The use of confocal microscopy in quantifying changes in membrane potential

Monitoring the plasma membrane potential and its changes can be a time consuming and challenging task especially when conventional electrophysiological techniques are used. The use of potentiometric fluorophores, namely tetramethylrhodamine methylester (TMRM), and digital imaging devices (laser scanning confocal microscopy) provides reliable and time efficient method. Two scorpion pore-forming peptides, namely PP and OP1, were used as a tool to induce depolarization of the plasma membrane potential of neuroblastoma cell line and cardiac myocytes. Alternative methods for the neuroblastoma cells and cardiac myocytes were used. Depolarization of the neuroblastoma cells was calibrated with 140 mM KCl solution with 1 µM valinomycin, after which intensity readers were substituted in the Nernst equation for quantification. Calibration of the alternative method used of the cardiac myocytes' plasma membrane potential changes was calibrated with the use of 5, 20, 40, and 80 mM KCl solutions with 1 µM valinomycin. A calibration curve was then constructed from which plasma membrane potential could be calculated

Target-specific expression of presynaptic NMDA receptors in neocortical microcircuits.

Traditionally, NMDA receptors are located postsynaptically; yet, putatively presynaptic NMDA receptors (preNMDARs) have been reported. Although implicated in controlling synaptic plasticity, their function is not well understood and their expression patterns are debated. We demonstrate that, in layer 5 of developing mouse visual cortex, preNMDARs specifically control synaptic transmission at pyramidal cell inputs to other pyramidal cells and to Martinotti cells, while leaving those to basket cells unaffected. We also reveal a type of interneuron that mediates ascending inhibition. In agreement with synapse-specific expression, we find preNMDAR-mediated calcium signals in a subset of pyramidal cell terminals. A tuned network model predicts that preNMDARs specifically reroute information flow in local circuits during high-frequency firing, in particular by impacting frequency-dependent disynaptic inhibition mediated by Martinotti cells, a finding that we experimentally verify. We conclude that postsynaptic cell type determines presynaptic terminal molecular identity and that preNMDARs govern information processing in neocortical columns

Structural drivers and social protection:Mechanisms of HIV risk and HIV prevention for South African adolescents

Introduction: Social protection is high on the HIV-prevention agenda for youth in sub-Saharan Africa. However, questions remain: How do unconditional cash transfers work? What is the effect of augmenting cash provision with social care? And can “cash plus care” social protection reduce risks for adolescents most vulnerable to infection? This study tackles these questions by first identifying mediated pathways to adolescent HIV risks and then examining potential main and moderating effects of social protection in South Africa. Methods: This study was a prospective observational study of 3515 10-to-17-year-olds (56.7% female; 96.8% one-year retention). Within randomly selected census areas in four rural and urban districts in two South African provinces, all homes with a resident adolescent were sampled between 2009/2010 and 2011/2012. Measures included 1) potential structural drivers of HIV infection such as poverty and community violence; 2) HIV risk behaviours; 3) hypothesized psychosocial mediating factors; and 4) types of social protection involving cash and care. Using gender-disaggregated analyses, longitudinal mediation models were tested for potential main and moderating effects of social protection. Results: Structural drivers were associated with increased onset of adolescent HIV risk behaviour (p<0.001, B=0.06, SE=0.01), fully mediated by increased psychosocial problems. Both cash and care aspects of social protection were associated with reductions in HIV risk behaviour and psychosocial deprivations. In addition, cash social protection moderated risk pathways: for adolescent girls and boys experiencing more acute structural deprivation, social protection had the greatest associations with HIV risk prevention (e.g. moderation effects for girls: B=−0.08, p<0.002 between structural deprivation and psychosocial problems, and B=−0.07, p<0.001 between psychosocial problems and HIV risk behaviour). Conclusions: Adolescents with the greatest structural deprivation are at higher risk of HIV, but social protection has the greatest prevention effects for the most vulnerable. Social protection comprising unconditional cash plus care was associated with reduced risk pathways through moderation and main effects, respectively. Our findings suggest the importance of social protection within a combination package of HIV-prevention approaches

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2,3,4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Ion selectivity and membrane potential effects of two scorpion pore-forming peptides

Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2006.Parabutoporin (PP) and opistoporin 1 (OP1) are cation, a-helical antimicrobial peptides isolated from the southern African scorpion species, Parabuthus schlechteri and Opistophthalmus carinatus, respectively. Along with their antimicrobial action against bacteria and fungi, these peptides show pore-forming properties in the membranes of mammalian cells. Pore-formation and ion selectivity in cardiac myocytes were investigated by measuring the whole cell leak current by means of the patch clamp technique. Pore-formation was observed as the induction of leak currents. Ion selectivity of the pores was indicated by the shift of the reversal potential (Erev) upon substitution of intra (K⁺ with CS⁺ and CI- with aspartate) and extracellular (Na' with NMDG⁺) ions. Results were compared with the effect of gramicidin A used as a positive control for monovalent cation selective pores. PP and OP I induced a fluctuating leak current and indicate non-selectivity of PP and OP1-induced pores. An osmotic protection assay to determine estimated pore size was performed on the cardiac myocytes. PP and OP1-induced pores had an estimate pore size of 1.38-1.78 nm in diameter. The effect of PP and OP1 on the membrane potential (MP) of a neuroblastoma cell line and cardiac myocytes was investigated. TMRM was used to mark the MP fluorescently and a confocal microscope used to record the data digitally. The resting membrane potential (RMP) of the neuroblastoma cells was calculated at -38.3 f 1.9 mV. PP (0.5 μM) and OP1 (0.5-1 μM) depolarized the entire cell uniformly to a MP of -1 1.9 k 3.9 mV and -9.4 k 1.9 mV, respectively. This occurred after 20-30 min of peptide exposure. In the case of the cardiac myocytes depolarization was induced to -39.7 f 8.4 mV and -32.6 f 5.2 mV by 0.5-1 μM PP and 1.5-2.5 μM OPl, respectively.Master

A toxicity profile of the Pheroid® technology in rodents

The Pheroid® drug delivery system is now on the threshold of progressing into human clinical trials for various patented pharmaceutical applications and a systematic investigation of its toxicological properties in vitro and in vivo is thus a priority. Colloidal dispersions (nano- and microemulsions) demonstrate the ability to be adapted to accommodate either lipophilic, hydrophilic or amphiphilic drug molecules. The colloidal dispersions investigated during this evaluation has a general size of 200 nm - 2 μm, a zeta-potential of -25 mV and the main ingredient was ethyl esters of essential fatty acids. The Ames mutagenicity assay was performed on selected Salmonella thyphimurium strains TA98, TA100 and TA102. The Ames assay included S9 metabolic activation and no mutagenicity was present during the assay. The effect of acute and subchronic administration on a biological system was investigated in two species of rodent (BALB/c mice and Sprague-Dawley rats). Observations focused on the physical condition, blood biochemical analysis and the haematological profiles. Gross necropsy was performed on all the test animals. Organ weights followed by histopathology of selected organ tissues were recorded. During the acute evaluation animals showed tolerance of the maximum prescribed dose of 2000 mg/kg (according to OECD guidelines) in two rodent species after intravenous administration (absolute bioavaibility). The oral formulation was tolerated without incidents in both acute and subchronic studies. Although valuable baseline safety data was obtained regarding the Pheroid® system, future studies with the entrapped active pharmaceutical ingredients is necessary to provide a definitive safety profil