Characterization of Glutathione Dithiophosphates as Long-Acting H2S Donors.

Considering the important cytoprotective and signaling roles but relatively narrow therapeutic index of hydrogen sulfide (H2S), advanced H2S donors are required to achieve a therapeutic effect. In this study, we proposed glutathione dithiophosphates as new combination donors of H2S and glutathione. The kinetics of H2S formation in dithiophosphate solutions suggested a continuous H2S release by the donors, which was higher for the dithiophosphate of reduced glutathione than oxidized glutathione. The compounds, unlike NaHS, inhibited the proliferation of C2C12 myoblasts at submillimolar concentrations due to an efficient increase in intracellular H2S. The H2S donors more profoundly affected reactive oxygen species and reduced glutathione levels in C2C12 myocytes, in which these parameters were elevated compared to myoblasts. Oxidized glutathione dithiophosphate as well as control donors exerted antioxidant action toward myocytes, whereas the effect of reduced glutathione dithiophosphate at (sub-)micromolar concentrations was rather modulating. This dithiophosphate showed an enhanced negative inotropic effect mediated by H2S upon contraction of the atrial myocardium, furthermore, its activity was prolonged and reluctant for washing. These findings identify glutathione dithiophosphates as redox-modulating H2S donors with long-acting profile, which are of interest for further pharmacological investigation.

Enhancing Structural Rigidity via a Strategy Involving Protons for Creating Water-Resistant Mn4+-Doped Fluoride Phosphors.

The poor water resistance property of a commercial Mn4+-activated narrow-band red-emitting fluoride phosphor restricts its promising applications in high-performance white LEDs and wide-gamut displays. Herein, we develop a structural rigidity-enhancing strategy using a novel KHF2:Mn4+ precursor as a Mn source to construct a proton-containing water-resistant phosphor K2(H)TiF6:Mn4+ (KHTFM). The parasitic [HMnF6]- complexes in the interstitial site from the fall off the KHF2:Mn4+ are also transferred to the K2TiF6 host by ion exchange to form KHTFM with rigid bonding networks, improving the water resistance and thermostability of the sample. The KHTFM sample retains at least 92% of the original emission value after 180 min of water immersion, while the non-water-resistant K2TiF6:Mn4+(KTFM) phosphor maintains only 23%. Therefore, these findings not only illustrate the effect of protons on fluoride but also provide a novel insight into commercial water-resistant fluoride phosphors.

Efficacy and Safety of a 3-Antigen (Pre-S1/Pre-S2/S) Hepatitis B Vaccine: Results of a Phase 3 Randomized Clinical Trial in the Russian Federation.

BACKGROUND: This study compares the immunogenicity and safety of a 3-antigen (S/pre-S1/pre-S2) hepatitis B (HepB) vaccine (3AV), to a single antigen vaccine (1AV) in adults to support the registration of 3AV in Russia. METHODS: We conducted a randomized, double-blind, comparative study of 3-dose regimens of 3AV (10 µg) and 1AV (20 µg) in adults aged 18-45 years. We evaluated immunogenicity based on hepatitis B surface (HBs) antibody titers at days 1, 28, 90, 180, and 210, adverse and serious adverse events (SAEs) to study day 210. The primary outcome was based on the difference in rates of seroconversion at day 210 (lower bound 95% confidence interval [CI]: > - 4%). Secondary outcomes were seroprotection rates (SPR), defined as anti-HBs ≥10 mIU/mL and anti-HBs geometric mean concentration (GMC). RESULTS: Rate of seroconversion in 3AV (100%) was noninferior to 1AV (97.9%) at study day 210 (difference: 2.1%, 95% CI: -2.0, 6.3%]) but significantly higher at study day 28. SPR at study day 210 was >97% in both arms. Anti-HBs titers were significantly higher at study days 90 (P = .001) and 180 (P = .0001) with 3AV. Sex, age, and body mass index (BMI) had no impact on anti-HBs titers. The rates of local reactions related to vaccination were similar between vaccine arms (3AV vs 1AV) after the first (30% vs 18.8%, P = .15), second (20.0% vs 14.6%, P = .33), and third vaccination (14.9% vs 23.4%, P = .22). No SAEs were reported. CONCLUSIONS: 3AV was noninferior to 1AV. 3AV induced high SPR, and there were no safety concerns. Clinical Trials Registration. NCT04209400.

Alcohol metabolite acetic acid activates BK channels in a pH-dependent manner and decreases calcium oscillations and exocytosis of secretory granules in rat pituitary GH3 cells.

Acetaldehyde and acetic acid/acetate, the active metabolites of alcohol (ethanol, EtOH), generate actions of their own ranging from behavioral, physiological, to pathological/cancerogenic effects. EtOH and acetaldehyde have been studied to some depth, whereas the effects of acetic acid have been less well explored. In this study, we investigated the effect of acetic acid on big conductance calcium-activated potassium (BK) channels present in GH3 rat pituitary tumor cells in more detail. In whole cell voltage clamp recordings, extracellular application of acetic acid increased total outward currents in a dose-dependent manner. This effect was prevented after the application of the specific BK channel blocker paxilline. Acetic acid action was pH-dependent-in whole cell current and single BK channel recordings, open probability (Po) was significantly increased by extracellular pH reduction and decreased by neutral or base pH. Acetic acid hyperpolarized the membrane potential, whereas acidic physiological solution had a depolarizing effect. Moreover, acetic acid reduced calcium (Ca2+) oscillations and exocytosis of growth hormone contained secretory granules from GH3 cells. These effects were partially prevented by BK inhibitors-tetraethylammonium or paxillin. In conclusion, our experiments indicate that acetic acid activates BK channels in GH3 cells which eventually contribute to acetic acid-induced membrane hyperpolarization, cessation of Ca2+ oscillations, and decrease of growth hormone release.

Hydrogen Sulfide Alleviates Anxiety, Motor, and Cognitive Dysfunctions in Rats with Maternal Hyperhomocysteinemia via Mitigation of Oxidative Stress.

Hydrogen sulfide (H2S) is endogenously produced from sulfur containing amino acids, including homocysteine and exerts neuroprotective effects. An increase of homocysteine during pregnancy impairs fetal growth and development of the offspring due to severe oxidative stress. We analyzed the effects of the H2S donor-sodium hydrosulfide (NaHS) administered to female rats with hyperhomocysteinemia (hHcy) on behavioral impairments and levels of oxidative stress of their offspring. Rats born from females fed with control or high methionine diet, with or without H2S donor injections were investigated. Rats with maternal hHcy exhibit increased levels of total locomotor activity and anxiety, decreased muscle endurance and motor coordination, abnormalities of fine motor control, as well as reduced spatial memory and learning. Oxidative stress in brain tissues measured by activity of glutathione peroxidases and the level of malondialdehyde was higher in rats with maternal hHcy. Concentrations of H2S and the activity and expression of the H2S generating enzyme-cystathionine-beta synthase-were lower compared to the control group. Administration of the H2S donor to females with hHcy during pregnancy prevented behavioral alterations and oxidative stress of their offspring. The acquisition of behavioral together with biochemical studies will add to our knowledge about homocysteine neurotoxicity and proposes H2S as a potential agent for therapy of hHcy associated disorders.

Phase Transformation of a K2GeF6 Polymorph for Phosphors Driven by a Simple Precipitation-Dissolution Equilibrium and Ion Exchange.

Tuning crystal phase transformations is very important for obtaining polymorphs for phosphors with the ideal optical properties and stability. Mn4+-doped K2GeF6 (KGF) is a typical polymorphic phosphor, but the thermodynamic and kinetic mechanism of its phase transformation is still unclear. Herein, the phase transformation of polymorphs varying from P63mc KGF and trigonal KGF to P63mc Si4+-doped KGF is realized by introducing the synergistic action of an HF solution and Si4+ ions. The full structural refinements of KGF polymorphs at room temperature and the electronic band structure calculations were performed. The results show that the Si4+-doped hexagonal KGF polymorph with good photoluminescence properties is the most stable phase according to the calculated total energy landscape and relative formation energy. The morphologic changes were monitored in situ to clearly understand the rapid phase transformation mechanism, which proves that the phase transformation is driven by a simple precipitation-dissolution equilibrium and ionic exchange.

Prenatal hyperhomocysteinemia induces oxidative stress and accelerates 'aging' of mammalian neuromuscular synapses.

Enhanced levels of homocysteine during pregnancy induce oxidative stress and contribute to many age-related diseases. In this study, we analyzed age-dependent synaptic modifications in developing neuromuscular synapses of rats with prenatal hyperhomocysteinemia (hHCY). One of the main findings indicate that the intensity and the timing of transmitter release in synapses of neonatal (P6 and P10) hHCY rats acquired features of matured synaptic transmission of adult rats. The amplitude and frequency of miniature end-plate currents (MEPCs) and evoked transmitter release were higher in neonatal hHCY animals compared to the control group. Analysis of the kinetics of neurotransmitter release demonstrated more synchronized release in neonatal rats with hHCY. At the same time lower release probability was observed in adults with hHCY. Spontaneous transmitter release in neonates with hHCY was inhibited by hydrogen peroxide (H2O2) whereas in controls this oxidant was effective only in adult animals indicating a higher susceptibility of motor nerve terminals to oxidative stress. The morphology and the intensity of endocytosis of synaptic vesicles in motor nerve endings was assessed using the fluorescence dye FM 1-43. Adult-like synapses were found in neonates with hHCY which were characterized by a larger area of presynaptic terminals compared to controls. No difference in the intensity of FM 1-43 fluorescence was observed between two groups of animals. Prenatal hHCY resulted in reduced muscle strength assessed by the Paw Grip Endurance test. Using biochemical assays we found an increased level of H2O2 and lipid peroxidation products in the diaphragm muscles of hHCY rats. This was associated with a lowered activity of superoxide dismutase and glutathione peroxidase. Our data indicate that prenatal hHCY induces oxidative stress and apparent faster functional and morphological "maturation" of motor synapses. Our results uncover synaptic mechanisms of disrupted muscle function observed in hHCY conditions which may contribute to the pathogenesis of motor neuronal diseases associated with enhanced level of homocysteine.

Simulation of patient flow in multiple healthcare units using process and data mining techniques for model identification.

INTRODUCTION: An approach to building a hybrid simulation of patient flow is introduced with a combination of data-driven methods for automation of model identification. The approach is described with a conceptual framework and basic methods for combination of different techniques. The implementation of the proposed approach for simulation of the acute coronary syndrome (ACS) was developed and used in an experimental study. METHODS: A combination of data, text, process mining techniques, and machine learning approaches for the analysis of electronic health records (EHRs) with discrete-event simulation (DES) and queueing theory for the simulation of patient flow was proposed. The performed analysis of EHRs for ACS patients enabled identification of several classes of clinical pathways (CPs) which were used to implement a more realistic simulation of the patient flow. The developed solution was implemented using Python libraries (SimPy, SciPy, and others). RESULTS: The proposed approach enables more a realistic and detailed simulation of the patient flow within a group of related departments. An experimental study shows an improved simulation of patient length of stay for ACS patient flow obtained from EHRs in Almazov National Medical Research Centre in Saint Petersburg, Russia. CONCLUSION: The proposed approach, methods, and solutions provide a conceptual, methodological, and programming framework for the implementation of a simulation of complex and diverse scenarios within a flow of patients for different purposes: decision making, training, management optimization, and others.

Age-Dependent, Subunit Specific Action of Hydrogen Sulfide on GluN1/2A and GluN1/2B NMDA Receptors.

Hydrogen sulfide (H2S) is an endogenously produced neuroactive gas implicated in many key processes in the peripheral and central nervous system. Whereas the neuroprotective role of H2S has been shown in adult brain, the action of this messenger in newborns remains unclear. One of the known targets of H2S in the nervous system is the N-methyl-D-aspartate (NMDA) glutamate receptor which can be composed of different subunits with distinct functional properties. In the present study, using patch clamp technique, we compared the effects of the H2S donor sodium hydrosulfide (NaHS, 100 µM) on hippocampal NMDA receptor mediated currents in rats of the first and third postnatal weeks. This was supplemented by testing effects of NaHS on recombinant GluN1/2A and GluN1/2B NMDA receptors expressed in HEK293T cells. The main finding is that NaHS action on NMDA currents is age-dependent. Currents were reduced in newborns but increased in older juvenile rats. Consistent with an age-dependent switch in NMDA receptor composition, in HEK239T cells expressing GluN1/2A receptors, NaHS increased NMDA activated currents associated with acceleration of desensitization and decrease of the deactivation rate. In contrast, in GluN1/2B NMDA receptors, which are prevalent in newborns, NaHS decreased currents and reduced receptor deactivation without effect on the desensitization rate. Adenylate cyclase inhibitor MDL-12330A (10 µM) did not prevent the age-dependent effects of NaHS on NMDA evoked currents in pyramidal neurons of hippocampus. The reducing agent dithiothreitol (DTT, 2 mM) applied on HEK293T cells prevented facilitation induced by NaHS on GluN1/2A NMDA receptors, however in GluN1/2B NMDA receptors the inhibitory effect of NaHS was still observed. Our data indicate age-dependent effect of H2S on NMDA receptor mediated currents determined by glutamate receptor subunit composition. While the inhibitory action of H2 on GluN1/2B receptors could limit the excessive activation in early age, the enhanced functionality of GluN1/2A receptor in the presence of this gasotransmitter can enlarge synaptic efficacy and promote synaptic plasticity in adults.

Receptor Mechanisms Mediating the Pro-Nociceptive Action of Hydrogen Sulfide in Rat Trigeminal Neurons and Meningeal Afferents.

Hydrogen sulfide (H2S), a well-established member of the gasotransmitter family, is involved in a variety of physiological functions, including pro-nociceptive action in the sensory system. Although several reports have shown that H2S activates sensory neurons, the molecular targets of H2S action in trigeminal (TG) nociception, implicated in migraine, remains controversial. In this study, using suction electrode recordings, we investigate the effect of the H2S donor, sodium hydrosulfide (NaHS), on nociceptive firing in rat meningeal TG nerve fibers. The effect of NaHS was also explored with patch-clamp and calcium imaging techniques on isolated TG neurons. NaHS dramatically increased the nociceptive firing in TG nerve fibers. This effect was abolished by the TRPV1 inhibitor capsazepine but was partially prevented by the TRPA1 blocker HC 030031. In a fraction of isolated TG neurons, NaHS transiently increased amplitude of capsaicin-induced currents. Moreover, NaHS by itself induced inward currents in sensory neurons, which were abolished by the TRPV1 inhibitor capsazepine suggesting involvement of TRPV1 receptors. In contrast, the inhibitor of TRPA1 receptors HC 030031 did not prevent the NaHS-induced currents. Imaging of a large population of TG neurons revealed that NaHS induced calcium transients in 41% of tested neurons. Interestingly, this effect of NaHS in some neurons was inhibited by the TRPV1 antagonist capsazepine whereas in others it was sensitive to the TRPA1 blocker HC 030031. Our data suggest that both TRPV1 and TRPA1 receptors play a role in the pro-nociceptive action of NaHS in peripheral TG nerve endings in meninges and in somas of TG neurons. We propose that activation of TRPV1 and TRPA1 receptors by H2S during neuro-inflammation conditions contributes to the nociceptive firing in primary afferents underlying migraine pain.

Hydrogen sulfide inhibits giant depolarizing potentials and abolishes epileptiform activity of neonatal rat hippocampal slices.

Hydrogen sulfide (H2S) is an endogenous gasotransmitter with neuroprotective properties that participates in the regulation of transmitter release and neuronal excitability in various brain structures. The role of H2S in the growth and maturation of neural networks however remains unclear. The aim of the present study is to reveal the effects of H2S on neuronal spontaneous activity relevant to neuronal maturation in hippocampal slices of neonatal rats. Sodium hydrosulfide (NaHS) (100µM), a classical donor of H2S produced a biphasic effect with initial activation and subsequent concentration-dependent suppression of network-driven giant depolarizing potentials (GDPs) and neuronal spiking activity. Likewise, the substrate of H2S synthesis l-cysteine (1mM) induced an initial increase followed by an inhibition of GDPs and spiking activity. Our experiments indicate that the increase in initial discharge activity by NaHS is evoked by neuronal depolarization which is partially mediated by a reduction of outward K+ currents. The subsequent decrease in the neuronal activity by H2S appears to be due to the rightward shift of activation and inactivation of voltage-gated Na+ currents, thus preventing network activity. NaHS also reduced N-methyl-d-aspartate (NMDA)-mediated currents, without essential effect on AMPA/kainate or GABAA-mediated currents. Finally, H2S abolished the interictal-like events induced by bicuculline. In summary, our results suggest that through the inhibitory action on voltage-gated Na+ channels and NMDA receptors, H2S prevents the enhanced neuronal excitability typical to early hippocampal networks.

Homocysteine augments BK channel activity and decreases exocytosis of secretory granules in rat GH3 cells.

In this study, we investigated the effects of L-homocysteine (Hcy) on maxi calcium-activated potassium (BK) channels and on exocytosis of secretory granules in GH3 rat pituitary-derived cells. A major finding of our study indicates that short-term application of Hcy increased the open probability of oxidized BK channels in inside-out recordings. Whole-cell recordings show that extracellular Hcy also augmented BK currents during long-term application. Furthermore, Hcy decreased the exocytosis of secretory granules. This decrease was partially prevented by the BK channel inhibitor paxilline and fully prevented by N-acetylcysteine, a reactive oxygen species scavenger. Taken together, our data show that elevation of cellular Hcy level induces oxidative stress, increases BK channel activity, and decreases exocytosis of secretory granules. These findings may provide insight into some of the developmental impairments and neurotoxicity associated with Hyperhomocysteinemia (HHcy), a disease arising due to abnormally elevated levels of Hcy in the plasma.

Hydrogen sulfide induces hyperpolarization and decreases the exocytosis of secretory granules of rat GH3 pituitary tumor cells.

The aim of the present study was to evaluate the effects of hydrogen sulfide (H2S) on the membrane potential, action potential discharge and exocytosis of secretory granules in neurosecretory pituitary tumor cells (GH3). The H2S donor - sodium hydrosulfide (NaHS) induced membrane hyperpolarization, followed by truncation of spontaneous electrical activity and decrease of the membrane resistance. The NaHS effect was dose-dependent with an EC50 of 152 µM (equals effective H2S of 16-19 µM). NaHS effects were not altered after inhibition of maxi conductance calcium-activated potassium (BK) channels by tetraethylammonium or paxilline, but were significantly reduced after inhibition or activation of ATP-dependent potassium channels (KATP) by glibenclamide or by diazoxide, respectively. In whole-cell recordings NaHS increased the amplitude of KATP currents, induced by hyperpolarizing pulses and subsequent application of glibenclamide decreased currents to control levels. Using the fluorescent dye FM 1-43 exocytosis of secretory granules was analyzed in basal and stimulated conditions (high K(+) external solution). Prior application of NaHS decreased the fluorescence of the cell membrane in both conditions which links with activation of KATP currents (basal secretion) and activation of KATP currents and BK-currents (stimulated exocytosis). We suggest that H2S induces hyperpolarization of GH3 cells by activation of KATP channels which results in a truncation of spontaneous action potentials and a decrease of hormone release.

Cell-penetrating nanobiosensors for pointillistic intracellular Ca2+-transient detection.

Small-molecule chemical calcium (Ca(2+)) indicators are invaluable tools for studying intracellular signaling pathways but have severe shortcomings for detecting local Ca(2+) entry. Nanobiosensors incorporating functionalized quantum dots (QDs) have emerged as promising alternatives but their intracellular use remains a major challenge. We designed cell-penetrating FRET-based Ca(2+) nanobiosensors for the detection of local Ca(2+) concentration transients, using commercially available CANdot565QD as a donor and CaRuby, a custom red-emitting Ca(2+) indicator, as an acceptor. With Ca(2+)-binding affinities covering the range of 3-20 µM, our CaRubies allow building sensors with a scalable affinity for detecting intracellular Ca(2+) transients at various concentrations. To facilitate their cytoplasmic delivery, QDs were further functionalized with a small cell-penetrating peptide (CPP) derived from hadrucalcin (HadUF1-11: H11), a ryanodine receptor-directed scorpion toxin identified within the venom of Hadrurus gertschi. Efficient internalization of QDs doubly functionalized with PEG5-CaRuby and H11 (in a molar ratio of 1:10:10, respectively) is demonstrated. In BHK cells expressing a N-methyl-d-aspartate receptor (NMDAR) construct, these nanobiosensors report rapid intracellular near-membrane Ca(2+) transients following agonist application when imaged by TIRF microscopy. Our work presents the elaboration of cell-penetrating FRET-based nanobiosensors and validates their function for detection of intracellular Ca(2+) transients.

Calcium rubies: a family of red-emitting functionalizable indicators suitable for two-photon Ca2+ imaging.

We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca(2+)) indicators as new tools for biological Ca(2+) imaging. The specificity of this Ca(2+)-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca(2+)-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 µM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca(2+) uncaging or optogenetic stimulation with Ca(2+) imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca(2+) transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca(2+)-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca(2+) chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca(2+)-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca(2+) concentrations are feasible.

Quantum dot peptide biosensors for monitoring caspase 3 proteolysis and calcium ions.

The nanoscale size and unique optical properties of semiconductor quantum dots (QDs) have made them attractive as central photoluminescent scaffolds for a variety of biosensing platforms. In this report we functionalize QDs with dye-labeled peptides using two different linkage chemistries to yield Förster resonance energy transfer (FRET)-based sensors capable of monitoring either enzymatic activity or ionic presence. The first sensor targets the proteolytic activity of caspase 3, a key downstream effector of apoptosis. This QD conjugate utilized carbodiimide chemistry to covalently link dye-labeled peptide substrates to the terminal carboxyl groups on the QD's surface hydrophilic ligands in a quantitative manner. Caspase 3 cleaved the peptide substrate and disrupted QD donor-dye acceptor FRET providing signal transduction of enzymatic activity and allowing derivation of relevant Michaelis-Menten kinetic descriptors. The second sensor was designed to monitor Ca2+ ions that are ubiquitous in many biological processes. For this sensor, Cu+-catalyzed [3 + 2] azide-alkyne cycloaddition was exploited to attach a recently developed azide-functionalized CalciumRuby-Cl indicator dye to a cognate alkyne group present on the terminus of a modified peptide. The labeled peptide also expressed a polyhistidine sequence, which facilitated its subsequent metal-affinity coordination to the QD surface establishing the final FRET sensing construct. Adding exogenous Ca2+ to the sensor solution increased the dyes fluorescence, altering the donor-acceptor emission ratio and manifested a dissociation constant similar to that of the native dye. These results highlight the potential for combining peptides with QDs using different chemistries to create sensors for monitoring chemical compounds and biological processes.

Wrapping nanocrystals with an amphiphilic polymer preloaded with fixed amounts of fluorophore generates FRET-based nanoprobes with a controlled donor/acceptor ratio.

Colloidal nanocrystal (NC) donors wrapped with a polymer coating including multiple organic acceptor molecules are promising scaffolds for fluorescence resonance energy transfer (FRET)-based nanobiosensors. Over other self-assembling donor-acceptor configurations, our preloaded polymers have the virtue of producing compact assemblies with a fixed donor/acceptor distance. This property, together with the possibility of stoichiometric polymer loading, allowed us to directly address how the FRET efficiency depended on the donor/acceptor. At the population level, nanoprobes based on commercial as well as custom CdSe/ZnS donors displayed the expected dose-dependent rise in transfer efficiency, saturating from about five ATTO dyes/NC. However, for a given acceptor concentration, both the intensity and lifetime of single-pair FRET data revealed a large dispersion of transfer efficiencies, highlighting an important heterogeneity among nominally identical FRET-based nanoprobes. Rigorous quality check during synthesis and shell assembly as well as postsynthesis sorting and purification are required to make hybrid semiconductor-organic nanoprobes a robust and viable alternative to organic or genetically encoded nanobiosensors.

Molecular epidemiology of HIV-1 in St Petersburg, Russia: predominance of subtype A, former Soviet Union variant, and identification of intrasubtype subclusters.

OBJECTIVES: To examine HIV-1 genetic diversity in St. Petersburg. METHODS: Partial HIV-1 pol sequences from 102 plasma samples collected in 2006 were analyzed with a Bayesian phylogeny inference method. RESULTS: Subtype A, former Soviet Union (FSU) variant (AFSU), was the predominant clade (89.3%); other clades were subtypes B (9.7%) and F1 (1%). AFSU was predominant both among injecting drug users (98.2%) and heterosexually infected individuals (91.4%), whereas subtype B was more prevalent among homosexual men (75%). Within the AFSU variant, most sequences (93.5%) branched within 1 of 4 strongly supported subclusters. The largest comprised 63% AFSU viruses and was uncommon outside St Petersburg. A second subcluster (17.4% AFSU viruses) corresponds to the variant with the V77I substitution in protease, which is widely circulating in different FSU countries. Two minor subclusters comprised 8.7% and 6.5% AFSU viruses, respectively. There was no correlation between risk exposure and AFSU subclusters. Six of 8 subtype B sequences, 4 of them from homosexual men, grouped in a monophyletic subcluster. CONCLUSIONS: The results of this study show a great predominance of AFSU viruses in St Petersburg and point to a few phylogenetically identifiable introductions as the origin of most current HIV-1 AFSU infections in the city.

Measuring mitochondrial and cytoplasmic Ca2+ in EGFP expressing cells with a low-affinity calcium Ruby and its dextran conjugate.

The limited choice and poor performance of red-emitting calcium (Ca(2+)) indicators have hampered microfluorometric measurements of the intracellular free Ca(2+) concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca(2+) indicator would also permit a better discrimination against cellular autofluorescence than the commonly used fluorescein-based probes. Here, we report an improved synthesis and characterization of Calcium Ruby, a red-emitting probe consisting of an extended rhodamine chromophore (578/602 nm peak excitation/emission) conjugated to BAPTA and having an additional NH(2) linker arm. The low-affinity variant (K(D,Ca) approximately 30 microM) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca(2+) transients. While Calcium Ruby is a mitochondrial Ca(2+)probe, its conjugation, via the NH(2) tail, to a 10,000 MW dextran abolishes the sub-cellular compartmentalization and generates a cytosolic Ca(2+) probe with an affinity matched to microdomain Ca(2+) signals. As an example, we show depolarization-evoked Ca(2+) signals triggering the exocytosis of individual chromaffin granules. Calcium Ruby should be of use in a wide range of applications involving dual- or triple labeling schemes or targeted sub-cellular Ca(2+) measurements.

New insights into the origin of the HIV type 1 subtype A epidemic in former Soviet Union's countries derived from sequence analyses of preepidemically transmitted viruses.

The HIV-1 subtype A epidemic affecting injecting drug users (IDU) in former Soviet Union (FSU) countries started dramatically in Odessa, southern Ukraine, in 1995, and is caused by a variant of monophyletic origin, often designated IDU-A. We phylogenetically analyzed one near full-length genome and two partial sequences of three HIV-1 subtype A viruses collected in St. Petersburg, Russia, heterosexually transmitted in 1992-1994. The sequences branched basally to the IDU-A clade, together with eight viruses from Odessa collected in 1993, all presumably acquired heterosexually, and two viruses from the Democratic Republic of Congo. Of all other FSU sequences in databases, only those from three recently collected viruses, one from Ukraine and two from northwestern Russia, at least one of them acquired heterosexually, branched basally to the IDU-A cluster. The results indicate that the FSU IDU-A variant derives from a strain that initially propagated heterosexually in Ukraine and originated in central Africa.