Detection and typing of herpes simplex DNA in genital swabs by real-time polymerase chain reaction

The LightCycler polymerase chain reaction (PCR) is a sensitive assay for the detection of Herpes simplex virus (HSV) DNA in muco-cutaneous swabs. Software-based analysis of the probe melting temperature (Tm) can be used to discriminate between HSV types (HSV-1 and HSV-2). Among 76 HSV DNA positive genital swabs, atypical Tms were observed in 14 (18%). The 14 samples were all typed as HSV-2 by sequence alignment. In 4/14 samples, the atypical Tm was associated with sequence variation at the probe-binding site. Among 10 samples with conserved sequences, Tms were influenced by the specimen preparation method prior to PCR. These findings indicate that multiple factors including, but not limited to sequence variation complicate melting curve analysis following real-time PCR. Alternative typing methods are recommended for specimens with atypical melting curves.     

Anti-Tumor Activity of Four Ayurvedic Herbs in Dalton Lymphoma Ascites Bearing Mice and Their Short-Term In Vitro Cytotoxicity on DLA-Cell-Line

The anti-tumor activity and chemopreventive potential of four Ayurvedic herbs viz. Curcuma longa L., Ocimum sanctum L., Tinospora cordifolia (Wild) Miers ex Hook.f & Thomas and Zizyphus mauritiana Lam. were evaluated using Dalton Lymphoma ascites (DLA) tumor model in Swiss Albino mice. The outcome was assessed using survival time, peritoneal ascitic fluid (Tumor volume) and hematological indices as parameters. Animals were divided into five groups (n = 6) viz. one DLA control and four Herb + DLA treated groups. All the four herb + DLA groups were pre-treated with respective herbs for 7 days and hematological indices were measured for entire five groups. On day-8 animals were inoculated with 1×10⁶ DLA cells i.p., and Herb + DLA groups were continued with oral herbal treatment for 21-days. Hematological parameters and tumor volume were assessed to find the effects of herbs. Short term in vitro cytotoxicity was determined by Trypan Blue exclusion method and LDH leakage assay using different concentrations of herbal extracts and 5-FU as a positive control and IC₅₀ for each herbal extract and 5-FU were determined. Oral administration of crude herb increased the survival time and decreased the peritoneal ascitic fluid content significantly. Hb, RBCs and total WBC which were altered by DLA inoculation were restored significantly by all the herbs except O. sanctum. All the four herbs showed in vitro cytotoxic activity against DLA cell-line. Moreover inter group comparison of all the four herbs for anti-tumor activity showed efficacy in the following order- T. cordifolia > Z. mauritiana ≥ C. longa > O. sanctum respectively.     

Continuous Glucose Monitoring Use in Clinical Trials for On-Market Diabetes Drugs

To the best of our knowledge, there are no published data on the historical and recent use of CGM in clinical trials of pharmacological agents used in the treatment of diabetes. We analyzed 2,032 clinical trials of 40 antihyperglycemic therapies currently on the market with a study start date between 1 January 2000 and 31 December 2019. According to ClinicalTrials.gov, 119 (5.9%) of these trials used CGM. CGM usage in clinical trials has increased over time, rising from <5% before 2005 to 12.5% in 2019. However, it is still low given its inclusion in the American Diabetes Association’s latest guidelines and known limitations of A1C for assessing ongoing diabetes care.

The availability of reliable continuous glucose monitoring (CGM) systems has proven to be a major innovation in diabetes management and research. Most current CGM systems are approved for 7- to 14-day use and use a wire-tipped glucose oxidase sensor inserted in subcutaneous tissue to monitor glucose concentrations in interstitial fluid. One implanted CGM system is approved for longer-term use (90–180 days); it operates with fluorescence-based technology. CGM sensors record a glucose data point every 1–15 minutes (depending on the system), collecting far more granular data and information on glycemic patterns than self-monitoring of blood glucose (SMBG) alone. Real-time CGM or intermittently scanned CGM systems send data continuously or intermittently to dedicated receivers or smartphones, whereas professional CGM systems provide retrospective data, either blinded or unblinded, for analysis and can be used to identify patterns of hypo- and hyperglycemia. Professional CGM can be helpful to evaluate patients when other CGM systems are not available to the patient or the patient prefers a blinded analysis or a shorter experience with unblinded data.In the 20 years since CGM systems first became available to people with diabetes, technological improvements, particularly pertaining to accuracy and form factor, have made CGM increasingly viable for both patient use and clinical investigation (1,2). Average sensor MARD (mean absolute relative difference; a summary accuracy statistic) has decreased from >20 to <10% (3–10), including two systems that do not require fingerstick calibrations and three that are approved to be used for insulin dosing (11). Concurrently, size, weight, and cost of CGM systems have all decreased, while user-friendliness and convenience have increased (12).To encourage use of CGM-derived data, researchers and clinicians have worked to develop a standard set of glycemic metrics beyond A1C. In 2017, two international groups of leading diabetes clinical and research organizations published consensus definitions for key metrics, including clinically relevant glycemic cut points for hypoglycemia (<70 and <54 mg/dL), hyperglycemia (>180 and >250 mg/dL), and time in range (TIR; 70–180 mg/dL) (13,14).CGM-derived metrics provide far greater precision and granularity than is possible with SMBG or A1C data alone (Table 1), enabling clinicians and investigators to better represent inter- and intraday glycemic differences with metrics such as TIR, glycemic variability, and time in hypoglycemia and hyperglycemia (15). Crucially, CGM also allows for the accurate measurement and detection of nocturnal glycemia (16). The use of these metrics enables a more comprehensive understanding of glycemic management that can facilitate individualized treatment for people with diabetes or prediabetes. Although A1C is a useful estimate of mean glucose over the previous 2–3 months, especially when evaluating population health, it is important to include other glycemic outcomes in clinical trials. Furthermore, there is emerging evidence suggesting that TIR predicts the development of microvascular complications at least as well as A1C (17,18).TABLE 1Benefits of CGM Compared With A1C Alone in Assessing Glycemia

Lipid-altering therapy and atrial fibrillation

Atrial fibrillation (AF) is a common cardiac arrhythmia with significant morbidity and public health cost. Because of limitations of efficacy and safety of conventional antiarrhythmic agents, alternative therapies for AF are needed. The potential antiarrhythmic properties of lipid-altering therapy, including the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors and fish oils, are increasingly recognized, particularly in light of their potential anti-inflammatory properties. This review examines the known effects of lipid-altering therapy on atrial arrhythmias in both experimental and clinical settings. Inflammatory states, such as post-cardiac surgery and AF of recent onset, show promise as targets. In contrast, lipid-lowering therapy is less likely to affect longstanding persistent AF. Current recommendations for the use of lipid-altering therapy for prevention and treatment of AF are summarized.     

Molecular basis of multistep voltage activation in plant two-pore channel 1

Voltage-gated ion channels confer excitability to biological membranes, initiating and propagating electrical signals across large distances on short timescales. Membrane excitation requires channels that respond to changes in electric field and couple the transmembrane voltage to gating of a central pore. To address the mechanism of this process in a voltage-gated ion channel, we determined structures of the plant two-pore channel 1 at different stages along its activation coordinate. These high-resolution structures of activation intermediates, when compared with the resting-state structure, portray a mechanism in which the voltage-sensing domain undergoes dilation and in-membrane plane rotation about the gating charge–bearing helix, followed by charge translocation across the charge transfer seal. These structures, in concert with patch-clamp electrophysiology, show that residues in the pore mouth sense inhibitory Ca²⁺ and are allosterically coupled to the voltage sensor. These conformational changes provide insight into the mechanism of voltage-sensor domain activation in which activation occurs vectorially over a series of elementary steps.

Voltage-gated ion channels (VGICs) use voltage-sensing domains (VSDs) to sense changes in electrical potential across biological membranes (1, 2). VSDs are composed of a four-helix bundle, in which one helix carries charged residues that move in response to changes in transmembrane electric field (3, 4). VSDs usually adopt a “resting state” when the membrane is at “resting potential”: ∼ −80 mV for animal and ∼ −150 mV for plant plasma membranes (5). In comparison, much lower resting membrane voltages are set for intracellular endo-membranes: ∼ −30 mV across the plant vacuole (6) and mammalian lysosome (7). As the membrane potential vanishes during depolarization, so does the downward electrostatic force on the cationic side chains causing them to relax toward the outside of the membrane across a hydrophobic constriction site (HCS) or hydrophobic seal (8). This conformational change is conveyed to the central pore, formed by four pore domains in a quasi-fourfold arrangement, which dilates to allow the diffusion of ions down their electrochemical gradients. The exact nature of the conformational change in VSDs has been the subject of decades of biophysical investigation (9–12), though to this date, only a few structural examples exist of voltage sensors in resting or multiple conformations (13–18).Two-pore channels (TPCs) are defined by their two tandem Shaker-like cassette subunits in a single polypeptide chain, which dimerize to form a C2-symmetric channel with four subunits and 24 (4 × 6) transmembrane helices (19–21). There are three TPC channels, TPC1, 2, and 3, each with different voltage or ligand gating and ion selectivity. Among the voltage-gated TPCs (all except lipid-gated TPC2), only the second VSD (VSD2) is electrically active (18, 22–24), while VSD1 is insensitive to voltage changes and is likely static under all changes in potential.In plants, the vacuole comprises up to 90% of the plant cell volume and provides for a dynamic storage organelle that, in addition to metabolites, is a repository for ions including Ca²⁺. TPC1 channels confer excitability to this intracellular organelle (25) and, unlike other TPCs, are calcium regulated: external Ca²⁺ (in the vacuolar lumen) inhibits the channel by binding to multiple luminal sites, while cytosolic Ca²⁺ is required to open the channel by binding to EF hands, although the exact mechanism by which this activation occurs is unknown (22). These electrical properties allowed our group and Youxing Jiang’s group to determine the first structure of an electrically resting VGIC by cocrystallizing the channel with 1 mM Ca²⁺, which maintains the VSD in a resting configuration at 0 mV potential (16, 22).Previously (17), we used a gain-of-function mutant of AtTPC1 with three luminal Ca²⁺-binding acidic residues on VSD2 neutralized (D240N/D454N/E528N) termed AtTPC1_DDE (abbreviated here as DDE) to visualize channel activation at the level of atomic structure, but we were unable to sufficiently resolve details of the electrically active VSD2 due to structural heterogeneity. In addition, the intracellular activation gate remained closed. We now present multiple structures of intermediately activated states of AtTPC1 determined by extensive image processing. In order to visualize such states, we modulated the channel’s luminal Ca²⁺ sensitivity using a well-studied gain-of-function single-point mutant, D454N (fou2), and also the triple mutant DDE for comparison. fou2 is known to desensitize the channel to inhibitory, external (luminal) calcium ions (26, 27). Mutations in D454 and closely related luminal Ca²⁺-binding carboxyls to alanine D240A, D454A, E528A (termed AtTPC1ΔCa_i) were previously shown to effectively attenuate the Ca²⁺-induced shift of the voltage activation threshold of AtTPC1 to depolarizing potentials at high luminal Ca²⁺ (28).The fou2 and DDE mutations lie in the coordination sphere of the inhibitory Ca²⁺ site on the luminal side of the VSD2–pore interface formed by D454, D240, and E528. The D454N mutation in the fou2 channel enhances the defense capacity of plants against fungal or herbivore attack due to increased production of the wounding hormone jasmonate (29). These effects on plant performance and defense are probably due to short circuiting of the vacuolar membrane (26, 27, 30) in which TPC1 has increased open probability at resting potential. Compared to wild-type (WT) TPC1, the activation threshold in the fou2 channel is shifted to more negatively polarized potentials and also has significantly lower sensitivity to inhibitory Ca²⁺ in addition to exhibiting faster activation kinetics than its WT counterpart that was originally named the “slow vacuolar” (SV) channel due to its slow conductance onset (20, 21, 30). Therefore, D454N confers more than just reduced sensitivity to external Ca²⁺ but intrinsic hyperactivity as well. Our structures of these AtTPC1 mutants attempt to explain how the voltage sensor functions during electrical activation and how exactly luminal Ca²⁺ affects this process.