ELISA genotyping of hepatitis B virus in China with antibodies specific for genotypes B and C.

Hepatitis B virus (HBV) causes hepatitis B (HB) and distinct HBV genotypes can lead to different prognoses. However, HBV genotyping is rarely done in clinics, because the traditional method by PCR-based DNA sequencing is impractical for clinical diagnosis with tedious process and low success rate. Herein, we have established an ELISA-based genotyping method to quickly determine the HBV genotypes of HB patients in China. First, two commercial antibodies, 16D12 and 6H3 specific for HBV genotypes B and C respectively, are chosen as capture antibodies, since these two genotypes dominate in China. Then two home-made genotype-specific antibodies, B19 and C04, are used as the detection antibodies for genotypes B and C in sandwiched ELISA. The ELISA kit shows high sensitivity (> 95%) and specificity (> 95%) in detecting genotypes B and C of Chinese HB patients. Moreover, the ELISA kit has demonstrated higher success rate (98.7%) than PCR-based DNA sequencing (93.5%) and a commercial PCR-based genotyping kit (92.2%) for sera with HBV DNA ≥ 1000 IU/mL and HBsAg ≥ 250 IU/mL. Such an advantage is more obvious for the sera with HBV DNA < 1000 IU/mL. The kappa analysis between the ELISA and PCR-based DNA sequencing results exhibits a kappa of 0.836, indicating a good correlation.

Discovery of sandwich type COVID-19 nucleocapsid protein DNA aptamers.

Here, we report for the first time DNA aptamers targeted toward the COVID-19 nucleocapsid protein (Np). Np is one of the most abundant structural proteins and it serves as a diagnostic marker for the accurate and sensitive detection of COVID-19. After five rounds of selection, we obtained four DNA sequences with an affinity below 5 nM. The best one displayed a superb binding performance toward Np with a Kd value of 0.49 nM. Interestingly, we found that the four pairs of aptamers could bind to Np successively, suggesting a sandwich-type interaction. Using these sandwiched aptamers in ELISA and colloidal gold immunochromatographic strips, we were able to detect Np at the tens of pM level. The results demonstrate that aptamers are powerful molecular tools for virus detection, diagnosis, and antiviral therapy.

Feasibility of an internet-based HIV testing service: anonymous urine collection from men who have sex with men.

A large proportion of people who are HIV positive do not know their serostatus because facility-based provider-initiated HIV testing and counseling, and voluntary counseling and testing, have not been efficiently implemented in China. Therefore, a new HIV testing strategy must be developed to improve testing services so that more HIV infections can be detected earlier. In this study, we established an anonymous internet-aided urine-based HIV testing service for men who have sex with men (MSM) from 1 April 2016 to 20 January 2017. In total, 3092 urine sample collection packs were distributed by grassroots organizations to MSM; 1977 (69.3%) packs were mailed back to the laboratory; and 1911 (96.7%) eligible samples were tested for HIV antibody. The rate of HIV antibody positivity was 7.1% (135/1901), excluding 10 previously-identified HIV infections. Of those tested, 65.4% (1243/1901) participants obtained their results from our website, 94 (69.6%) of 135 newly-identified urine HIV antibody-positive participants were contacted by CDC staff, and 61.7% (58/94) reported undergoing blood HIV antibody confirmation testing after learning of their urine HIV antibody test results. Of those who were tested for venous HIV antibody, 84.5% (49/58) reported being confirmed HIV antibody positive. Thirty-six of the newly diagnosed participants were successfully referred to a hospital to receive antiretroviral therapy. The rate of confirmed HIV antibody positivity was estimated to be 72.8-89.2 times of that of routine HIV antibody testing. In conclusion, this approach offers an alternative efficient HIV testing strategy to identify HIV positive persons in vulnerable populations.

An innovative HIV testing service using the internet: Anonymous urine delivery testing service at drugstores in Beijing, China.

Innovative human immunodeficiency virus (HIV) testing services will be needed to achieve the first 90 (90% of HIV-positive persons aware of their infection status) of the 90-90-90 target in China. Here, we describe an internet-based urine delivery testing service delivered through three pilot drugstores in Beijing that send specimens to a designated laboratory for HIV. From May 2016 to January 2017, we provided 500 HIV urine-testing service packs for display at the drugstores, and a total of 430 (86.0%) urine specimens were mailed back. All of the 430 urine specimens were of good quality and were tested. 70 urine specimens were HIV positive, showing a 16.3% (70/430) positivity rate. A total of 94.3% (66/70) of the HIV-positive participants obtained their test results through the internet, and 69.7% (46/66) of these participants received follow-up care. A total of 40 out of 46 (87.0%) participants agreed to have their results confirmed by a blood test, and 39 out of 40 (97.5%) participants were confirmed as HIV-1 positive, including two individuals that were previously diagnosed. Lastly, 28 out of 37 (75.7%) of the study participants were referred to the hospital and provided free antiviral treatment. Our data indicate that this innovative HIV testing service is effective and play an important role in HIV testing and surveillance.

STIM1 plays an important role in TGF-ß-induced suppression of breast cancer cell proliferation.

Store-operated calcium entry (SOCE) signaling is involved in cancer progression. Stromal interaction molecule 1 (STIM1) triggers store-operated calcium channels to induce SOCE. Transforming growth factor-ß (TGF-ß) influences a wide range of cellular behaviors, including cell proliferation. However, little is known about the relationship between calcium signaling and TGF-ß signaling in cancer cell proliferation. Here, we found that TGF-ß induced cell cycle arrest at the G0/G1 phase and suppressed cell proliferation in MDA-MB-231 and MCF-7 breast cancer cells. These effects were impaired by extracellular Ca2+ chelator EGTA or SOCE specific inhibitor SKF96365 in MDA-MB-231 cells. Treating MDA-MB-231 cells with TGF-ß for 24 and 48 h markedly decreased STIM1 expression and thapsigargin-induced SOCE. A transcriptional inhibitor of STIM1, Wilm's tumor suppressor 1 (WT1), was upregulated in TGF-ß-treated MDA-MB-231 cells, and knockdown of WT1 expression partially restored the TGF-ß-induced downregulation of STIM1. Stably overexpressing STIM1 in MDA-MB-231 cells restored the TGF-ß-induced effects. The p21 mRNA level increased in SKF96365- or TGF-ß-treated MDA-MB-231 cells, whereas that for cyclin E1 decreased. Our findings demonstrate for the first time that STIM1 and SOCE are involved in the TGF-ß-induced suppression of cell proliferation. Furthermore, our studies also provide a new approach to inhibit breast cancer cell proliferation with small molecules targeting STIM1 and SOCE.

Developments of the Physical and Electrical Properties of NiCr and NiCrSi Single-Layer and Bi-Layer Nano-Scale Thin-Film Resistors.

In this study, commercial-grade NiCr (80 wt % Ni, 20 wt % Cr) and NiCrSi (55 wt % Ni, 40 wt % Cr, 5 wt % Si) were used as targets and the sputtering method was used to deposit NiCr and NiCrSi thin films on Al2O3 and Si substrates at room temperature under different deposition time. X-ray diffraction patterns showed that the NiCr and NiCrSi thin films were amorphous phase, and the field-effect scanning electronic microscope observations showed that only nano-crystalline grains were revealed on the surfaces of the NiCr and NiCrSi thin films. The log (resistivity) values of the NiCr and NiCrSi thin-film resistors decreased approximately linearly as their thicknesses increased. We found that the value of temperature coefficient of resistance (TCR value) of the NiCr thin-film resistors was positive and that of the NiCrSi thin-film resistors was negative. To investigate these thin-film resistors with a low TCR value, we designed a novel bi-layer structure to fabricate the thin-film resistors via two different stacking methods. The bi-layer structures were created by depositing NiCr for 10 min as the upper (or lower) layer and depositing NiCrSi for 10, 30, or 60 min as the lower (or upper) layer. We aim to show that the stacking method had no apparent effect on the resistivity of the NiCr-NiCrSi bi-layer thin-film resistors but had large effect on the TCR value.

The effect of TGF-beta-induced epithelial-mesenchymal transition on the expression of intracellular calcium-handling proteins in T47D and MCF-7 human breast cancer cells.

The contribution of Ca(2+) in TGF-ß-induced EMT is poorly understood. We aimed to confirm the effect of TGF-ß on the gene expression of intracellular calcium-handling proteins and to investigate the potential underlying mechanisms in TGF-ß-induced EMT. T47D and MCF-7 cells were cultured in vitro and treated with TGF-ß. The mRNA expression of EMT marker genes and intracellular calcium-handling proteins were quantified by qRT-PCR. qRT-PCR and Western blot analysis results verified the changes of EMT marker gene expression. Furthermore, we found that TGF-ß induced cell morphological changes significantly with an increase of cell surface area and cell length. These results indicated that TGF-ß induced EMT. The mRNA expression levels of SPCA1, SPCA2 and MCU were not influenced by TGF-ß treatment, while NCX1 expression was decreased in T47D cells. In addition, the mRNA levels of SERCAs and IP3Rs were significantly changed due to TGF-ß-induced EMT. The TGF-ß-treated T47D cells exhibited markedly greater response to ATP than the control cells, and the descent velocity of cytosolic calcium concentration was faster in TGF-ß-treated cells than in control cells. This is the first report to demonstrate that TGF-ß-induced EMT in human breast cancer cells is associated with alterations in endoplasmic reticulum calcium homeostasis.

Effect of Annealing Process on the Properties of Ni(55%)Cr(40%)Si(5%) Thin-Film Resistors.

Resistors in integrated circuits (ICs) are implemented using diffused methods fabricated in the base and emitter regions of bipolar transistor or in source/drain regions of CMOS. Deposition of thin films on the wafer surface is another choice to fabricate the thin-film resistors in ICs' applications. In this study, Ni(55%)Cr(40%)Si(5%) (abbreviated as NiCrSi) in wt % was used as the target and the sputtering method was used to deposit the thin-film resistors on Al2O3 substrates. NiCrSi thin-film resistors with different thicknesses of 30.8 nm~334.7 nm were obtained by controlling deposition time. After deposition, the thin-film resistors were annealed at 400 °C under different durations in N2 atmosphere using the rapid thermal annealing (RTA) process. The sheet resistance of NiCrSi thin-film resistors was measured using the four-point-probe method from 25 °C to 125 °C, then the temperature coefficient of resistance could be obtained. We aim to show that resistivity of NiCrSi thin-film resistors decreased with increasing deposition time (thickness) and the annealing process had apparent effect on the sheet resistance and temperature coefficient of resistance. We also aim to show that the annealed NiCrSi thin-film resistors had a low temperature coefficient of resistance (TCR) between 0 ppm/°C and +50 ppm/°C.