Alcohol and marijuana use while driving-an unexpected crash risk in Pakistani commercial drivers: a cross-sectional survey

Background:A significant proportion of road traffic crashes are attributable to alcohol and marijuana use while driving globally. Sale and use of both substances is illegal in Pakistan and is not considered a threat for road traffic injuries. However literature hints that this may not be the case. We did this study to assess usage of alcohol and marijuana in Pakistani commercial drivers.Methods:A sample of 857 commercial bus and truck drivers was interviewed in October 2008 at the largest commercial vehicle station in Rawalpindi and Islamabad, Pakistan. Time location cluster sampling was used to select the subjects and a structured questionnaire was used to assess the basic demographic profile, substance abuse habits of the drivers while on the road, and reasons for usage of illicit substances while driving were recorded. Self reported information was collected after obtaining informed consent. Chi square and fisher exact tests were used to assess differences between groups and logistic regression was used to identify significant associations between driver characteristics and alcohol and marijuana use.Results:Almost 10% of truck drivers use alcohol while driving on Pakistani roads. Marijuana use is almost 30% in some groups. Statistically different patterns of usage are seen between population subgroups based on age, ethnicity, education, and marital status. Regression analysis shows association of alcohol and marijuana use with road rage and error behaviours, and also with an increased risk of being involved in road crashes. The reported reasons for using alcohol or marijuana show a general lack of awareness of the hazardous nature of this practice among the commercial driver population.Conclusion:Alcohol and marijuana use is highly prevalent in Pakistani commercial drivers. The issue needs to be recognized by concerned authorities and methods such as random breath tests and sobriety check points need to be employed for proper law enforcement

Direct Ionic Liquid Extractant Injection for Volatile Chemical Analysis - A Gas Chromatography Sampling Technique

A green sampling approach by direct injection of ionic liquid (IL) solvent containing a variety of analytes using programmable temperature vaporisation (PTV) injection with gas chromatography (GC) is presented. The method was developed using test mixtures of n-alkanes, n-alcohols and polyaromatic compounds, whilst back extraction of isolated compounds from the IL with organic solvent is not required. In the final method, 2 μL of IL, 1-butyl-3 methylimidazolium bis(trifluoromethylsulfonyl)amide containing analytes, was diluted to different volumes (ranging from 10 to 70%) with solvent then injected into the system. Several PTV injector parameters were investigated to ensure analyte volatilisation and transfer into the GC column. Concentration calibration curves (10–150 μg mL−1 and 10–100 μg mL−1 for n-alkanes and n-alcohols, respectively) were constructed, and showed addition of IL increased the peak area of each analyte, with good precision, and acceptable linearity with correlation coefficient, r2 > 0.93. This method was successfully applied in analysis of a polynuclear aromatic hydrocarbons (PAHs) mixture, with addition of IL in the mixture and suitable operation of the PTV injector. The method was also applied to eucalyptus leaf essential oil compounds as a test sample in a single drop microextraction experiment

Določanje benzodiazepinov v urinu preko benzofenonskih derivatov z uporabo tekočinske kromatografije sklopljene s tandemsko masno spektrometrijo

The aim of this study was to validate a new method for determining benzodiazepines in urine via their benzophenone derivatives, based on liquid chromatography-tandem mass spectrometry (LC-MS/MS). Selected benzodiazepines were analysed after acid hydrolysis of urine and extraction by ethyl acetate in the presence of an internal standard. Samples were analysed using electrospray ionization LC-MS/MS in a multiple reaction monitoring mode. The chromatographic run time on a reversed phase C18 analytical column was set for 9 min. This method was validated in 21 patients receiving methadone. Benzodiazepines intake was established in two out of three patients. LC-MS/MS results were also compared with the rapid immunoassay and the methods showed good agreement. However, in three cases benzodiazepines were detected by LC-MS/MS, but not by the immunoassay. The sensitivity of the developed LC-MS/MS method is comparable to or even higher than of previously reported methods, which makes it suitable as a confi rmatory method.Razvili smo selektivno in občutljivo metodo za določanje nekaterih benzodiazepinov v urinu preko določanja njihovih benzofenonov. Metoda temelji na tekočinski kromatografi ji, sklopljeni s tandemsko masno spektrometrijo (LC-MS/MS). Izbrane benzodiazepine smo analizirali po kisli hidrolizi urinskih vzorcev in ekstrakciji z etilacetatom v prisotnosti internega standarda. Vzorce smo analizirali z elektrorazprševalno ionizacijo z MRM načinom detekcije. Čas kromatografske ločbe na reverznofazni (C18) analitski koloni je bil 9 min. Metoda je bila validirana in preizkušena na 21 pacientih, ki so prejemali metadonsko terapijo. Pri dveh tretjinah primerov je bil vnos benzodiazepinov tudi potrjen. Vzorce smo testirali tudi s hitro imunokemijsko metodo in rezultate primerjali z rezultati pridobljenimi z LC-MS/MS metodo. Ugotovili smo dobro ujemanje med rezultati pridobljenimi z obe a metodama. Kljub temu smo v treh primerih določili prisotnost benzodiazepinov z LC-MS/MS metodo, ki je z imunokemijsko metodo nismo. Občutljivost razvite metode je primerljiva ali celo boljša od predhodno opisanih metod, zato jo lahko uporabimo kot potrditveno metodo

A new panel of epitope mapped monoclonal antibodies recognising the prototypical tetraspanin CD81

Background: Tetraspanins are small transmembrane proteins, found in all higher eukaryotes, that compartmentalize cellular membranes through interactions with partner proteins. CD81 is a prototypical tetraspanin and contributes to numerous physiological and pathological processes, including acting as a critical entry receptor for hepatitis C virus (HCV). Antibody engagement of tetraspanins can induce a variety of effects, including actin cytoskeletal rearrangements, activation of MAPK-ERK signaling and cell migration. However, the epitope specificity of most anti-tetraspanin antibodies is not known, limiting mechanistic interpretation of these studies. Methods: We generated a panel of monoclonal antibodies (mAbs) specific for CD81 second extracellular domain (EC2) and performed detailed epitope mapping with a panel of CD81 mutants. All mAbs were screened for their ability to inhibit HCV infection and E2-CD81 association. Nanoscale distribution of cell surface CD81 was investigated by scanning electron microscopy. Results: The antibodies were classified in two epitope groups targeting opposing sides of EC2. We observed a wide range of anti-HCV potencies that were independent of their epitope grouping, but associated with their relative affinity for cell-surface expressed CD81. Scanning electron microscopy identified at least two populations of CD81; monodisperse and higher-order assemblies, consistent with tetraspanin-enriched microdomains. Conclusions: These novel antibodies provide well-characterised tools to investigate CD81 function, including HCV entry, and have the potential to provide insights into tetraspanin biology in general

The SR-BI Partner PDZK1 Facilitates Hepatitis C Virus Entry

Entry of hepatitis C virus (HCV) into hepatocytes is a multi-step process that involves a number of different host cell factors. Following initial engagement with glycosaminoglycans and the low-density lipoprotein receptor, it is thought that HCV entry proceeds via interactions with the tetraspanin CD81, scavenger receptor class B type I (SR-BI), and the tight-junction proteins claudin-1 (CLDN1) and occludin (OCLN), culminating in clathrin-dependent endocytosis of HCV particles and their pH-dependent fusion with endosomal membranes. Physiologically, SR-BI is the major receptor for high-density lipoproteins (HDL) in the liver, where its expression is primarily controlled at the post-transcriptional level by its interaction with the scaffold protein PDZK1. However, the importance of interaction with PDZK1 to the involvement of SR-BI in HCV entry is unclear. Here we demonstrate that stable shRNA-knockdown of PDZK1 expression in human hepatoma cells significantly reduces their susceptibility to HCV infection, and that this effect can be reversed by overexpression of full length PDZK1 but not the first PDZ domain of PDZK1 alone. Furthermore, we found that overexpression of a green fluorescent protein chimera of the cytoplasmic carboxy-terminus of SR-BI (amino acids 479–509) in Huh-7 cells resulted in its interaction with PDZK1 and a reduced susceptibility to HCV infection. In contrast a similar chimera lacking the final amino acid of SR-BI (amino acids 479–508) failed to interact with PDZK1 and did not inhibit HCV infection. Taken together these results indicate an indirect involvement of PDZK1 in HCV entry via its ability to interact with SR-BI and enhance its activity as an HCV entry factor

Understanding pore formation and the effect on mechanical properties of high speed sintered polyamide-12 parts: A focus on energy input

High Speed Sintering is a novel powder-bed fusion Additive Manufacturing technique that uses an infrared lamp to provide intensive thermal energy to sinter polymer powders. The amount of thermal energy is critical to particle coalescence related defects such as porosity. This study investigates the effect of energy input on porosity and the resulting mechanical properties of polyamide-12 parts. Samples were produced at different lamp speeds, generating varying amount of energy input from a low to a high level. They were then scanned using X-ray Computed Tomography technique, following which they were subject to tensile testing. A strong correlation between energy input, porosity and mechanical properties was found, whereby pore formation was fundamentally caused by insufficient energy input. A greater amount of energy input resulted in a reduced porosity level, which in turn led to improved mechanical properties. The porosity, ultimate tensile strength and elongation achieved were 0.58%, 42.4 MPa and 10.0%, respectively, by using the standard parameters. Further increasing the energy input resulted in the lowest porosity of 0.14% and the highest ultimate tensile strength and elongation of 44.4 MPa and 13.5%, respectively. Pore morphology, volume, number density and spatial distribution were investigated, which were found to be closely linked with energy input and mechanical properties

Hepatocyte Permissiveness to Plasmodium Infection Is Conveyed by a Short and Structurally Conserved Region of the CD81 Large Extracellular Domain

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria infection, and thus represents an attractive target for anti-malarial interventions. Still, the molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that the tetraspanin CD81, a known receptor for the hepatitis C virus (HCV), is required on hepatocytes for infection by sporozoites of several Plasmodium species. Here we have characterized CD81 molecular determinants required for infection of hepatocytic cells by P. yoelii sporozoites. Using CD9/CD81 chimeras, we have identified in CD81 a 21 amino acid stretch located in a domain structurally conserved in the large extracellular loop of tetraspanins, which is sufficient in an otherwise CD9 background to confer susceptibility to P. yoelii infection. By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain. A mAb that requires this region for optimal binding did not block infection, in contrast to other CD81 mAbs. This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein

High Rates of Hepatitis C Virus Reinfection and Spontaneous Clearance of Reinfection in People Who Inject Drugs: A Prospective Cohort Study

Hepatitis C virus reinfection and spontaneous clearance of reinfection were examined in a highly characterisedcohort of 188 people who inject drugs over a five-year period. Nine confirmed reinfections and 17 possiblereinfections were identified (confirmed reinfections were those genetically distinct from the previous infection andpossible reinfections were used to define instances where genetic differences between infections could not beassessed due to lack of availability of hepatitis C virus sequence data). The incidence of confirmed reinfection was28.8 per 100 person-years (PY), 95%CI: 15.0-55.4; the combined incidence of confirmed and possible reinfectionwas 24.6 per 100 PY (95%CI: 16.8-36.1). The hazard of hepatitis C reinfection was approximately double that ofprimary hepatitis C infection; it did not reach statistical significance in confirmed reinfections alone (hazard ratio [HR]:2.45, 95%CI: 0.87-6.86, p=0.089), but did in confirmed and possible hepatitis C reinfections combined (HR: 1.93,95%CI: 1.01-3.69, p=0.047) and after adjustment for the number of recent injecting partners and duration of injecting.In multivariable analysis, shorter duration of injection (HR: 0.91; 95%CI: 0.83-0.98; p=0.019) and multiple recentinjecting partners (HR: 3.12; 95%CI: 1.08-9.00, p=0.035) were independent predictors of possible and confirmedreinfection. Time to spontaneous clearance was shorter in confirmed reinfection (HR: 5.34, 95%CI: 1.67-17.03,p=0.005) and confirmed and possible reinfection (HR: 3.10, 95%CI: 1.10-8.76, p-value=0.033) than primary infection.Nonetheless, 50% of confirmed reinfections and 41% of confirmed or possible reinfections did not spontaneouslyclear.Conclusions: Hepatitis C reinfection and spontaneous clearance of hepatitis C reinfection were observed at highrates, suggesting partial acquired natural immunity to hepatitis C virus. Public health campaigns about the risks ofhepatitis C reinfection are required

RNA Interference and Single Particle Tracking Analysis of Hepatitis C Virus Endocytosis

Hepatitis C virus (HCV) enters hepatocytes following a complex set of receptor interactions, culminating in internalization via clathrin-mediated endocytosis. However, aside from receptors, little is known about the cellular molecular requirements for infectious HCV entry. Therefore, we analyzed a siRNA library that targets 140 cellular membrane trafficking genes to identify host genes required for infectious HCV production and HCV pseudoparticle entry. This approach identified 16 host cofactors of HCV entry that function primarily in clathrin-mediated endocytosis, including components of the clathrin endocytosis machinery, actin polymerization, receptor internalization and sorting, and endosomal acidification. We next developed single particle tracking analysis of highly infectious fluorescent HCV particles to examine the co-trafficking of HCV virions with cellular cofactors of endocytosis. We observe multiple, sequential interactions of HCV virions with the actin cytoskeleton, including retraction along filopodia, actin nucleation during internalization, and migration of internalized particles along actin stress fibers. HCV co-localizes with clathrin and the ubiquitin ligase c-Cbl prior to internalization. Entering HCV particles are associated with the receptor molecules CD81 and the tight junction protein, claudin-1; however, HCV-claudin-1 interactions were not restricted to Huh-7.5 cell-cell junctions. Surprisingly, HCV internalization generally occurred outside of Huh-7.5 cell-cell junctions, which may reflect the poorly polarized nature of current HCV cell culture models. Following internalization, HCV particles transport with GFP-Rab5a positive endosomes, which is consistent with trafficking to the early endosome. This study presents technical advances for imaging HCV entry, in addition to identifying new host cofactors of HCV infection, some of which may be antiviral targets