H⁺ Permeation and pH Regulation at a Mammalian Serotonin Transporter

The rat serotonin transporter expressed in Xenopusoocytes displays an inward current in the absence of 5-HT when external pH is lowered to 6.5 or below. The new current differs from the leakage current described previously in two ways. (1) It is ∼10-fold larger at pH 5 than the leakage current at pH 7.5 and reaches 1000 H⁺/sec per transporter at extremes of voltage and pH with no signs of saturation. (2) It is selective for H⁺ by reversal potential measurements. Similar H⁺-induced currents are also observed in several other ion-coupled transporters, including the GABA transporter, the dopamine transporter, and the Na⁺/glucose transporter. The high conductance and high selectivity of the H⁺-induced current suggest that protons may be conducted via a hydrogen-bonded chain (a “proton-wire mechanism”) formed at least partially by side chains within the transporter. In addition, pH affects other conducting states of rat serotonin transporter. Acidic pH potentiates the 5-HT-induced, transport-associated current and inhibits the hyperpolarization-activated transient current. The dose–response relationships for these two effects suggest that two H⁺ binding sites, with pK_a values close to 5.1 and close to 6.3, govern the potentiation of the 5-HT-induced current and the inhibition of the transient current, respectively. These results are important for developing structure-function models that explain permeation properties of neurotransmitter transporters

H⁺ Permeation and pH Regulation at a Mammalian Serotonin Transporter

The rat serotonin transporter expressed in Xenopusoocytes displays an inward current in the absence of 5-HT when external pH is lowered to 6.5 or below. The new current differs from the leakage current described previously in two ways. (1) It is ∼10-fold larger at pH 5 than the leakage current at pH 7.5 and reaches 1000 H⁺/sec per transporter at extremes of voltage and pH with no signs of saturation. (2) It is selective for H⁺ by reversal potential measurements. Similar H⁺-induced currents are also observed in several other ion-coupled transporters, including the GABA transporter, the dopamine transporter, and the Na⁺/glucose transporter. The high conductance and high selectivity of the H⁺-induced current suggest that protons may be conducted via a hydrogen-bonded chain (a “proton-wire mechanism”) formed at least partially by side chains within the transporter. In addition, pH affects other conducting states of rat serotonin transporter. Acidic pH potentiates the 5-HT-induced, transport-associated current and inhibits the hyperpolarization-activated transient current. The dose–response relationships for these two effects suggest that two H⁺ binding sites, with pK_a values close to 5.1 and close to 6.3, govern the potentiation of the 5-HT-induced current and the inhibition of the transient current, respectively. These results are important for developing structure-function models that explain permeation properties of neurotransmitter transporters

A novel assay based on DNA melting temperature for multiplexed identification of SARS-CoV-2 and influenza A/B viruses

IntroductionThe severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and influenza viruses can cause respiratory illnesses with similar clinical symptoms, making their differential diagnoses challenging. Additionally, in critically ill SARS-CoV-2–infected patients, co-infections with other respiratory pathogens can lead to severe cytokine storm and serious complications. Therefore, a method for simultaneous detection of SARS-CoV-2 and influenza A and B viruses will be clinically beneficial.MethodsWe designed an assay to detect five gene targets simultaneously via asymmetric PCR-mediated melting curve analysis in a single tube. We used specific probes that hybridize to corresponding single-stranded amplicons at low temperature and dissociate at high temperature, creating different detection peaks representing the targets. The entire reaction was conducted in a closed tube, which minimizes the risk of contamination. The limit of detection, specificity, precision, and accuracy were determined.ResultsThe assay exhibited a limit of detection of <20 copies/μL for SARS-CoV-2 and influenza A and <30 copies/μL for influenza B, with high reliability as demonstrated by a coefficient of variation for melting temperature of <1.16% across three virus concentrations. The performance of our developed assay and the pre-determined assay showed excellent agreement for clinical samples, with kappa coefficients ranging from 0.98 (for influenza A) to 1.00 (for SARS-CoV-2 and influenza B). No false-positive, and no cross-reactivity was observed with six common non-influenza respiratory viruses.ConclusionThe newly developed assay offers a straightforward, cost-effective and nucleic acid contamination-free approach for simultaneous detection of the SARS-CoV-2, influenza A, and influenza B viruses. The method offers high analytical sensitivity, reliability, specificity, and accuracy. Its use will streamline testing for co-infections, increase testing throughput, and improve laboratory efficacy

A genetic study and meta-analysis of the genetic predisposition of prostate cancer in a Chinese population.

Prostate cancer predisposition has been extensively investigated in European populations, but there have been few studies of other ethnic groups. To investigate prostate cancer susceptibility in the under-investigated Chinese population, we performed single-nucleotide polymorphism (SNP) array analysis on a cohort of Chinese cases and controls and then meta-analysis with data from the existing Chinese prostate cancer genome-wide association study (GWAS). Genotyping 211,155 SNPs in 495 cases and 640 controls of Chinese ancestry identified several new suggestive Chinese prostate cancer predisposition loci. However, none of them reached genome-wide significance level either by meta-analysis or replication study. The meta-analysis with the Chinese GWAS data revealed that four 8q24 loci are the main contributors to Chinese prostate cancer risk and the risk alleles from three of them exist at much higher frequencies in Chinese than European populations. We also found that several predisposition loci reported in Western populations have different effect on Chinese men. Therefore, this first extensive single-nucleotide polymorphism study of Chinese prostate cancer in comparison with European population indicates that four loci on 8q24 contribute to a great risk of prostate cancer in a considerable large proportion of Chinese men. Based on those four loci, the top 10% of the population have six- or two-fold prostate cancer risk compared with men of the bottom 10% or median risk respectively, which may facilitate the design of prostate cancer genetic risk screening and prevention in Chinese men. These findings also provide additional insights into the etiology and pathogenesis of prostate cancer.This work was conducted on behalf of the CHIPGECS and The PRACTICAL consortia (see Supplementary Consortia). We acknowledge the contribution of doctors, nurses and postgraduate research students at the CHIPGENCS sample collecting centers. We thank Orchid and Rosetrees for funding support. This work was also supported by National Natural Science foundation of China for funding support to H Zhang (Grant No: 30671793 and 81072377), N Feng (Grant No: 81272831), X Zhang (Grant No: 30572139, 30872924 and 81072095), S Zhao (Grant No: 81072092 and 81328017), Y Yu (Grant No: 81172448) and Program for New Century Excellent Talents in University from Department of Education of China (NCET-08-0223) and the National High Technology Research and Development Program of China (863 Program 2012AA021101) to X Zhang.This is the final version of the article. It first appeared from Impact Journals via http://dx.doi.org/10.18632/oncotarget.725