InterPrEP: internet-based pre-exposure prophylaxis with generic tenofovir disoproxil fumarate/emtrictabine in London - analysis of pharmacokinetics, safety and outcomes.

OBJECTIVES: The National Health Service in England (NHS England) does not provide pre-exposure prophylaxis (PrEP) against HIV, forcing people to purchase generic versions on the internet. However, there are concerns about the authenticity of medicines purchased online. We established an innovative service offering plasma tenofovir (TFV) and emcitrabine (FTC) therapeutic drug monitoring for people buying generic PrEP online, to ensure that drug concentrations in vivo were consistent with those of propriety brands and previously published data. METHODS: TFV/FTC concentrations were measured by ultra-performance liquid chromatography ultraviolet detection. Evaluation of renal function and testing for HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) were also carried out, at baseline and every 3-6 months, with risk reduction advice. RESULTS: A total of 293 individuals presented having purchased PrEP on the internet: 85% were white, 84% were taking daily PrEP, and 16% were event-driven. Most were on generic TFV disoproxil fumarate (TDF)/FTC from Cipla Ltd. Median (range) TFV and FTC plasma concentrations were 104 (21-597) ng/mL and 140 (17-1876) ng/mL, respectively. All concentrations were above our established plasma TFV and FTC targets, based on previously published data. Renal function was normal in all evaluable individuals and no new cases of HIV, HBV or HCV infection were seen. CONCLUSIONS: In a population at high risk of HIV acquisition, who cannot yet access PrEP on the NHS, concentrations of TFV and FTC in generic formulations purchased over the internet were similar to (or slightly higher than) those measured in phase I studies with the original formulation from Gilead (Truvada™), which has demonstrated high levels of protection against HIV infection in previous PrEP clinical trials.

Completeness of West Nile virus testing in patients with meningitis and encephalitis during an outbreak in Arizona, USA.

Accurate data on West Nile virus (WNV) cases help guide public health education and control activities, and impact regional WNV blood product screening procedures. During an outbreak of WNV disease in Arizona, records from patients with meningitis or encephalitis were reviewed to determine the proportion tested for WNV. Of 60 patients identified with meningitis or encephalitis, 24 (40%) were tested for WNV. Only 12 (28%) of 43 patients aged <50 years were tested for WNV compared to 12 (71%) of 17 patients aged ≥50 years (P<0·01). Patients with clinical signs of weakness or paralysis, elevated CSF protein, admitted to an inpatient facility, or discharged to a rehabilitation facility were also more likely to have WNV testing performed. The lack of testing in younger age groups and in those with less severe disease probably resulted in substantial underestimates of WNV neuroinvasive disease burden.

Cytokinesis without myosin II.

The ability of substrate-anchored Dictyostelium cells to divide without myosin II has opened the possibility of analysing the formation of cleavage furrows in the absence of a contractile ring made of filamentous myosin and actin. Similar possibilities exist in mutants of budding yeast and, less strictly, also in drug-treated mammalian cells. Myosin-II-independent activities in Dictyostelium include the microtubule-induced programming of the cell surface into ruffling areas and regions that are converted into a concave furrow, as well as the translocation of cortexillins and cross-linked membrane proteins towards the cleavage furrow. A centripetal flow of actin filaments followed by their disassembly in the cleavage furrow is proposed to underlie the translocation.

Molecular epidemiological investigation of a typhoid fever outbreak in South Africa, 2005: the relationship to a previous epidemic in 1993.

In 2005, over 600 clinically diagnosed typhoid fever cases occurred in South Africa, where an outbreak had been previously described in 1993. Case-control and molecular investigations, including Salmonella enterica serovar Typhi (S. Typhi) isolates from that area from 1993, 2005 and later, were undertaken. Controls were significantly older than cases (P=0·003), possibly due to immunity from previous infection, and a significantly larger proportion had attended a gathering (P=0·035). Exposure to commercial food outlets and person-to-person transmission was not significant. Pulsed-field gel electrophoresis and multi-locus tandem repeat analysis revealed common clusters of S. Typhi strains identified in 1993 and 2005 as well as in 2007 and 2009. This outbreak probably occurred in a non-immune population due to faecally contaminated water. S. Typhi strains appeared to be related to strains from 1993; failure to address unsafe water may lead to further outbreaks in the area if the current population immunity wanes or is lost.

Talin-null cells of Dictyostelium are strongly defective in adhesion to particle and substrate surfaces and slightly impaired in cytokinesis.

Dictyostelium discoideum contains a full-length homologue of talin, a protein implicated in linkage of the actin system to sites of cell-to-substrate adhesion in fibroblasts and neuronal growth cones. Gene replacement eliminated the talin homologue in Dictyostelium and led to defects in phagocytosis and cell-to-substrate interaction of moving cells, two processes dependent on a continuous cross talk between the cell surface and underlying cytoskeleton. The uptake rate of yeast particles was reduced, and only bacteria devoid of the carbohydrate moiety of cell surface lipopolysaccharides were adhesive enough to be recruited by talin-null cells in suspension and phagocytosed. Cell-to-cell adhesion of undeveloped cells was strongly impaired in the absence of talin, in contrast with the cohesion of aggregating cells mediated by the phospholipid-anchored contact site A glycoprotein, which proved to be less talin dependent. The mutant cells were still capable of moving and responding to a chemoattractant, although they attached only loosely to a substrate via small areas of their surface. With their high proportion of binucleated cells, the talin-null mutants revealed interactions of the mitotic apparatus with the cell cortex that were not obvious in mononucleated cells.

DAip1, a Dictyostelium homologue of the yeast actin-interacting protein 1, is involved in endocytosis, cytokinesis, and motility.

The 64-kD protein DAip1 from Dictyostelium contains nine WD40-repeats and is homologous to the actin-interacting protein 1, Aip1p, from Saccharomyces cerevisiae, and to related proteins from Caenorhabditis, Physarum, and higher eukaryotes. We show that DAip1 is localized to dynamic regions of the cell cortex that are enriched in filamentous actin: phagocytic cups, macropinosomes, lamellipodia, and other pseudopodia. In cells expressing green fluorescent protein (GFP)-tagged DAip1, the protein rapidly redistributes into newly formed cortical protrusions. Functions of DAip1 in vivo were assessed using null mutants generated by gene replacement, and by overexpressing DAip1. DAip1-null cells are impaired in growth and their rates of fluid-phase uptake, phagocytosis, and movement are reduced in comparison to wild-type rates. Cytokinesis is prolonged in DAip1-null cells and they tend to become multinucleate. On the basis of similar results obtained by DAip1 overexpression and effects of latrunculin-A treatment, we propose a function for DAip1 in the control of actin depolymerization in vivo, probably through interaction with cofilin. Our data suggest that DAip1 plays an important regulatory role in the rapid remodeling of the cortical actin meshwork.

Three-dimensional patterns and redistribution of myosin II and actin in mitotic Dictyostelium cells.

Myosin II is not essential for cytokinesis in cells of Dictyostelium discoideum that are anchored on a substrate (Neujahr, R., C. Heizer, and G. Gerisch. 1997. J. Cell Sci. 110:123-137), in contrast to its importance for cell division in suspension (DeLozanne, A., and J.A. Spudich. 1987. Science. 236:1086-1091; Knecht, D.A., and W.F. Loomis. 1987. Science. 236: 1081-1085.). These differences have prompted us to investigate the three-dimensional distribution of myosin II in cells dividing under one of three conditions: (a) in shaken suspension, (b) in a fluid layer on a solid substrate surface, and (c) under mechanical stress applied by compressing the cells. Under the first and second conditions outlined above, myosin II does not form patterns that suggest a contractile ring is established in the furrow. Most of the myosin II is concentrated in the regions that flank the furrow on both sides towards the poles of the dividing cell. It is only when cells are compressed that myosin II extensively accumulates in the cleavage furrow, as has been previously described (Fukui, Y., T.J. Lynch, H. Brzeska, and E.D. Korn. 1989. Nature. 341:328-331), i.e., this massive accumulation is a response to the mechanical stress. Evidence is provided that the stress-associated translocation of myosin II to the cell cortex is a result of the dephosphorylation of its heavy chains. F-actin is localized in the dividing cells in a distinctly different pattern from that of myosin II. The F-actin is shown to accumulate primarily in protrusions at the two poles that ultimately form the leading edges of the daughter cells. This distribution changes dynamically as visualized in living cells with a green fluorescent protein-actin fusion.

Molecular modeling of the HIV-1 protease and its substrate binding site.

The human immunodeficiency virus (HIV-1) encodes a protease that is essential for viral replication and is a member of the aspartic protease family. The recently determined three-dimensional structure of the related protease from Rous sarcoma virus has been used to model the smaller HIV-1 dimer. The active site has been analyzed by comparison to the structure of the aspartic protease, rhizopuspepsin, complexed with a peptide inhibitor. The HIV-1 protease is predicted to interact with seven residues of the protein substrate. This information can be used to design protease inhibitors and possible antiviral drugs.

Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease.

The rational design of drugs that can inhibit the action of viral proteases depends on obtaining accurate structures of these enzymes. The crystal structure of chemically synthesized HIV-1 protease has been determined at 2.8 angstrom resolution (R factor of 0.184) with the use of a model based on the Rous sarcoma virus protease structure. In this enzymatically active protein, the cysteines were replaced by alpha-amino-n-butyric acid, a nongenetically coded amino acid. This structure, in which all 99 amino acids were located, differs in several important details from that reported previously by others. The interface between the identical subunits forming the active protease dimer is composed of four well-ordered beta strands from both the amino and carboxyl termini and residues 86 to 94 have a helical conformation. The observed arrangement of the dimer interface suggests possible designs for dimerization inhibitors.

Strategic considerations for the sustainable remediation of nuclear installations.

Nuclear sites around the world are being decommissioned and remedial actions are being undertaken to enable the sites or parts of the sites to be reused. Although this is relatively straightforward for most sites, experience has suggested that preventative action is needed to minimise the impact of remediation activities on the environment and the potential burden to future generations. Removing all contamination in order to make a site suitable for any use generates waste and has associated environmental, social and economic detriments and benefits that should be taken into account. Recent experience of OECD Nuclear Energy Agency (NEA) member countries in the remediation of contaminated land, predominantly contaminated soil and groundwater, on nuclear sites during decommissioning has been assessed by an NEA task group. The experience was used to identify strategic considerations for nuclear site remediation, to consider the application of sustainability principles to nuclear site remediation, to describe good practice, and to make recommendations for further research and development. The key aspects that were identified were that 1) site remediation should be sustainable by resulting in an overall net benefit; and 2) an adaptive approach is essential in order to take into account the inherent uncertainty associated with the decommissioning and site remediation timescales. A report describing the findings was published by OECD/NEA in 2016. The conclusions provide insights to decision makers, regulators, implementers and stakeholders involved in nuclear site decommissioning so that they can achieve sustainable remediation of nuclear sites, now and in the future.

Graves' disease and radioiodine therapy. Is success of ablation dependent on the choice of thyreostatic medication?

AIM: This study was performed to analyse the impact of the choice of antithyroid drugs (ATD) on the outcome of ablative radioiodine therapy (RIT) in patients with Graves' disease. PATIENTS, MATERIAL, METHODS: A total of 571 consecutive patients were observed for 12 months after RIT between July 2001 and June 2004. Inclusion criteria were the confirmed diagnosis of Graves' disease, compensation of hyperthyroidism and withdrawal of ATD two days before preliminary radioiodine-testing and RIT. The intended dose of 250 Gy was calculated from the results of the radioiodine test and the therapeutically achieved dose was measured by serial uptake measurements. The end-point measure was thyroid function 12 months after RIT; success was defined as elimination of hyperthyroidism. The pretreatment ATD was retrospectively correlated with the results achieved. RESULTS: Relief from hyperthyroidism was achieved in 96% of patients. 472 patients were treated with carbimazole or methimazole (CMI) and 61 with propylthiouracil (PTU). 38 patients had no thyrostatic drugs (ND) prior to RIT. The success rate was equal in all groups (CMI 451/472; PTU 61/61; ND 37/38; p = 0.22). CONCLUSION: Thyrostatic treatment with PTU achieves excellent results in ablative RIT, using an accurate dosimetric approach with an achieved post-therapeutic dose of more than 200 Gy.

Graves' disease and radioiodine therapy. Is success of ablation dependent on the achieved dose above 200 Gy?

AIM: This study was performed to determine the results of ablative radioiodine therapy (RIT) when the achieved dose in the thyroid was above 200 Gy and to characterize predictive factors for treatment outcome. PATIENTS, METHODS: A total of 571 consecutive patients were observed for 12 months between July 2001 and June 2004. Inclusion criteria were a confirmed diagnosis Graves' disease, compensation of hyperthyroidism and withdrawal of antithyroid drugs two days before preliminary radioiodine-testing and RIT. The intended dose was 250 Gy and the therapeutically achieved dose was calculated from serial uptake measurements. The end-point measure was thyroid function 12 months after RIT; success was defined as elimination of hyperthyroidism. The relation between success rate and the achieved dose, thyroid volume, age and sex of patients, TSH- and TRAb-values and presence of ophthalmopathy was analysed. RESULTS: Relief from hyperthyroidism was achieved in 96% of patients who received more than 200 Gy, even for thyroid volumes >40 ml. The success of ablative RIT was not influenced by age or sex of patients, or by TSH- or TRAb values or concomitant ophthalmopathy. The mean achieved dose in the thyroid was 298 Gy with a standard deviation of 74.6 Gy. CONCLUSION: To achieve a dose of over 200 Gy with the above standard deviation, we recommend calculating an intended dose of 250 Gy and using a dosimetric approach with early and late uptake values in the radioiodine test, to allow early therapeutic intervention should the posttherapeutic thyroid dose fall unexpectedly below 200 Gy.

Two-step positioning of a cleavage furrow by cortexillin and myosin II.

BACKGROUND: Myosin II, a conventional myosin, is dispensable for mitotic division in Dictyostelium if the cells are attached to a substrate, but is required when the cells are growing in suspension. Only a small fraction of myosin II-null cells fail to divide when attached to a substrate. Cortexillins are actin-bundling proteins that translocate to the midzone of mitotic cells and are important for the formation of a cleavage furrow, even in attached cells. Here, we investigated how myosin II and cortexillin I cooperate to determine the position of a cleavage furrow. RESULTS: Using a green fluorescent protein (GFP)-cortexillin I fusion protein as a marker for priming of a cleavage furrow, we found that positioning of a cleavage furrow occurred in two steps. In the first step, which was independent of myosin II and substrate, cortexillin I delineated a zone around the equatorial region of the cell. Myosin II then focused the cleavage furrow to the middle of this cortexillin I zone. If asymmetric cleavage in the absence of myosin II partitioned a cell into a binucleate and an anucleate portion, cell-surface ruffles were induced along the cleavage furrow, which led to movement of the anucleate portion along the connecting strand towards the binucleate one. CONCLUSIONS: In myosin II-null cells, cleavage furrow positioning occurs in two steps: priming of the furrow region and actual cleavage, which may proceed in the middle or at one border of the cortexillin ring. A control mechanism acting at late cytokinesis prevents cell division into an anucleate and a binucleate portion, causing a displaced furrow to regress if it becomes aberrantly located on top of polar microtubule asters.

A role for myosin VII in dynamic cell adhesion.

BACKGROUND: The initial stages of phagocytosis and cell motility resemble each other. The extension of a pseudopod at the leading edge of a migratory cell and the formation of a phagocytic cup are actin dependent, and each rely on the plasma membrane adhering to a surface during dynamic extension. RESULTS: A myosin VII null mutant exhibited a drastic loss of adhesion to particles, consistent with the extent of an observed decrease in particle uptake. Additionally, cell-cell adhesion and the adhesion of the leading edge to the substratum during cell migration were defective in the myosin VII null cells. GFP-myosin VII rescued the phagocytosis defect of the null mutant and was distributed in the cytosol and recruited to the cortical cytoskeleton, where it appeared to be enriched at the tips of filopods. It was also localized to phagocytic cups, but only during the initial stages of particle engulfment. During migration, GFP-myosin VII is found at the leading edge of the cell. CONCLUSIONS: Myosin VII plays an important role in mediating the initial binding of cells to substrata, a novel role for an unconventional myosin.

A search for a mutation of the Aiolos phosphorylation domain in lymphocytes from patients with leukemia.

We examined whether phosphorylation of Aiolos in primary human lymphocytes is part of the malignant transformation in leukemia. By analyzing mutations at a restriction site we show here that impairment of Aiolos activity in human leukemia is not based on deficient phosphorylation as had been demonstrated in experiments in vitro.

Structure of Bacillus subtilis superoxide dismutase.

The sodA gene of Bacillus subtilis was expressed in Escherichia coli, purified and crystallized. The crystal structure of MnSOD was solved by molecular replacement with four dimers per asymmetric unit and refined to an R factor of 21.1% at 1.8 A resolution. The dimer structure is very similar to that of the related enzyme from B. anthracis. Larger structural differences were observed with the human MnSOD, which has one less helix in the helical domain and a longer loop between two beta-strands and also showed differences in three amino acids at the intersubunit interface in the dimer compared with the two bacterial MnSODs. These structural differences can be exploited in the design of drugs that selectively target the Bacillus enzymes.

The effects of inhalation anaesthetics on common clinical pathology parameters in laboratory rats.

UNLABELLED: Effects of common anaesthetics such as ether, methoxyflurane, isoflurane, carbon dioxide (at 100%, 80% or 60% admixed with O(2)) on toxicity and clinical pathology parameters in rats were investigated. Ether, methoxyflurane and 100% CO(2) induced toxicity in some animals. Erythrocyte, haemoglobin and haematocrit were reduced in females by 100% CO(2), methoxyflurane and isoflurane. Glucose was increased by 60% CO(2), 80% CO(2), ether, isoflurane and methoxyflurane in males. Chloride was reduced by isoflurane and all CO(2) concentrations in females. Serum proteins were reduced by isoflurane and methoxyflurane. Sodium, inorganic phosphate, calcium and magnesium were reduced by methoxyflurane and isoflurane, but increased by all CO(2) concentrations. Potassium was reduced by ether, methoxyflurane or isoflurane. Triiodothyronine and thyroxine were reduced by all anaesthetics. Prolactin was reduced by methoxyflurane, but raised by ether and isoflurane. Erythrocyte cholinesterase (E-ChE) activity is markedly reduced (20-40%) after anaesthesia with all CO(2) concentrations in both sexes. E-ChE was unaffected by ether, methoxyflurane, or isoflurane. Serum and brain cholinesterase activities were not affected. E-ChE inhibition correlated with decreased blood pH, suggesting that this was caused by acidosis. This is of practical relevance in the risk assessment of cholinesterase inhibitors. CONCLUSIONS: Clinical pathology data were affected by all anaesthetics. CO(2)/O(2) (80%/20%) and isoflurane are the most suitable anaesthetics. If E-ChE activity is to be determined, isoflurane is the anaesthetic of choice.

Effects of varying dietary forage particle size in two concentrate levels on chewing activity, ruminal mat characteristics, and passage in dairy cows.

The objective of this study was to investigate the effects of varying dietary forage particle size on chewing activity, ruminal mat characteristics, passage, and in situ ruminal and total tract digestion in dairy cows at a low- and high-concentrate inclusion. The experiment was designed as a 4 x 4 Latin square with a 2 x 2 factorial arrangement of treatments. Four ruminally cannulated late-lactating dairy cows were restrictively fed (17 kg of dry matter/d), in four 23-d periods, 1 of 4 different diets varying in the theoretical particle size (6 and 30 mm) of hay (56.6% NDF of dry matter) and in the levels (approximately 20 and 60%, dry matter basis) of a cereal-based concentrate. Ingredients of the ration were offered separately to the cows; dietary hay and low-level concentrate were offered twice daily at 0800 and 1600 h, whereas concentrate of the high-level treatment was offered in 4 meals a day at 0800, 1200, 1600, and 1900 h. This study showed that altering the forage particle size from 6 to 30 mm in a low-concentrate diet significantly increased the rumination time and ruminal mat consistency without affecting ruminal fermentation and passage. Further, particle breakdown and proportion of mat in the rumen increased, and in situ hay dry matter degradability improved, which in turn indicated a higher capacity of ruminal digesta to degrade fiber. On the other hand, increasing the forage particle size in a diet containing a high amount of concentrate increased the proportion of dry matter retained on a 1.18-mm screen from 37.5 to 42.0% and extended the rumination time by 100 min/d, as well as increasing the ruminal mat consistency. However, ruminal particle breakdown, short-term ruminal pH, fibrolytic capacity of the digesta, and proportion of mat in the rumen decreased. This was also reflected in a higher bailable liquid pool, increased fractional passage rate of solid digesta from the reticulorumen, and increased retention time in the hindgut, which in turn indicated a shift of fiber digestion from the rumen to the lower digestive tract. This study showed that the response of chewing or ruminating activity alone seemed to be insufficient to assess the dietary physical effectiveness or fiber adequacy in limit-fed dairy cows when high-concentrate diets were fed separately. Based on the results of this study, we concluded that inclusion of coarsely chopped hay in the high-concentrate diet did not appear to further improve rumen conditions and digestion when the rations were formulated to exceed the fiber requirements in limit-fed dairy cows.

Use of luminescent gunshot residues markers in forensic context-Part II.

Gunshot residues (GSR) are important physical evidence in firearm-related crimes. Recently developed non-toxic ammunition, however, requires a new methodology for its characterization. To overcome this drawback, the introduction of noncommercial luminescent markers in ammunition was proposed. These markers, synthesized and added to the gunpowder, presented as a versatile tool for GSR analysis, since they require UV radiation alone to visualize the luminescent GSR (LGSR). This has opened up new perspectives for understanding GSR behavior at a crime scene. This work aims to expand previous studies performed with the luminescent markers in forensic contexts, exploring four different important aspects related to GSR behavior. Using LGSR amount/dispersion and a series of blind tests with marked ammunition, we tried to (1) identify the shooter position; (2) estimate the shooting distance; (3) evaluate the influence of the pistol type on the LGSR distribution on the shooter's hands and guns; and (4) study the transference of LGSR by a chain of handshaking. For this purpose, a portable UV lamp (λ=254nm) and/or techniques such as video spectral comparator (VSC) and scanning electron microscopy with energy dispersive X-ray analysis (SEM/EDS) were used to visualize and analyze the residues. As a result, the observation of the LGSR enabled accurate determination of the shooter position and the firing distance without any chemicals. Besides, the LGSR were visualized on different kinds of pistols, regardless of firing mechanism. And finally, this study evidenced transference of residues from shaking hands with the shooter, which should be carefully considered when interpreting the results of a GSR analysis.

Identification of ANFO: Use of luminescent taggants in post-blast residues.

This work describes the incorporation of luminescent taggants in cartridges of ammonium nitrate-fuel oil (ANFO) to allow easy identification and collection of post-blast residues. Metal-Organic Frameworks taggants proved to be appropriate for explosive marking when ANFO proportions were above 3.0wt.%. Lanthanide-based light emission enabled in situ retrieval of explosive residues as well as chemical identification, allowing the creation of a coding system using Energy Dispersive Spectroscopy (EDS). The proposed method will accelerate laboratory analysis and support forensic investigations, connecting evidence to suspects and/or to the explosive origin.