The trajectory patterns of single HIV-1 virus-like particle in live CD4 cells: A real time three-dimensional multi-resolution microscopy study using encapsulated nonblinking giant quantum dot.

BACKGROUND: The exploration of virology knowledge was limited by the optical technology for the observation of virus. Previously, a three-dimensional multi-resolution real-time microscope system (3D-MRM) was developed to observe the uptake of HIV-1-tat peptide-modified nanoparticles in cell membrane. In this study, we labeled HIV-1 virus-like particles (VLPs) with passivated giant quantum dots (gQDs) and recorded their interactive trajectories with human Jurkat CD4 cells through 3D-MRM. METHODS: The labeled of gQDs of the HIV-1 VLPs in sucrose-gradient purified viral lysates was first confirmed by Cryo-electronic microscopy and Western blot assay. After the infection with CD4 cells, the gQD-labeled VLPs were visualized and their extracellular and intracellular trajectories were recorded by 3D-MRM. RESULTS: A total of 208 prime trajectories was identified and classified into three distinct patterns: cell-free random diffusion pattern, directional movement pattern and cell-associated movement pattern, with distributions and mean durations were 72.6%/87.6 s, 9.1%/402.7 s and 18.3%/68.7 s, respectively. Further analysis of the spatial-temporal relationship between VLP trajectories and CD4 cells revealed the three stages of interactions: (1) cell-associated (extracellular) diffusion stage, (2) cell membrane surfing stage and (3) intracellular directional movement stage. CONCLUSION: A complete trajectory of HIV-1 VLP interacting with CD4 cells was presented in animation. This encapsulating method could increase the accuracy for the observation of HIV-1-CD4 cell interaction in real time and three dimensions.

Quantitative analysis of progesterone using isotope dilution-matrix-assisted laser desorption ionization-time of flight mass spectrometry as a reference procedure for radioimmunoassay.

BACKGROUND: Progesterone is one of the female steroid hormones and plays an important role in the menstrual cycle and during pregnancy. It is especially important in preparing the uterus for the implantation of the blastocyst and maintaining pregnancy. The concentration in human serum is measured to determine the ovarian function retroactively and the cause of abortion in early pregnancy. METHODS: A quantification assay based on isotope dilution mass spectrometry to determine the concentration of progesterone in human serum is reported. Incorporated with 13C3-progesterone, serum samples were subjected to progesterone extraction and clean-up by C4 solid-phase-extraction columns and hexane-based liquid/liquid extraction, respectively. The cleaned-up serum samples were then subjected to MALDI-TOF mass spectrometry for the quantification of progesterone. RESULTS: Progesterone and the internal standard, 13C3-progesterone, were measured in the selected reaction monitoring mode for the transitions m/z 315.4 to 108.9 and m/z 318.4 to 111.9, respectively. We calculated the peak area ratio of progesterone to 13C3-progesterone. The progesterone concentration in human serum was calculated by substituting the peak area ratio into an isotope dilution calibration curve, and then compared with the radioimmunoassay. CONCLUSIONS: In the study, the concentrations of serum progesterone were measured, and the recovered progesterone concentration determined by the assay showed good robustness and consistency in comparison to the conventional radioimmunologic assay. We concluded that the 13C3-progesterone-based quantification assay is a robust method for the measurement of serum progesterone.

Risk of Diffuse Infiltrative Lymphocytosis Syndrome in HIV-Infected Patients: A Nationwide Population-Based Cohort Study.

OBJECTIVE: Diffuse infiltrative lymphocytosis syndrome (DILS) is the term used for sicca syndrome in HIV patients and has similar clinical manifestations as Sjögren syndrome. In this nationwide population-based study, we aimed to determine the association between HIV infection and DILS in the Taiwanese population. METHODS: The National Health Insurance Research Database was searched for cases of DILS in HIV-infected individuals diagnosed between January 1, 2000, and December 31, 2012. The incidence of DILS and the factors associated with DILS in people living with HIV/AIDS (PLWHA) were determined. RESULTS: A total of 20,364 PLWHA were followed, and 57 (0.28%) individuals had new-onset DILS. The incidence rate of DILS in PLWHA was 0.56/1000 person-years. One (0.11%) female HIV patient with highly active antiretroviral therapy (HAART) and 24 (2.99%) without HAART had incident DILS, whereas 22 (0.17%) male HIV patients with HAART and 10 (0.17%) without HAART had incident DILS. Hypertension increased the risk of incident DILS. HAART decreased the risk of DILS, but this relationship somewhat attenuated in an adjusted model. None of the patients taking emtricitabine, raltegravir, darunavir, enfuvirtide, or tipranavir developed DILS. Lopinavir was associated with a decreased risk of DILS (adjusted hazard ratio = 0.10, 95% confidence interval: 0.01 to 0.84), whereas zalcitabine was associated with an increased risk of DILS (adjusted hazard ratio = 13.7, 95% confidence interval: 2.18 to 85.9). CONCLUSIONS: DILS is a rare disease found in PLWHA. Hypertension is a risk factor for incident DILS, and HAART could affect the pathogenesis of DILS. Zalcitabine was the only antiretroviral agent found to increase the risk of DILS.

Association of highly active antiretroviral treatment with incident tuberculosis in people living with HIV/AIDS.

PURPOSE: To determine the short-term and long-term effects of highly active antiretroviral therapy (HAART) on incident tuberculosis (TB) in people living with HIV/AIDS (PLWHA). METHODS: From 2000 to 2012, we identified adult PLWHA from Taiwan Centers for Disease Control HIV Surveillance System. All PLWHA were followed up until December 31, 2012, and observed for TB occurrence. Time-dependent Cox proportional hazards models were used to determine the short-term and long-term effects of HAART on incident TB. RESULTS: Of 20,072 PLWHA, 628 (3.13%) had incident TB, corresponding to an incident rate of 701/100,000 person-years. After adjusting for potential confounders, PLWHA receiving HAART were more likely to develop TB than those not receiving the drugs (adjusted hazard ratio [AHR] 1.56; 95% confidence interval [CI] 1.18-2.05). While the short-term and long-term effects of HAART on incident TB were considered, HAART was a risk factor for TB development within the first 90 days (AHR 6.06; 95% CI 4.58-8.01) and between 90 and 180 days of treatment (AHR 1.80; 95% CI 1.11-2.94) but was a protective factor after 180 days of HAART use (AHR 0.51; 95% CI 0.39-0.66). CONCLUSIONS: HAART is a risk factor for the development of TB in the short term but a protective factor in the long term.

Utilizing glycine N-methyltransferasegene knockout mice as a model for identification of missing proteins in hepatocellular carcinoma.

Glycine N-methyltransferase is a tumor suppressor gene for hepatocellular carcinoma, which can activate DNA methylation by inducing the S-adenosylmethionine to S-adenosylhomocystine. Previous studies have indicated that the expression of Glycine N-methyltransferase is inhibited in hepatocellular carcinoma. To confirm and identify missing proteins, the pathologic analysis of the tumor-bearing mice will provide critical histologic information. Such a mouse model is applied as a screening tool for hepatocellular carcinoma as well as a strategy for missing protein discovery. In this study we designed an analysis platform using the human proteome atlas to compare the possible missing proteins to human whole chromosomes. This will integrate the information from animal studies to establish an optimal technique in the missing protein biomarker discovery.

HIV Infection Increases the Risk of Incident Psoriasis: A Nationwide Population-Based Cohort Study in Taiwan.

BACKGROUND: HIV can cause an imbalance of T lymphocytes, which may contribute to the onset of psoriasis. However, the association of HIV with incident psoriasis has not been extensively studied. OBJECTIVES: The aim of this nationwide population-based cohort study was to determine the association of HIV with incident psoriasis. METHODS: Since January 1, 2000, we identified adult people living with HIV/AIDS (PLWHA) from the Taiwan National Health Insurance Research Database. A control cohort without HIV infection, matched for age and sex, was selected for comparison. All patients were followed until December 31, 2012, and observed for the occurrence of psoriasis. The time-dependent Cox proportional hazards model was used to determine the association of HIV with incident psoriasis, while considering death as a competing risk event. RESULTS: Of the 102,070 patients (20,294 PLWHA and 81,776 matched controls), 248 (0.24%) had incident psoriasis during a mean follow-up period of 5.53 years, including 81 (0.40%) PLWHA and 171 (0.21%) controls. After adjusting for age, sex, and comorbidities, HIV infection was found to be an independent risk factor for incident psoriasis (adjusted hazard ratio, 1.80; 95% confidence interval: 1.38 to 2.36). CONCLUSIONS: The population of PLWHA is living longer; clinicians need to be aware of their higher risk of psoriasis.

Characterization of the GNMT-HectH9-PREX2 tripartite relationship in the pathogenesis of hepatocellular carcinoma.

The pathogenesis of hepatocellular carcinoma (HCC) involves many molecular pathways. Glycine N-methyltransferase (GNMT) is downregulated in almost all HCC and its gene knockout mice developed HCC with high penetrance. We identified PREX2, a novel PTEN inhibitor, as a GNMT-interacting protein. Such interaction enhanced degradation of PREX2 through an E3 ligase HectH9-mediated proteasomal ubiquitination pathway. Depletion of GNMT or HectH9 resulted in AKT activation in a PREX2 dependent manner and enhanced cell proliferation. An elevated PREX2 protein expression accompanied by activation of AKT was observed in the liver of Gnmt knockout mice. PREX2 protein expression was upregulated in 54.9% of human HCC samples, while its mRNA level was comparable in tumor and tumor-adjacent tissue, suggesting a post-translational alteration of PREX2 expression. Higher level of PREX2 in the tumor tissues was associated with poorer survival. These results reveal a novel mechanism in which GNMT participates in AKT signaling and HCC tumorigenesis by promoting HectH9-mediated PREX2 degradation.

Generation and characterization of new monoclonal antibodies against swine origin 2009 influenza A (H1N1) virus and evaluation of their prophylactic and therapeutic efficacy in a mouse model.

In 2009, a swine-origin influenza A virus - A(H1N1)pdm09 - emerged and has became a pandemic strain circulating worldwide. The hemagglutinin (HA) of influenza virus is a potential target for the development of anti-viral therapeutic agents. Here, we generated mAbs by immunization of baculovirus-insect expressing trimeric recombinant HA of the A(H1N1)pdm09 strain. Results indicated that the mAbs recognized two novel neutralizing and protective epitopes-"STAS" and "FRSK" which located near Cb and Ca1 antigenic regions respectively and were conserved in almost 2009-2016 influenza H1N1 stains. The mAb 12E11 demonstrated higher protective efficacy than mAb 8B10 in mice challenge assay. Both mAb pretreatments significantly reduced virus titers and pro-inflammatory cytokines in mice lung postinfection (p < 0.01), and showed prophylactic and therapeutic efficacies even 48 h postinfection (p < 0.05). Combination therapy using the mAbs with oseltamivir pre- and post-treatment showed synergistic therapeutic effect in mice model (p < 0.01). Further investigation for clinical application in humans is warranted.

Niemann-Pick type C2 protein regulates liver cancer progression via modulating ERK1/2 pathway: Clinicopathological correlations and therapeutical implications.

Primary hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the third leading cause of cancer-related death. It is important to identify new targets for early diagnosis and treatment of HCC. Niemann-Pick type C2 (NPC2) plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in HCC tumorigenesis. In this study, we showed that NPC2 is abundantly expressed in normal liver, but is downregulated in human HCC tissues. The patients with NPC2 downregulation expressed much higher α-fetoprotein, multiple tumor type, vascular invasion, later pathological stage and shorter survival rate. Knockdown NPC2 in liver cancer cell lines promote cell proliferation, migration and xenograft tumorigenesis. In contrast, NPC2 overexpression inhibits HuH7 promoted tumor growth. Furthermore, administration of hepatotropic adeno-associated virus 8 (AAV8) delivered NPC2 decreased the inflammatory infiltration, the expression of two early HCC markers-glypican 3 and survivin and suppressed the spontaneous HCC development in mice. To identify the NPC2-dependent mechanism, we emphasized on the status of MAPK/ERK signaling. MEK1/2 inhibitor treatment demonstrated that the expression of NPC2 affected the activation of ERK1/2 but not MEK1/2. In addition, cholesterol trafficking inhibitor treatment did not alter the cell proliferation and the activation of MEK/ERK. In conclusion, our study demonstrates that NPC2 may play an important role in negatively regulate cell proliferation and ERK1/2 activation that were independent of cholesterol accumulation. AAV-NPC2 may thus represent a new treatment strategy for liver cancer.

Galectin-3 promotes HIV-1 budding via association with Alix and Gag p6.

Galectin-3 has been reported to regulate the functions of a number of immune cell types. We previously reported that galectin-3 is translocated to immunological synapses in T cells upon T-cell receptor engagement, where it associates with ALG-2-interacting protein X (Alix). Alix is known to coordinate with the endosomal sorting complex required for transport (ESCRT) to promote human immunodeficiency virus (HIV)-1 virion release. We hypothesized that galectin-3 plays a role in HIV-1 viral budding. Cotransfection of cells of the Jurkat T line with galectin-3 and HIV-1 plasmids resulted in increased HIV-1 budding, and suppression of galectin-3 expression by RNAi in Hut78 and primary CD4+ T cells led to reduced HIV-1 budding. We used immunofluorescence microscopy to observe the partial colocalization of galectin-3, Alix and Gag in HIV-1-infected cells. Results from co-immunoprecipitation experiments indicate that galectin-3 expression promotes Alix-Gag p6 association, whereas the results of Alix knockdown suggest that galectin-3 promotes HIV-1 budding through Alix. HIV-1 particles released from galectin-3-expressing cells acquire the galectin-3 protein in an Alix-dependent manner, with proteins primarily residing inside the virions. We also found that the galectin-3 N-terminal domain interacts with the proline-rich region of Alix. Collectively, these results suggest that endogenous galectin-3 facilitates HIV-1 budding by promoting the Alix-Gag p6 association.

Cancer incidence in a Nationwide HIV/AIDS patient cohort in Taiwan in 1998-2009.

BACKGROUND: The aims of this study were to investigate the cancer incidence and risk in HIV/AIDS patients relative to the general population in Taiwan. METHODS: Using Taiwan's National Health Insurance Research Database, 15,269 HIV/AIDS patients were identified between 1998 and 2009. Gender-specific incidence densities (IDs) of both AIDS-defining cancers (ADC) and non-AIDS-defining cancers (NADC) after HIV infection were calculated. Age-, sex-, and period-adjusted standardized incidence rates (SIRs) were obtained using 1.8 million people from the general population as controls. RESULTS: A total of 1117 male and 165 female HIV/AIDS patients were diagnosed with cancer. Non-Hodgkin lymphoma (n = 196; ID = 328.79/100,000 person-years) and cervical cancer (n = 50; ID = 712.08/100,000 person-years) were the most common ADCs, whereas liver cancer (n = 125; ID = 184.52/100,000 person-years) and colon cancer (n = 11; ID = 156.66/100,000 person-years) were the most common NADCs in males and females, respectively. Period-adjusted gender-specific ADC and NADC rates decreased from more than 1500 cases/100,000 person-years to less than 500 cases/100,000 person-years (P < 0.001 for trend). SIRs of ADCs and NADCs also decreased. However, relative to the general population, increased SIRs were still seen for most cancers, many of which had an infectious etiology. The highest SIRs in ADCs and NADCs were seen in Kaposi sarcoma [SIR = 298.0, 95% confidence interval (CI): 258.16 to 343.85] and anal cancer (SIR = 19.10, 95% CI: 12.80 to 27.50). CONCLUSION: This study showed that although the cancer incidence rates have significantly decreased in the highly active antiretroviral therapy era, HIV/AIDS patients were still at increased risk of ADCs and most NADCs. Cancer screening, especially for infection-related NADCs, should therefore be promoted.

Characterization of Niemann-Pick Type C2 protein expression in multiple cancers using a novel NPC2 monoclonal antibody.

Niemann-Pick Type C2 (NPC2) plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in cancer. In this study, we have pinpointed the impact of various different cancers on NPC2 expression. A series of anti-NPC2 monoclonal antibodies (mAbs) with the IgG2a isotype were generated and peptide screening demonstrated that the reactive epitope were amino acid residues 31-40 of the human NPC2 protein. The specificity of these mAbs was confirmed by Western blotting using shRNA mediated knock-down of NPC2 in human SK-Hep1 cells. By immunohistochemical staining, NPC2 is expressed in normal kidney, liver, breast, colon, lung, esophageal, uterine cervical, pancreatic and stomach tissue. Strong expression of NPC2 was found in the distal and proximal convoluted tubule of kidney and the hepatocytes of liver. Normal esophageal, uterine cervical, pancreatic, stomach, breast, colon and lung tissue stained moderately to weakly. When compared to their normal tissue equivalents, NPC2 overexpression was observed in cancers of the breast, colon and lung. Regarding to breast cancer, NPC2 up-regulation is associated with estrogen receptor (-), progesterone receptor (-) and human epidermal growth factor receptor (+). On the other hand, NPC2 was found to be down-regulated in renal cell carcinoma, liver cirrhosis and hepatoma tissues. By antigen-capture enzyme immunoassay ELISA, the serum NPC2 is increased in patients with cirrhosis and liver cancer. According to western blot data, the change of glycosylated pattern of NPC2 in serum is associated with cirrhosis and liver cancer. To the best of our knowledge, this is the first comprehensive immunohistochemical and serological study investigating the expression of NPC2 in a variety of different human cancers. These novel monoclonal antibodies should help with elucidating the roles of NPC2 in tumor development, especially in liver and breast cancers.

Androgen response element of the glycine N-methyltransferase gene is located in the coding region of its first exon.

Androgen plays an important role in the pathogenesis of PCa (prostate cancer). Previously, we identified GNMT (glycine N-methyltransferase) as a tumour susceptibility gene and characterized its promoter region. Besides, its enzymatic product-sarcosine has been recognized as a marker for prognosis of PCa. The goals of this study were to determine whether GNMT is regulated by androgen and to map its AREs (androgen response elements). Real-time PCR analyses showed that R1881, a synthetic AR (androgen receptor) agonist induced GNMT expression in AR-positive LNCaP cells, but not in AR-negative DU145 cells. In silico prediction showed that there are four putative AREs in GNMT-ARE1, ARE2 and ARE3 are located in the intron 1 and ARE4 is in the intron 2. Consensus ARE motif deduced from published AREs was used to identify the fifth ARE-ARE5 in the coding region of exon 1. Luciferase reporter assay found that only ARE5 mediated the transcriptional activation of R1881. ARE3 overlaps with a YY1 [Yin and Yang 1 (motif (CaCCATGTT, +1118/+1126)] that was further confirmed by antibody supershift and ChIP (chromatin immunoprecipitation) assays. EMSA (electrophoretic mobility shift assay) and ChIP assay confirmed that AR interacts with ARE5 in vitro and in vivo. In summary, GNMT is an AR-targeted gene with its functional ARE located at +19/+33 of the first exon. These results are valuable for the study of the influence of androgen on the gene expression of GNMT especially in the pathogenesis of cancer.