Temperature and oxidation-sensitive dioleoylphosphatidylethanolamine liposome stabilized with poly(ethyleneimine)/(phenylthio)acetic acid ion pair

Temperature and oxidation-sensitive liposomes were prepared by stabilizing dioleoylphosphatidylethanolamine (DOPE) bilayers with the ion pair of poly(ethyleneimine)/(phenylthio)acetic acid (PEI/PTA). An upper critical solution temperature (UCST) behavior was observed when PEI/PTA ion pair was suspended in an aqueous solution. It was observed that the UCST increased with increasing PTA content. The ion pair was self-assembled into nanospheres owing to its amphiphilic property which was confirmed by transmission electron microscopy. The FT-IR spectroscopic spectrum showed that the ion pair formed a salt bridge between the amino group and the carboxyl group and the PTA content in the ion pair was readily oxidized by H2O2. Further, DOPE liposomal membranes could be stabilized with PEI/PTA ion pair. Due to the amphiphilic property, the ion pair played a role as a stabilizer for the formation of DOPE liposomes. The liposome released its payload in a temperature-responsive manner, possibly because when the temperature is raised, the ion pair loses its amphiphilic property and can be detached from the liposomal membrane. The liposome was also oxidation-responsive in terms of release, possibly because the amphiphilic property of the ion pair disappears when the PTA is oxidized. The schematic diagram of temperature and oxidation-sensitive DOPE liposome incorporating PEI/PTA ion pair.</p

Antioxidant cocktail attenuates Tat and cocaine-mediated augmentation of endothelial dysfunction.

<p>Effect of antioxidants (A) and catalase or SOD (B) on cocaine (1 µM) and Tat (25 ng/ml) mediated barrier dysfunction of HPMECs. Confluent monolayers were grown on collagen-coated Transwell inserts and treated with antioxidant cocktail followed by Tat and cocaine exposure for 6 or 24 hours. Monolayers were then treated with FITC-dextran and after 15 min the fluorescence in the lower compartment was measured and expressed as percentage of basal fluorescence. (C) Effect of antioxidants on Tat and/or cocaine mediated down-regulation of tight junction protein expression in pulmonary arterial endothelial cells. Cells grown on coverslip were immunostained for TJP-1. (D) Quantification of ZO-1 immunofluorescence using ImageJ software. (E) Western blot analysis of ZO-1 in various cellular compartments. Blot is representative of at least three independent experiments with histogram showing the average densitometry analysis normalized to β-integrin for membrane fraction, β-actin for cytosolic fraction and PCNA for nuclear compartment. The values shown are means (±S.E.M.). *P≤0.001 compared to control; ^@P≤0.001, compared to cocaine and Tat treatment.</p

Blocking Tat and cocaine-mediated ERK activation reverses ZO-1 disruption in HPMECs.

<p>Phosphorylated ERK was detected by western blot analysis of HPMECs treated with (A) Tat and cocaine for different time intervals as indicated and (B) with cocaine and/or Tat for 1.5 hours. (C) ZO-1 expression analysis in HPMECs pre-treated with U0126 for 30 min followed by Tat and cocaine treatment for 24 hours. Membrane fraction was isolated using compartment protein fractionation kit and ZO-1 was detected by western blot analysis. Lower panels show the average densitometry analysis normalized to β-actin for total cellular extract (A and B) and β-integrin in case of membrane fraction (C) of at least three independent experiments. Mean (±S.E.M.). *P≤0.05, **P≤0.01, ***P≤0.001 compared to control; ^#P≤0.001 compared to cocaine treatment. ^$p<0.001 compared to Tat treatment, ^@P≤0.01, compared to combined cocaine and Tat treatment.</p

Attenuation of Tat and cocaine mediated endothelial dysfunction in the presence of VEGFR-2 or sigma receptor antagonists.

<p>(A) Expression of sigma and dopamine receptors in HPMECs as analyzed by Western blot of total cellular extract. (B) HPMECs were treated with Tat (25 ng/ml) and cocaine (1 µM) for 6 or 24 hours in the presence or absence of SU5416 (antagonist of VEGFR-2) or BD1047 (antagonist of sigma receptor). FITC-Dextran permeability was assessed by using <i>in-vitro</i> vascular permeability assay kit. The values shown are means (±SD) of at least three independent experiments. (C) Membrane fraction was isolated and analyzed for ZO-1 by western blot analysis. Blot is representative of at least three independent experiments with histogram showing (lower panel) the average densitometry analysis normalized to β-integrin (mean ± S.E.M). (D) Representative images showing immunocyto-fluorescence staining of ZO-1. (E) Quantification of ZO-1 immunofluorescence using ImageJ software. The values are represented as fold change compared to untreated control. *P≤0.01, **P≤0.001 compared to untreated control; ^@P≤0.05, ^@@P≤0.01, ^@@@P≤0.001 compared to Tat and cocaine treatment.</p

Enhanced oxidative stress on treatment of pulmonary endothelial cells with Tat and cocaine.

<p>(A) Generation of ROS in HPMECs treated with Tat and/or cocaine was quantified by DCF assay at indicated time-points. (B) ROS production in cells treated with Tat and cocaine in the presence or absence of SU5416 (antagonist of VEGFR-2) or BD1047 (antagonist of sigma receptor) for 1 hour as analyzed by DCF assay. (C) Generation of H₂O₂ was quantified using Amplex red assay kit. HPMECs were treated with Tat and cocaine in the presence or absence of catalase (10U/ml) or SOD (100U/ml) for 1 hour. (D) Reduction of H₂O₂ formation on pre-treatment of Tat and cocaine exposed HPMECs with SU5416 or BD1047. (E) Changes in Tat and cocaine-mediated superoxide generation on SU5416 or BD1047 pre-treatment. Formation of superoxide (O₂⁻) was quantified by SOD-inducible cytochrome c reductase assay. The values shown are means (±SD) of at least three independent experiments. *P≤0.05, **P≤0.01, ***P≤0.001 compared to control; ^#P≤0.05, ^##P≤0.01,^###P≤0.001 compared to cocaine treatment; ^$P≤0.001 compared to Tat treatment;^@ P≤0.05, ^@@ P≤0.001, compared to Tat and cocaine combinational treatment; ^&P≤0.01, ^&&P≤0.001 compared to catalase-treated control; ^%‘P≤0.001, compared to SOD-treated control.</p

Activation of Ras/Raf/Erk pathway in Tat and cocaine exposed HPMECs.

<p>(A) Ras activation was assessed by pull-down assay in cells treated with Tat and cocaine for 30 or 60 min. (B) HPMECs were pre-treated with antioxidant cocktail, SU5416 or BD1047 for 5 min followed by Tat and cocaine treatment for 30 min. Representative western blot images are shown with histogram showing the average densitometry analysis of at least three independent experiments. Mean (±S.E.M.), *P≤0.001, compared to control; ^@P≤0.001, compared to cocaine and Tat treatment.</p

Data_Sheet_1_Antibiotic use, knowledge, and practices of milk vendors in India's informal dairy value chain.docx

BackgroundMilk vendors play an important role in India's dairy value chain; however, their food safety practices are poorly understood. From a milk safety perspective, vendor behavior is significant because it has the potential to affect both consumer and producer behavior. This study describes the types of milk vendors in two Indian states, in an attempt to investigate vendors' hygienic knowledge and practices toward safety and antimicrobial resistance (AMR).MethodsA cross-sectional study was conducted in the states of Assam and Haryana, India. In selected villages, all the milk vendors identified at the time of visit were interviewed. A questionnaire was used to assess the knowledge and practices on antibiotics, milk safety and hygiene. The milk samples were tested for presence of antibiotic resistant bacteria using antibiotic susceptibility testing.ResultsIn total, 244 milk vendors were interviewed during the survey. Out of these, 146 (59.8%) of the vendors traded raw milk, while 40.2% traded pasteurized milk. Vendors were categorized depending on whom they supplied milk to. Five categories were identified: (a) those who sold at grocery shops; (b) those who sold on roadside (roadside vendors); (c) those who sold from door to door; (d) those who sold to sweet makers/tea stalls, and (e) those who sold from own home/other entity. The level of training among vendors on milk hygiene was non-existent and the knowledge related to antibiotics was low. Most of them [210/244 (86.07%)] agreed that boiled milk is always safer than raw milk but almost half [119 (48.77%)] of them admitted that sometimes they drink milk without boiling it. Most vendors believed that they could identify whether milk is safe or not for consumption just by its appearance and smell. Out of 124 milk samples collected from surveyed milk vendors and tested for the presence of antibiotic-resistant bacteria, 80 (64.52%) were tested positive.ConclusionThis study highlights the low levels of knowledge regarding food safety among milk vendors. It shows the predominance of informal milk vendors in the surveyed states and prevalence of AMR bacteria in milk traded by them. Training may be a beneficial strategy for addressing the issue.</p

Epidemiology and Prognostic Implications of Coronary Artery Calcium in Asymptomatic Individuals with Prediabetes: A Multi-Cohort Study

Objectives: To describe the epidemiology and prognostic value of the coronary artery calcium (CAC) among individuals with prediabetes. Research Design and Methods: We pooled participants free of clinical ASCVD from 4 prospective cohorts Multi-Ethnic Study of Atherosclerosis (MESA), Heinz-Nixdorf Recall Study (HNR), Framingham Heart Study (FHS) and Jackson Heart Study (JHS). Two definitions were used for prediabetes: inclusive (fasting plasma glucose [FPG] ≥100-<126 mg/dL and hemoglobin A1c [HbA1c] ≥5.7-<6.5%, if available, among participants not taking glucose-lowering medications) and restrictive (FPG ≥110-<126 mg/dL and HbA1c ≥5.7-<6.5%, if available). Results: The study included 13,376 participants (mean age 58 years, 54% women, 57% White, 27% Black). The proportion with CAC≥100 was 17%, 22%, and 37% among those with euglycemia, prediabetes, and diabetes, respectively. Over a median (25th – 75th percentile) follow up time of 14.6 (7.8-16.4) years, individuals with prediabetes and CAC≥100 had higher unadjusted 10-year incidence of ASCVD (13.4%) than the overall group of those with diabetes (10.6%). In adjusted analyses, using the inclusive definition of prediabetes, compared to individuals with euglycemia the hazard ratio (HR) (95% confidence interval) for ASCVD was 0.79 (0.62, 1.01) for prediabetes and CAC=0, 0.70 (0.54, 0.89) for prediabetes and CAC 1-99, 1.54 (1.27, 1.88) for prediabetes and CAC≥100; and 1.64 (1.39, 1.93) for diabetes. Using the restrictive definition, the HR for ASCVD was 1.63 (1.29, 2.06) for prediabetes and CAC≥100. Conclusions: CAC≥100 is frequent among individuals with prediabetes, and identifies a high ASCVD risk subgroup in which the adjusted ASCVD risk is similar to people with diabetes.</p