an innovative strategy in the synthesis of Cu SACs but also represents a significant breakthrough in the stability of Cu SACs in acetylene hydration.

A simple, feasible, efficient and safe treatment strategy of sofosbuvir/velpatasvir for chronic HCV/HIV-1 coinfected patients regardless of HCV genotypes: a multicenter, open-label study in China.

Background: The direct-acting antiviral agents (DAAs) have revolutionized the treatment of Hepatitis C Virus (HCV) infection. However, a simple and feasible treatment strategy with high efficacy and safety for HCV in patients coinfected with Human Immunodeficiency Virus (HIV) remains an unmet medical need, especially in areas with limited health resource. This study aims to assess the efficacy and safety of 12 weeks of treatment with sofosbuvir and velpatasvir in patients with chronic HCV/HIV-1 coinfection. Methods: We conducted a multicenter, single-arm, open-label study in China, which involved chronic HCV/HIV-1 coinfected patients who are receiving an antiretroviral regimen of a combination tablet consisting of elvitegravir, cobicistat, emtricitabine, tenofovir alafenamide, (EVG/c/FTC/TAF) once daily. Patients with liver cirrhosis or experienced to DAAs treatment were excluded. All patients received combined sofosbuvir (400 mg) and velpatasvir (100 mg) tablet once daily for 12 weeks regardless of HCV genotype. The primary efficacy endpoint was sustained virologic response, defined as HCV RNA <15 IU/mL at 12 weeks after completion of treatment (SVR12). The primary safety endpoint was the proportion of patients who prematurely discontinued treatment because of adverse events. Safety and efficacy data were analyzed with an intention-to-treat (ITT) population (last observation carried forward) and per-protocol (PP) population. This trial is registered on ChiCTR.org.cn with number being ChiCTR1800020246. Findings: Of the 243 patients enrolled, 78% were male, 9% had been previously treated for HCV with interferon, and none had pre-defined cirrhosis, although 8% had Fibrosis 4 score (FIB-4) >3.25. A total of 233 patients completed 12-week post-treatment follow-up. Overall, 227/233 patients (97%) achieved SVR12: 100% (63/63) in those with HCV genotype 1, 67% (2/3) in those with genotype 2, 95% (84/88) in those with genotype 3, 99% (78/79) in those with genotype 6. Rates of SVR12 were lower among those with baseline FIB-4 >3.25 than those without (78% [14/18] vs. 99% [211/212], P < 0.001). HIV-1 suppression was not compromised. The most common adverse events were upper respiratory tract infection (5%), cough (3%), abnormal renal function (2%), abnormal liver function (2%), constipation (2%), urinary tract infection (2%) and sleep disorders (2%). No participant discontinued treatment because of adverse events or death. Interpretation: Twelve weeks of treatment with sofosbuvir/velpatasvir provide high rates of SVR and is well-tolerated in patients coinfected with HIV-1 and HCV regardless of HCV genotypes. Non-invasive liver fibrosis score may help to further distinguish patients at greater likelihood of a suboptimal response. Funding: The 13th Five Year Plan of the Ministry of Science and Technology of China for the prevention and treatment of major infectious diseases such as AIDS and viral hepatitis, the National Key Research and Development Program of China, Medical Key Discipline Program of Guangzhou-Viral Infectious Diseases (2021-2023), Basic research program on people's Livelihood Science and technology of Guangzhou, and National Natural Science Foundation of China.

The delayed clearance of Talaromyces marneffei in blood culture may be associated with higher MIC of voriconazole after antifungal therapy among AIDS patients with talaromycosis.

OBJECTIVES: This study aimed to investigate the influencing factors of delayed clearance of Talaromyces marneffei (T. marneffei) in blood culture of patients with acquired immune deficiency syndrome (AIDS) complicated with talaromycosis after antifungal therapy. METHODS: The patients with AIDS complicated with talaromycosis were retrospectively enrolled, and divided into two groups according to the blood T. marneffei culture results in two weeks after antifungal therapy. The baseline clinical data were collected and the antifungal susceptibility of T. marneffei was tested. RESULTS: A total of 190 patients with AIDS and talaromycosis were enrolled, of whom 101 cases remained positive for T. marneffei (Pos-group) while the other 89 cases were negative in blood culture (Neg-group) after two weeks' antifungal treatment. The Pos-group had a higher baseline Aspartate aminotransferase (AST, 78.5 vs. 105 U/L; P = 0.073) and lower CD4+ T cells level (11 vs. 7 cells/µl; P = 0.061). The percentage of isolates with higher MICs of voriconazole (VOR) and fluconazole (FLU) in the Pos-group were significantly higher than those in the Neg-group (χ2 = 12.623, P < 0.001 and χ2 = 9.356, P = 0.002, respectively). By multivariate logistic regression, the MIC value for VOR was identified as the prognostic variable that may influence the clearance of T. marneffei in blood culture after antifungal therapy among AIDS patients with talaromycosis. CONCLUSIONS: The delayed negative conversion of blood T. marneffei-culture may be associated with some factors especially higher MIC of VOR, indicating the possibility of drug resistance of T. marneffei.

Characteristics and clinical significance of plasma IL-18, sCD14, and sCD163 levels in patients with HIV-1 infection.

Biomarkers of monocyte-macrophages activation and inflammation in plasma such as interleukin-18 (IL-18), soluble leukocyte differentiation antigen 14 (sCD14), and sCD163 are associated with disease severity and prognosis in HIV-1 infected patients, however, their relationships with efficacy of antiretroviral therapy (ART) need further investigation. We aimed to characterize and explore the clinical significance of plasma IL-18, sCD14, and sCD163 in this population. This was a retrospective cohort study consisting of HIV-1 infected patients enrolled in a randomized, controlled, open-label, noninferiority trial (ALTERLL study), with follow-up time points including initiation of ART (baseline), 12-, 24- and 48-weeks of treatment. Plasma levels of IL-18, sCD14, and sCD163 were measured using the enzyme-linked immunosorbent assay method. Viral suppression was defined as HIV-1 RNA < 20 copies/ml. Among the 193 studied patients (median age of 29.0 years, 180 males), IL-18 and sCD163 had U-shaped regression curves and sCD14 had an inverted U-shaped regression curve while the virus was decreased and immune function recovered. Patients with higher levels of IL-18 or lower levels of sCD163 at baseline were less likely to achieve viral suppression at Week 12 or Week 24 of treatment, respectively. In multivariate analysis, baseline sCD163 ≤ 500 pg/ml (adjusted odds ratio 0.33, 95% confidence interval 0.16-0.68) was independently associated with a lower rate of viral suppression at Week 24 of treatment. In conclusion, we demonstrated different dynamic changes among IL-18, sCD14, and sCD163 after ART. Baseline sCD163 level could be a potential predictor of early virological response to ART. Further validation and mechanistic research are needed.

Persistently low CD4 cell counts are associated with hepatic events in HCV/HIV coinfected patients: data from the national free antiretroviral treatment program of China.

BACKGROUND: Chronic liver disease has emerged as a leading cause of non-acquired immune deficiency syndrome (AIDS)-related mortality in hepatitis C virus (HCV)/human immunodeficiency virus (HIV)-coinfected patients. The relationship between CD4 cell count and HIV-related opportunistic infections and tumors has been well characterized; however, it is unclear whether CD4 cell count is associated with HCV-related hepatic events. METHODS: This observational cohort study enrolled HCV/HIV-coinfected patients from the National Free Antiretroviral Treatment Program of China from 2004 to 2019 in Guangzhou. The primary outcome was a composite of hepatic events, including cirrhosis complications, hepatocellular carcinoma (HCC), and liver-related mortality. Kaplan-Meier survival and multivariate logistic regression analyses were performed. RESULTS: Among the 793 patients, 43 developed hepatic events during a median follow-up of 6.7 years, including 35 cirrhosis complications, 13 HCC cases, and 14 cases of liver-related mortality. The 5-year and 10-year cumulative incidences of hepatic events were 4.2% and 9.3%, respectively. Patients who developed hepatic events had a less satisfactory increase in CD4 cell count, lower peak CD4 (354.5 cells/µL vs. 560.0 cells/µL, P < 0.001), and lower percentage of peak CD4 > 500 cells/µL (30.2% vs. 60.7%, P < 0.001) after the initiation of antiretroviral therapy (ART) than those who did not. The cumulative incidences of hepatic events were higher in patients with lower peak CD4 levels with adjusted odds ratios of 3.96 (95% confidence interval [CI]: 1.51-10.40), 2.25 (95% CI: 0.87-5.86), and 0.98 (95% CI: 0.35-2.74) for patients with peak CD4 at <200 cells/µL, 200-350 cells/µL, and 351 to 500 cells/µL, respectively, relative to those with peak CD4 > 500 cells/µL. Peak CD4 was negatively associated with the risk of hepatic events in a dose-response manner ( P -value for trend = 0.004). CONCLUSION: Persistently low CD4 cell counts after ART are independently associated with a high risk of hepatic events in HCV/HIV-coinfected patients, highlighting the important role of immune reconstitution in improving liver outcomes.

Talaromyces marneffei Mp1p Antigen Detection may Play an Important Role in the Early Diagnosis of Talaromycosis in Patients with Acquired Immunodeficiency Syndrome.

Talaromycosis is a life-threatening fungal disease commonly seen in patients with acquired immunodeficiency syndrome (AIDS), which is endemic in Southern China and Southeast countries. The diagnostic methods available for talaromycosis are relatively time-consuming and yield a high mortality. Therefore, early diagnosis of talaromycosis is extremely important. We aimed to determine a potential method for assisting in its early diagnosis. A total of 283 patients with AIDS admitted to our hospital were prospectively included in this cross-sectional study and divided into those with Talaromyces marneffei (TSM group, n = 93) and those without Talaromyces marneffei (non-TSM group, n = 190). The diagnostic accuracy of the Mp1p enzyme immunoassay (EIA), galactomannan (GM) assay, and blood culture performed within 3 days of hospitalisation were evaluated, using talaromycosis confirmed by culture and/or pathology as the gold standard. The positivity rates in the Mp1p EIA, GM assay, and blood culture were 72%, 64.5%, and 81.7%, respectively, in the TSM group. The sensitivity, specificity, and positive and negative predictive values of the Mp1p EIA were 72.0% (67/93), 96.8% (184/190), 91.8% (67/73), and 87.6% (184/210), respectively. The Mp1p EIA showed a substantial agreement with the gold standard (kappa: 0.729) and superiority to the GM assay (kappa: 0.603); it also showed a superior diagnostic accuracy in the patients with CD4+ counts of < 50 cells/µL compared to those with CD4+ counts ranged from 50-100 cells/µL. The Mp1p EIA has the advantage of assisting in the early diagnosis of talaromycosis in patients with AIDS, especially those with low CD4+ counts.

Broadly neutralizing antibody-derived CAR T cells reduce viral reservoir in individuals infected with HIV-1.

BACKGROUNDChimeric antigen receptor (CAR) T cells have emerged as an approach to treat malignant tumors. This strategy has also been proposed for the treatment of HIV-1 infection. We have developed a broadly neutralizing antibody-derived (bNAb-derived) CAR T cell therapy that can exert specific cytotoxic activity against HIV-1-infected cells.METHODSWe conducted an open-label trial of the safety, side-effect profile, pharmacokinetic properties, and antiviral activity of bNAb-derived CAR T cell therapy in individuals infected with HIV-1 who were undergoing analytical interruption of antiretroviral therapy (ART).RESULTSA total of 14 participants completed only a single administration of bNAb-derived CAR T cells. CAR T cell therapy administration was safe and well tolerated. Six participants discontinued ART, and viremia rebound occurred in all of them, with a 5.3-week median time. Notably, the cell-associated viral RNA and intact proviruses decreased significantly after CAR T cell treatment. Analyses of HIV-1 variants before or after CAR T cell administration suggested that CAR T cells exerted pressure on rebound viruses, resulting in a selection of viruses with less diversity and mutations against CAR T cell-mediated cytotoxicity.CONCLUSIONNo safety concerns were identified with adoptive transfer of bNAb-derived CAR T cells. They reduced viral reservoir. All the rebounds were due to preexisting or emergence of viral escape mutations.TRIAL REGISTRATIONClinicalTrials.gov (NCT03240328).FUNDINGMinistry of Science and Technology of China, National Natural Science Foundation of China, and Department of Science and Technology of Guangdong Province.

Human umbilical cord mesenchymal stem cell transfusion in immune non-responders with AIDS: a multicenter randomized controlled trial.

We examined the safety and efficacy of human umbilical cord mesenchymal stem cell (hUC-MSC) infusion for immune non-responder (INR) patients with chronic HIV-1 infection, who represent an unmet medical need even in the era of efficient antiretroviral therapy (ART). Seventy-two INR patients with HIV were enrolled in this phase II randomized, double-blinded, multicenter, placebo-controlled, dose-determination trial (NCT01213186) from May 2013 to March 2016. They were assigned to receive high-dose (1.5 × 106/kg body weight) or low-dose (0.5 × 106/kg body weight) hUC-MSC, or placebo. Their clinical and immunological parameters were monitored during the 96-week follow-up study. We found that hUC-MSC treatment was safe and well-tolerated. Compared with baseline, there was a statistical increase in CD4+ T counts in the high-dose (P < 0.001) and low-dose (P < 0.001) groups after 48-week treatment, but no change was observed in the control group. Kaplan-Meier analysis revealed a higher cumulative probability of achieving an immunological response in the low-dose group compared with the control group (95.8% vs. 70.8%, P = 0.004). However, no significant changes in CD4/CD8+ T counts and CD4/CD8 ratios were observed among the three groups. In summary, hUC-MSC treatment is safe. However, the therapeutic efficacy of hUC-MSC treatment to improve the immune reconstitution in INR patients still needs to be further investigated in a large cohort study.

Persistent chronic immune activation in HIV/HBV-coinfected patients after antiretroviral therapy.

We studied the characteristics of immune activation and investigated the underlying mechanisms in patients with human immunodeficiency virus-1/hepatitis B virus (HIV/HBV) coinfection after receiving HBV-active antiretroviral therapy. Forty patients with HIV/HBV coinfection, 38 patients with HIV monoinfection and 20 healthy controls were enrolled. CD4+ count, HIV load, HBV load, markers of immune activation and regulatory T-cell (Treg cell) frequency were assessed and compared between HIV-monoinfected and HIV/HBV-coinfected patients at week 0 (baseline), 12, 24, 36 and 48 after the onset of HBV-active antiretroviral therapy. Before antiretroviral therapy, frequencies of CD4+ HLADR+ CD38+ , CD8+ HLADR+ CD38+ , and Treg cells, and sCD163 and sCD14 levels were significantly higher in both HIV/HBV-coinfected patients and HIV-monoinfected patients, compared with healthy controls. Frequencies of CD4+ HLADR+ CD38+ and CD8+ HLADR+ CD38+ cells decreased following antiretroviral therapy in both groups. sCD163 levels did not change significantly in both groups and no significant difference was observed between the two groups at each time point during the 48-week antiretroviral therapy. In week 24, levels of sCD14 and frequencies of Treg cells appeared significantly higher in HIV/HBV-coinfected patients than in HIV-monoinfected patients, in which sCD14 levels and Treg cell frequencies declined to those in healthy controls. The Treg cell frequency was consistent with that of sCD14 levels in HIV/HBV-coinfected patients. Coinfection with HBV significantly increases sCD14 levels in HIV-infected patients during HBV-active antiretroviral therapy, which may potentially contribute to liver inflammation.

The Association of TLR2, TLR3, and TLR9 Gene Polymorphisms With Susceptibility to Talaromycosis Among Han Chinese AIDS Patients in Guangdong.

Background: Talaromycosis (TM) caused by Talaromyces marneffei (T. marneffei) is a growing public health concern. Although Toll-like receptor (TLR) genes play a critical role in the host defense against fungal infection, the influence of polymorphisms in these genes on the susceptibility of acquired immune deficiency syndrome (AIDS) patients to TM remains unknown. This study aims to uncover the associations of single nucleotide polymorphisms (SNPs) in TLR genes with TM susceptibility among patients with AIDS. Methods: Altogether 200 AIDS patients complicated with TM, 200 matched AIDS patients without TM, and 76 healthy controls (HCs) were enrolled in this case-control study. In total, 23 SNPs in the TLR2, TLR4, and TLR9 genes, which may influence the susceptibility of AIDS patients to TM, were checked by the time of flight mass spectrometry (TOF/MS) method among these Han Chinese subjects. Results: No significant differences in genotype or allele frequencies of selected SNPs were found among the TM group, Non-TM group, and HC group. Haplotype analysis also demonstrated no correlation of these SNPs with TM. However, subgroup analysis showed that the genotype TT and the T allele in TLR2 SNP rs1339 were more frequent in typical TM cases than controls (50.0 vs. 35.8%, 70.5 vs. 59.7%); the frequency of the GT genotype in TLR2 SNP rs7656411 was markedly higher in severe TM cases compared to controls (57.8 vs. 34.4%). Conclusion: Our results demonstrate a genetic connection of TLR2 SNPs rs1339 and rs7656411 with an increased susceptibility and severity of TM among Han Chinese populations.

Antiretroviral Long-Term Efficacy and Resistance of Lopinavir/Ritonavir Plus Lamivudine in HIV-1-Infected Treatment-Naïve Patients (ALTERLL): 144-Week Results of a Randomized, Open-Label, Non-Inferiority Study From Guangdong, China.

Dual therapy with lopinavir/ritonavir (LPV/r) plus lamivudine (3TC) has been demonstrated to be non-inferior to the triple drug regimen including LPV/r plus two nucleoside reverse transcriptase inhibitors (NRTIs) in 48-week studies. However, little is known about the long-term efficacy and drug resistance of this simplified strategy. A randomized, controlled, open-label, non-inferiority trial (ALTERLL) was conducted to assess the efficacy, drug resistance, and safety of dual therapy with LPV/r plus 3TC (DT group), compared with the first-line triple-therapy regimen containing tenofovir (TDF), 3TC plus efavirenz (EFV) (TT group) in antiretroviral therapy (ART)-naïve HIV-1-infected adults in Guangdong, China. The primary endpoint was the proportion of patients with plasma HIV-1 RNA < 50 copies/ml at week 144. Between March 1 and December 31, 2015, a total of 196 patients (from 274 patients screened) were included and randomly assigned to either the DT group (n = 99) or the TT group (n = 97). In the primary intention-to-treat (ITT) analysis at week 144, 95 patients (96%) in the DT group and 93 patients (95.9%) in the TT group achieved virological inhibition with plasma HIV-1 RNA <50 copies/ml (difference: 0.1%; 95% CI, -4.6-4.7%), meeting the criteria for non-inferiority. The DT group did not show significant differences in the mean increase in CD4+ cell count (247.0 vs. 204.5 cells/mm3; p = 0.074) or the CD4/CD8 ratio (0.47 vs. 0.49; p = 0.947) from baseline, or the inflammatory biomarker levels through week 144 compared with the TT group. For the subgroup analysis, baseline high viremia (HIV-1 RNA > 100,000 copies/ml) and genotype BC did not affect the primary endpoint or the mean increase in CD4+ cell count or CD4/CD8 ratio from baseline at week 144. However, in patients with genotype AE, the DT group showed a higher mean increase in CD4+ cell count from baseline through 144 weeks than the TT group (308.7 vs. 209.4 cells/mm3; p = 0.038). No secondary HIV resistance was observed in either group. Moreover, no severe adverse event (SAE) or death was observed in any group. Nonetheless, more patients in the TT group (6.1%) discontinued the assigned regimen than those in the DT group (1%) due to adverse events. Dual therapy with LPV/r plus 3TC manifests long-term non-inferior therapeutic efficacy, low drug resistance, good safety, and tolerability compared with the first-line triple-therapy regimen in Guangdong, China, indicating dual therapy is a viable alternative in resource-limited areas. Clinical Trial Registration: [http://www.chictr.org.cn], identifier [ChiCTR1900024611].

Tolerable and curable treatment in HIV/HCV co-infected patients using anti-HCV direct antiviral agents: a real-world observation in China.

OBJECTIVE: No brand direct-acting antiviral agents (DAAs) are available for treatment of HIV-1/HCV co-infected patients in China. This study aimed to observe the therapeutic efficacy and safety of generic DAAs for affected Chinese patients. DESIGN: Real-world setting to elucidate whether DAAs were tolerated and effective in HIV-1/HCV co-infected patients. METHODS: 176 HIV-1/HCV co-infected patients received anti-HCV DAA treatment together with ART regimens for HIV infection. Among the 176 patients, 99 patients were treated with SOF + DCV ± RBV, 60 patients were treated with SOF + LDV ± RBV, and 17 patients received SOF + RBV ± Peg-IFN regimens, for 12 or 24 weeks, respectively. The primary endpoint was undetectable HCV RNA 12 weeks after therapy was completed (SVR12). Data pertaining to safety and adverse events were analyzed. RESULTS: 151/176 HIV-1/HCV co-infected patients finished the treatment and 12-week follow-up. SVR12 for the patients treated with regimens of SOF + DCV, SOF + DCV+RBV, SOF + Peg-IFN+RBV, SOF + RBV, SOF + LDV, and SOF + LDV+RBV for 12 or 24 weeks was 100% (75/75), 100% (11/11), 100% (14/14), 100% (2/2), 95.2% (40/42), and 100% (7/7), respectively. HIV-1/HCV co-infected patients with liver cirrhosis achieved well SRV12. Notably, there was no significant difference in adverse effects among patients with different baseline CD4+ T-cell count in those who received DAA regimens with or without Peg-IFN and RBV. CONCLUSION: We showed generic SOF + DCV and SOF + LDV regimens were well tolerated and with high efficiency. Patient's baseline CD4+ T-cell count did not exhibit significant difference in adverse effects.

Dual therapy with lopinavir/ritonavir plus lamivudine could be a viable alternative for antiretroviral-therapy-naive adults with HIV-1 infection regardless of HIV viral load or subgenotype in resource-limited settings: A randomised, open-label and non-inferiority study from China.

BACKGROUNDS: This randomised controlled, open-label, non-inferiority trial was conducted in antiretroviral-naïve HIV-1-infected patients to assess the efficacy and safety of 48-week dual therapy of LPV/r plus 3TC (DT group) compared with Chinese first-line triple-therapy regimen (TT group). METHODS: 198 were randomised to DT (n = 100) or TT (n = 98). RESULTS: Ninety-two DT patients (92%) and 88 TT patients (89.8%) achieved HIV-1 RNA <50 copies/ml at week 48 (P = 0.629). Moreover, the safety profile was similar between two groups, and no secondary HIV resistance was observed. CONCLUSION: The results suggest that dual therapy of LPV/r plus 3TC is non-inferior to the first-line triple-therapy regimen in China.

Expression and characterization of a Talaromyces marneffei active phospholipase B expressed in a Pichia pastoris expression system.

Phospholipase B is a virulence factor for several clinically important pathogenic fungi, including Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, but its role in the thermally dimorphic fungus Talaromyces marneffei remains unclear. Here, we provide the first report of the expression of a novel phospholipase gene, designated TmPlb1, from T. marneffei in the eukaryotic expression system of Pichia pastoris GS115. Sensitive real-time quantitative reverse-transcription PCR (qRT-PCR) demonstrated that the expression of TmPlb1 increased 1.85-fold in the yeast phase compared with the mycelial phase. TmPlb1 contains an open reading frame (ORF) of 732 bp that encodes a protein of 243 amino acids. The conserved serine, aspartate and histidine catalytic triad and the G-X-S-X-G domain of TmPLB1 provide the structural basis for its molecular activity. The ORF of TmPlb1 was successfully cloned into a pPIC9K vector containing an α-mating factor secretion signal that allowed the secretory expression of TmPLB1 in P. pastoris. The heterologous protein expression began 12 h after methanol induction and peaked at 96 h. Through analysis with SDS-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting and mass spectrometry, we confirmed that TmPLB1 was successfully expressed. Through Ni-affinity chromatography, TmPLB1 was highly purified, and its concentration reached 240.4 mg/L of culture medium. With specific substrates, the phospholipase A1 and phospholipase A2 activities of TmPLB1 were calculated to be 5.96 and 1.59 U/mg, respectively. The high purity and activity of the TmPLB1 obtained here lay a solid foundation for further investigation.

Molecular epidemiology of human immunodeficiency virus type 1 in Guangdong province of southern China.

BACKGROUND: Although the outbreak of human immunodeficiency virus type 1 (HIV-1) in Guangdong has been documented for more than a decade, the molecular characteristics of such a regional HIV-1 epidemic remained unknown. METHODOLOGY/PRINCIPAL FINDINGS: By sequencing of HIV-1 pol/env genes and phylogenetic analysis, we performed a molecular epidemiologic study in a representative subset (n  = 200) of the 508 HIV-1-seropositive individuals followed up at the center for HIV/AIDS care and treatment of Guangzhou Hospital of Infectious Diseases. Of 157 samples (54.1% heterosexual acquired adults, 20.4% needle-sharing drug users, 5.7% receivers of blood transfusion, 1.3% men who have sex with men, and 18.5% remained unknown) with successful sequencing for both pol and env genes, 105 (66.9%) HIV-1 subtype CRF01_AE and 24 (15.3%) CRF07_BC, 9 (5.7%) B', 5 (3.2%) CRF08_BC, 5 (3.2%) B, 1 (0.6%) C, 3 (1.9%) CRF02_AG, and 5 (3.2%) inter-region recombinants were identified within pol/env sequences. Thirteen (8.3%) samples (3 naïves, 6 and 5 received with antiretroviral treatment [ART] 1-21 weeks and ≥24 weeks respectively) showed mutations conferring resistance to nucleoside/nonnucleoside reverse transcriptase inhibitors or protease inhibitors. Among 63 ART-naïve patients, 3 (4.8%) showed single or multiple drug resistant mutations. Phylogenetic analysis showed 8 small clusters (2-3 sequences/cluster) with only 17 (10.8%) sequences involved. CONCLUSION/SIGNIFICANCE: This study confirms that sexual transmission with dominant CRF01_AE strain is a major risk for current HIV-1 outbreak in the Guangdong's general population. The transmission with drug-resistant variants is starting to emerge in this region.

[The therapeutic effects of highly active anti-retroviral therapy in 74 treatment-naive patients with AIDS in China].

OBJECTIVE: To evaluate the therapeutic effect of highly active anti-retroviral therapy (HAART) in treatment-naïve Chinese patients with AIDS, to provide evidences for standardizing HAART. METHODS: Seventy-four treatment-naive AIDS patients were initiated with HAART and followed up regularly for 3 years. The clinical and laboratory data, side effects and drug resistance were observed and analyzed during the follow-up period. RESULTS: Of the 74 patients, 46 were males and 28 were females, with the average age being 42 years. The mean HIV viral load was (2.2 ± 2.0) × 10(5) copies/ml and the baseline mean CD(4)(+)T lymphocyte count was (62 ± 71) cells/µl before treatment. After treatment for 3, 6, 12, 18, 24, 30 and 36 months, the percentage of undetectable HIV viral road (less than 50 copies/ml) was 71.6%, 83.8%, 75.7%, 77.0%, 82.4%, 81.1% and 79.7% respectively, and CD(4)(+)T lymphocyte count ascended to (167 ± 105), (177 ± 129), (238 ± 137), (290 ± 158), (304 ± 191), (331 ± 175) and (352 ± 202) cells/µl. The increase in amplitude of CD(4)(+)T lymphocyte count in different periods examined was different, with the period of 0-3 months post-treatment demonstrating the most obvious augmentation (P < 0.01). The most common adverse reactions were liver function injury (52/74, 70.3%), hyperlipemia (52/74, 70.3%), hematopoietic inhibition of the bone marrow (33/74, 44.6%), peripheral neuritis (32/74, 43.2%) and lipoatrophy (26/74, 35.1%). Clinical drug resistance were found in nine patients and HIV gene mutations were detected in these patients. CONCLUSIONS: Chinese treatment-naive AIDS patients have achieved good virological and immunological response to generic-drug-predominant HAART regimes with low drug resistance, but relatively more side effects.

Microsatellite analysis of clinical isolates of the opportunistic fungal pathogen Penicillium marneffei from AIDS patients in China.

BACKGROUND: Penicillium marneffei is an opportunistic fungus that may cause fatal disease, and usually infects acquired immune deficiency syndrome (AIDS) patients. The molecular epidemiology of this fungus remains enigmatic. METHODS: A multilocus microsatellite typing (MLMT) system based on 11 microsatellite loci was applied to 169 unrelated isolates of P. marneffei obtained from AIDS patients, in order to identify their genetic diversity. These patients came from the provinces of Guangdong and Guangxi, areas endemic for P. marneffei in China. RESULTS: For the overall population, the average number of alleles per locus ranged from 3 to 8 (mean 5.5), while the discriminatory power (DP) of each locus ranged from 0.235 to 0.651 (mean 0.512). By combining the information generated for 11 loci, MLMT detected 159 different multilocus genotypes (MTs), resulting in a high degree of discrimination (DP = 0.999). One hundred and sixty-nine isolates were further clustered into 9 types (from A to I) at the similarity coefficient of 0.80, with type A (80 isolates) and type B (60 isolates) being the most common types. Within 5 subpopulations from different regions of China, the distribution of MTs of P. marneffei isolates was diverse. Although 169 isolates shared a high genetic similarity (range 0.71-0.933), isolates from Guangxi and Guangdong provinces could be differentiated from each other and clustered into 2 categories by unweighted pair-group method with arithmetic mean (UPGMA) cluster analysis. CONCLUSIONS: By MLMT, the genetic diversity of clinical P. marneffei isolates could be discriminated, the dominant strain of P. marneffei cultured from AIDS patients in China could be identified, and clinical isolates of P. marneffei from Guangxi Province could be differentiated from those from Guangdong Province.

[An analysis of opportunistic infection in 762 inpatients with human immunodeficiency virus infection in Guangdong areas].

OBJECTIVE: To analyze the characteristics of opportunistic infection (OI) in patients with HIV/AIDS in Guangdong and the relationship between OI and the change in blood CD4+ T lymphocyte count (CD4+). METHODS: Seven hundred and sixty two patients with HIV/AIDS admitted were analyzed. RESULTS: Among all the 762 patients, 704 (92.39%) had more than one kind of OI, with 1428 episodes totally. Etiologically, fungus infection (38.38%) was most common, followed by bacteria (36.20%), and virus (7.77%) infection. Most OI occurred in the lungs (33.05%), mouth (26.89%), skin (10.29%) and gastro-intestine (8.96%). Septicemia and other systemic disseminated diseases accounted for 6.58% and 9.94% respectively. The incidence of OI in patients with CD4+≥200/µl (103/136, 75.74%) was significantly lower than that in patients with CD4+<200/µl (601/626, 96.01%), P<0.01. All the AIDS defining OI were found in patients with CD4+<200/µl. Among them, 81.97% of patients with pneumonia carinii pneumonia (PCP), 71.43% of patients with cytomegalovirus retinitis and all the patients with cryptococcal meningitis, disseminated cryptococcosis, disseminated histoplasmosis, mycobacterium avium intracellular complex (MAC), disseminated penicilliosis marneffei and toxoplasma cerebritis had the CD4+ less than 50/µl. CONCLUSIONS: The most common OI in patients with AIDS in Guangdong area are fungi, bacterial and viral infections. Lung, mouth, skin, gastro-intestine and systemic disseminated infections are the most prevalent infections. As the CD4+ decreased, the incidence of OI especially AIDS defining OI increased. Dynamic detection of CD4+ will be of great help for the prediction, prevention, early diagnosis and treatment of OI in patients with AIDS.

Identification of Penicillium marneffei in Paraffin-Embedded Tissue Using Nested PCR.

Penicillium marneffei is one of the unique thermally dimorphic fungi in Penicillium species that causes a disseminated, progressive and life threatening infection in immunocompromised patients. The diagnosis of Penicilliosis marneffei depends on culture that may delay the treatment due to the time-consuming process. In the present study, we evaluated the specificity and sensitivity of nested PCR to identify Penicillium marneffei from paraffin-embedded tissue. Two sets of oligonucleotide primers were derived from the sequence of 18S rRNA of Penicillium marneffei. The outer primers (RRF1 and RRH1) were specific to fungi. The inner primers (Pm1 and Pm2) were specific to Penicillium marneffei. The specific fragment of approximately 400 bp was amplified from all paraffin-embedded tissues from 14 patients with Penicilliosis marneffei and 10 bamboo rats. The detectable DNA concentration of single PCR and nested PCR were 14 pg/microl and 14 fg/microl, respectively. Further studies are required in order to use nested PCR for early diagnosis of the disease.