Brucella melitensis Invades Murine Erythrocytes during Infection

Brucella spp. are facultative intracellular Gram-negative coccobacilli responsible for brucellosis, a worldwide zoonosis. We observed that Brucella melitensis is able to persist for several weeks in the blood of intraperitoneally infected mice and that transferred blood at any time point tested is able to induce infection in naive recipient mice. Bacterial persistence in the blood is dramatically impaired by specific antibodies induced following Brucella vaccination. In contrast to Bartonella, the type IV secretion system and flagellar expression are not critically required for the persistence of Brucella in blood. ImageStream analysis of blood cells showed that following a brief extracellular phase, Brucella is associated mainly with the erythrocytes. Examination by confocal microscopy and transmission electron microscopy formally demonstrated that B. melitensis is able to invade erythrocytes in vivo. The bacteria do not seem to multiply in erythrocytes and are found free in the cytoplasm. Our results open up new areas for investigation and should serve in the development of novel strategies for the treatment or prophylaxis of brucellosis. Invasion of erythrocytes could potentially protect the bacterial cells from the host''s immune response and hamper antibiotic treatment and suggests possible Brucella transmission by bloodsucking insects in nature.     

The role of surgery for metastatic renal cell carcinoma in the era of targeted therapies

Purpose

With the emergence of targeted therapies, the indications of cytoreductive nephrectomy have to be redefined. This review article presents the evidence data guiding our current indications of surgery in the management of metastatic renal cell carcinoma (mRCC) in the era of targeted therapies.

Methods

A nonsystematic review of the electronic databases PubMed and MEDLINE from 1980 to 2012 was performed and limited to English language.

Results

Two studies based on immunotherapy (EORTC 30947 and SWOG 8949) were at the origins of the recommendations on initial nephrectomy for patients with mRCC. Since the introduction of angiogenesis inhibitors, there is still no high-level evidence from prospective studies assessing the indication of surgery for mRCC. However, surgery still has its importance in the management of primary tumors and metastasis with the objective of an optimal balance between morbidity, quality of life, and survival. The treatment sequence between surgery and targeted therapies is still to be established and two randomized prospective studies were then specifically designed and are currently recruiting.

Conclusions

Preliminary evidence data from retrospective series seem to be in favor of a benefit of surgery for patients with good and intermediate prognosis. However, patients’ inclusions in current prospective studies are highly recommended to clearly precise nephrectomy’s indications.     

IgG4-related systemic disease: features and treatment response in a French cohort: results of a multicenter registry

IgG4-related systemic disease is now recognized as a systemic disease that may affect various organs. The diagnosis is usually made in patients who present with elevated IgG4 in serum and tissue infiltration of diseased organs by numerous IgG4+ plasma cells, in the absence of validated diagnosis criteria. We report the clinical, laboratory, and histologic characteristics of 25 patients from a French nationwide cohort. We also report the treatment outcome and show that despite the efficacy of corticosteroids, a second-line treatment is frequently necessary. The clinical findings in our patients are not different from the results of previous reports from Eastern countries. Our laboratory and histologic findings, however, suggest, at least in some patients, a more broad polyclonal B cell activation than the skewed IgG4 switch previously reported. These observations strongly suggest the implication of a T-cell dependent B-cell polyclonal activation in IgG4-related systemic disease, probably at least in part under the control of T helper follicular cells.     

X-ray crystallographic and computational studies of the O2-tolerant [NiFe]-hydrogenase 1 from Escherichia coli

The crystal structure of the membrane-bound O(2)-tolerant [NiFe]-hydrogenase 1 from Escherichia coli (EcHyd-1) has been solved in three different states: as-isolated, H(2)-reduced, and chemically oxidized. As very recently reported for similar enzymes from Ralstonia eutropha and Hydrogenovibrio marinus, two supernumerary Cys residues coordinate the proximal [FeS] cluster in EcHyd-1, which lacks one of the inorganic sulfide ligands. We find that the as-isolated, aerobically purified species contains a mixture of at least two conformations for one of the cluster iron ions and Glu76. In one of them, Glu76 and the iron occupy positions that are similar to those found in O(2)-sensitive [NiFe]-hydrogenases. In the other conformation, this iron binds, besides three sulfur ligands, the amide N from Cys20 and one Oε of Glu76. Our calculations show that oxidation of this unique iron generates the high-potential form of the proximal cluster. The structural rearrangement caused by oxidation is confirmed by our H(2)-reduced and oxidized EcHyd-1 structures. Thus, thanks to the peculiar coordination of the unique iron, the proximal cluster can contribute two successive electrons to secure complete reduction of O(2) to H(2)O at the active site. The two observed conformations of Glu76 are consistent with this residue playing the role of a base to deprotonate the amide moiety of Cys20 upon iron binding and transfer the resulting proton away, thus allowing the second oxidation to be electroneutral. The comparison of our structures also shows the existence of a dynamic chain of water molecules, resulting from O(2) reduction, located near the active site.     

Impact of vitamin D-related serum PTH reference values on the diagnosis of mild primary hyperparathyroidism, using bivariate calcium/PTH reference regions

Background, objective An international consensus conference underlined the importance of defining upper parathyroid hormone (PTH) reference values based on 25‐OH‐vitamin D [25(OH)D] to diagnose mild primary hyperparathyroidism. We determined the importance of this factor in a Belgian population. Design, patients, methods Intact PTH and 25(OH)D were measured in 261 healthy controls (18–65 years, winter/summer). They were classified as 25(OH)D replete (50–153 nmol/l; n = 129) or deplete (8–50 nmol/l; n = 132). PTH was determined in 49 patients with surgically proven primary hyperparathyroidism. PTH thresholds for 95% specificities and corresponding sensitivities were computed from both 25(OH)D replete and deplete receiver operating characteristic (ROC) curves. The 95% bivariate reference ellipses, relating PTH to calcium for 25(OH)D replete and deplete controls, were compared to the PTH/calcium pairs of patients with primary hyperparathyroidism. Results Parathyroid hormone correlated with 25(OH)D (r = ?0·3232; P < 0·0001). PTH normative values were 20% lower in 25(OH)D replete than deplete controls (P < 0·0001). PTH thresholds, providing 95% specificities for primary hyperparathyroidism diagnosis, were 7·6 pmol/l and 5·8 pmol/l, using ROC curves derived from 25(OH)D deplete or replete controls, respectively. Corresponding sensitivities were of 56%vs 88%, respectively (P < 0·05). The 95% PTH/calcium bivariate reference ellipses for?deplete and replete 25(OH)D controls differed, but the PTH/calcium pairs of patients with primary hyperparathyroidism did not overlap these ellipses. Conclusion For a given specificity, primary hyperparathyroidism diagnostic parathyroid hormone thresholds were lower and sensitivities higher using ROC curves, derived from 25(OH)D replete vs deplete controls. The 25(OH)D status does not affect the efficiency of primary hyperparathyroidism diagnosis, using bivariate PTH/calcium reference density ellipses.     

In situ formation of low molecular weight organogelators for slick solidification

We have investigated the in situ formation of Low Molecular Weight Organogelator (LMWO) molecules in oil-on-water slicks through dual reactive precursor injection. This method alleviates the need for any carrier solvent or prior heating, therefore reducing the environmental impact of LMWOs, giving instantaneous gelation, even at low temperatures (−5 °C). We show minimal leaching from our gels into the water layer.

Instantaneous gelation: a reactive precursors approach for the near-instant gelation of oil-on-water slicks down to −5 °C.

Low molecular weight organogelators (LMWOs or LMOGs) are small molecules designed to form supramolecular networks on addition to oil, turning the oil into a solid gel.^1–5 Once gelled, the oil can then be more easily removed. This makes LMWOs of great interest in the clean-up of marine oil and fuel spills, especially close to the shoreline or on bodies of inland water. A key advantage of LMWOs is that their properties can be designed to some extent at a molecular level⁴ insofar as one must ensure that the molecule will be able to form a supramolecular network with itself as well as ensuring solubility in the oil. Fig. 1(a) and (b) show the urea-based LMWOs used in this work, with hydrogen bonds between urea groups and π–π stacking between aromatic groups as examples of gelling intermolecular forces shown in Fig. 1(c). The lipophilic part of the LMWO grants the molecule solubility in oil as typically, the gelling intermolecular forces are polar in nature. Therefore, the synthesis of LMWOs often relies on balancing solubility in oil with polar intermolecular forces; too much hydrogen bonding, the compound will not be easily soluble, too little, and the compound simply will not gel.Open in a separate windowFig. 1(a) Reaction scheme of p-tolyl isocyanate “core” with a diisopropylamine “tail” forming N′-(4-methylphenyl)-N,N-dipropan-2-ylurea (referred to as compound 1), (b) reaction of p-tolyl isocyanate with dodecylamine forming N-dodecyl-N′-(4-methylphenyl)-urea (referred to as compound 2) (c) a scheme demonstrating the hypothetical self-assembly of urea-based LMWOs into supramolecular networks.Tolyl-isocyanates (“core” groups) are attractive precursors for urea based LMWOs, due to their ready availability from widespread use in poly(urethane) manufacture. They produce urea/urethane moieties on reaction with a nucleophile such as an amine or an alcohol, which readily hydrogen bond, giving the gelator the required intermolecular forces to form the supramolecular network in oils. These urea-based LMWOs have been widely characterised and explored.^1–4 The choice of nucleophilic “tail” (amine) is critical in order to impart solubility to the LMWO whilst still allowing the self-assembled structure to form. Previously, we have demonstrated a selection of p, m and o-tolyl isocyanates forming stable oil binding gels in sea water.⁹ In this study we examined the properties of a diisopropylamine/p-tolyl isocyanate system (Fig. 1(a)), and a dodecylamine/p-tolyl isocyanate system (Fig. 1(b)) with an eye to in situ application of the gel to an oil spill.It is the delivery of the LMWO to the hydrocarbon slick and gelation of the spill that remains a key challenge in environmental oil-spill remediation with LMWOs. The application of the LMWO to the oil has proven to be highly exacting simply due to the strength of the gelling intermolecular forces, and a successful LMWO will most commonly manifest itself as a solid. Therefore, in order to solubilise a solid LMWO in oil, energy (heat) has to be applied to the system to overcome these intermolecular forces and force dissolution. On mixing with the oil and cooling, the intermolecular forces can re-form and the oil will gel.This is a severe limitation of LMWOs in the oil-spill clean-up role, as they must either: (a) be applied hot, increasing deployment difficulty and energetic cost or (b) to hasten dissolution in oil, they would have to be deployed in a carrier solvent, increasing potential environmental consequences. Potentially, this is also a reason why LMWOs would only be suited to inshore clean-up as the cooling on aerial deployment would mean the LMWO would solidify before reaching the oil, preventing dissolution and gelation.Several groups have published investigations along these lines, such as: using heated solutions of LMWOs,^5,6 or supergelators dissolved in flammable ethanol/ethyl acetate blends of solvents to aid gel dissolution.^7–9 Sureshan et al. reported alkyl 4,6-O-benzylidene-glucopyranoside derivatives which can be applied as a powder and will gel oil mixtures on seawater⁵ and more recently, Zhang et al. reported d-gluconic acetal-based powder gelators able to gel oil slicks at room temperature.¹⁰Another emerging school of thought involved the combinatorial approach of LMWOs coupled with sorbents.¹¹ For example, the use of a supergelator (definition: a critical gelation concentration (CGC) of <0.1 wt%)¹² contained within a cellulose pulp matrix has been shown to be very effective at absorbing oil and affecting the release of the gelator into the oil.¹³ This approach alleviates the need for a carrier solvent and solves the problem of dissolution, but does require the presence of a solid matrix to work.In an alternative solution to the aforementioned challenges, we utilised the rapid reaction of isocyanate and amine to form a LMWO in situ, a method first utilised by Suzuki et al., who used the in situ synthesis of urea-based LMWOs to gel a variety of solvents in 2004. Here we extend the method to oil-on-sea water slicks.¹⁴ We also explore for the first time, the influence of temperature on in situ gelation, going below room temperature to more accurately simulate oceanic conditions. Herein we can report the successful, rapid gelation of 1-octadecene using compound 1 as a slick on cold seawater (−5 °C), an experiment essential for validating this method in cold environments.If two liquid precursors, such as diisopropylamine and p-tolyl isocyanate were sprayed into an oil in close proximity or one after another, a gel can form in the oil in situ, thus alleviating the need for elevated temperature or a carrier solvent. We examined this hypothesis by simulating an oil slick utilising 1-octadecene on deionised water, before rapid injection of equimolar amounts of p-tolyl isocyanate and amine (diisopropylamine (forming N′-(4-methylphenyl)-N,N-dipropan-2-ylurea), henceforth referred to as compound 1 or dodecylamine (forming N-dodecyl-N′-(4-methylphenyl)-urea), henceforth referred to as compound 2). On injection, either the isocyanate after the amine or vice versa (or indeed simultaneously), urea fibres began to form rapidly, completely gelling the 1-octadecene within 60 seconds (Fig. 2, ​,33 and ESI Video^†), even on slicks at low temperature on seawater at −5 °C (see Fig. S2^†). Rapid gelation within 60 seconds occurred with LMWO concentrations down to 2 wt% with the diisopropylamine/p-tolyl isocyanate system (1), and 5 wt% dodecylamine/p-tolyl isocyanate system (2), below which the reaction was slower with weaker gel consistency, and did not survive the “inversion test” (Fig. S1^†). We successfully managed to form an LMWO in situ in an oil on water slick, through rapid reaction of isocyanate and amine precursors, as shown by nuclear magnetic resonance (NMR) spectroscopy in the ESI.^† Indeed, water should compete for reaction with the isocyanate, but the localised high concentration precluded this. The NMR of the resulting gels did not show evidence of the isocyanate reaction with water in the oil slick, and no sequestered water in the oil. The resultant ureas and gelators were isolable from oil and could be extracted by distillation or centrifugation, as demonstrated in our previous work.⁴ We successfully repeated these experiments with kerosene and motor oil and several other oils at −5 °C (details in Table S1 ESI^†).Open in a separate windowFig. 2Scanning electron microscope images of xerogels of: (a) and (b) 10 wt% diisopropylamine/p-tolyl isocyanate system (compound 1), and (c) and (d) 10 wt% dodecylamine/p-tolyl isocyanate system (compound 2).Open in a separate windowFig. 3Photographs demonstrating the experimental protocol. (a) is gelator 1, the product from the reaction p-tolyl isocyanate with diisopropylamine, (b), (d) and (f) show the process of simultaneous injection of p-tolyl isocyanate and diisopropylamine into a 1-octadecene on water slick forming a gel in (f), (c) a 100 ml round bottom flask filled with water held back by a 1/1-octadecene gel and (e) illustrates complete separation of a 1/1-octadecene gel and water.The selected “cores” and “tails” are low molecular weight, with neither exceeding 320 g mol⁻¹. Precursors are all inexpensive (p-tolyl isocyanate is a poly(urethane) precursor) and alkyl amines which are readily available and inexpensive. The synthesis of ureas/carbamate from isocyanates and primary amines/alcohols is an instantaneous, facile one-pot reaction giving a pure product in high yield.⁴ Purification involved removal of the solvent (if any) and drying in vacuo. The process can easily be adapted to the kilogram scale, with prima facae evidence being the poly(urethane) industry. The LMWOs formed by this reaction are of unknown toxicity, but neither exhibited significant solubility in water, so are unlikely to pose a long-term threat to aquatic life.The in situ reaction was also very effective at gelling thin oil slicks (ca. 2 mm) and facilitated the collection of oil as evidenced in Fig. S3^† and ​and3e3e respectively. SEM images in Fig. 2 (xerogels of dodecylamine/p-tolyl isocyanate and diisopropylamine/p-tolyl isocyanate formed from gelling cyclohexane) demonstrate the formation of urea “tapes” in the same fashion as our previous work.⁴ The dodecylamine/p-tolyl isocyanate gel however formed more plate-like structures (Fig. 2(c) and (d)).The rapidity of reaction is very important for deployment of the in situ method system for fast clean-up of oil spills. Indeed, the use of diisopropylamine over dodecylamine facilitates a more rapid reaction, as evidenced by Video S2 in the ESI.^† We postulate that this is simply due to the small size of the diisopropylamine versus the dodecylamine, with the smaller diisopropylamine being able to react faster. Further to this, pump oil on river water (obtained from the River Thames near Kingston) was gelled successfully with both the dodecylamine/p-tolyl isocyanate and diisopropylamine/p-tolyl isocyanate systems. In terms of concentrations of precursors applied, we were able to invert 1 ml of 1-octadecene gels formed with the precursors applied against 2 ml of deionised water (see Fig. S4^†). The integrity of gels of 1 was maintained down to 1 wt%, whereas for 2, integrity was maintained down to 2 wt% of gelator.NMR spectroscopy confirmed that both gelators 1 and 2 were formed in high yield on reaction of p-tolyl isocyanate and diisopropylamine/dodecylamine respectively, in the absence of solvent (ESI^†). Furthermore, the same reagents reacted together successfully and in quantitative yields when added separately or together to a 1-octadecene layer on an aqueous layer. The gel formed in those cases dissolved fully in CDCl₃ with no significant amount of water present in the gel. This seemed to indicate no sequestration of water by the gel as it formed.One of the main problems with the approach outlined herein is toxicity. The safety datasheets (SDS) for both diisopropylamine and p-tolylisocyanate class them as irritants and as toxic. In order to ascertain the amount of precursors and gelator that leached into the aqueous phase, various slicks of 1-octadecene were created on seawater and river water before injection of precursors into the oil layer. The aqueous phases were then analysed for leachates by NMR. The results are summarised in

Neurochemical characterization of the hypothalamus of the early fetal and newborn alpaca Vicugna pacos

In this study we performed a neurochemical characterization of the hypothalamus in the developing alpaca (Vicugna pacos) with the aim of revealing the distributions of immunoreactive (-ir) cells containing parvalbumin (PV), calbindin (CB), calretinin (CR), the somatostatin (SOM), the enzyme aromatase P450 (P450Arom), the estrogen receptor α (ER-α), and estrogen receptor β (ER-β) in embryonal stages, early fetal age, and in the newborn. This analysis has been carried out on embryos at 20, 30, 45 days, fetuses at 90 days, and newborn alpaca. Our immunohistochemical results revealed no cells-ir throughout the embryonic hypothalami of 20, 30, and 45 days. On the fetal stage of 90 days, SOM-ir cells were observed in the lateral hypothalamus and the ventromedial nuclei of the tuberal region. We checked for the presence of P450Arom-ir cells in the periventricular area and dorsomedial hypothalamic nucleus of the tuberal region. In these fetal stages, no PV-ir, CB-ir, CR-ir or ERs-ir cells were identified. In the newborn, the PV-ir, CB-ir, CR-ir, and SOM-ir cells were detected in both the anterior and tuberal hypothalamic area. The P450Arom-ir cells the ER-α–ir and ER-β–ir cells were found in the anterior hypothalamus. Our results offer a contribution in the future purpose to obtain a time-expression pattern of the considered markers in alpaca during gestation and represents a foundation for future investigations on the alpaca brain to define the cross talk between PV, CB, CR, P450Arom, SOM, and ERs in the hypothalamus, the strategic region for the control of the reproductive behavior.     

Empowering HIV testing as a prevention tool: targeting interventions for high-risk men who have sex with men

In France, HIV testing can be easily performed in free and anonymous voluntary counselling testing (VCT) centres. The recent national study among French men who have sex with men (MSM) showed that 73% of those already tested for HIV had been tested in the previous two years. Nothing is known about the risk behaviours of MSM attending VCT centres. This study aimed to characterize sexual risk behaviours of MSM tested for HIV in such centres and identify factors associated with inconsistent condom use (ICU). A cross-sectional study was conducted from March to December 2009 in four VCT centres where a self-administered questionnaire was proposed to all MSM about to have a HIV test. ICU was defined as reporting non-systematic condom use during anal intercourse with casual male partners. Among the 287 MSM who fully completed their questionnaire, 44% reported ICU in the previous six months. Among those who had been already tested, 63% had had their test in the previous two years. Factors independently associated with ICU included: never avoiding one-night stands, not having been recently HIV tested, experiencing difficulty in using condoms when with a HIV negative partner or when under the influence of drugs or alcohol and finally, reporting to have had a large number of casual male partners in the previous six months. The rate of recently tested MSM was high in our study. Nevertheless, this rate was lower than that found in the last national study. Furthermore those not recently tested were significantly more likely to report high risk behaviours. We therefore recommend that further efforts be made to adapt the offer of both HIV testing and counselling to meet the specific needs of hard-to-reach MSM. Accordingly, an additional community-based offer of HIV testing to reach most-at-risk MSM is forthcoming in France.     

Validating the use of a stereoscopic robotized teleophthalmic drone slit lamp

ObjectiveTo validate the use of a mechanized remotely operated stereoscopic drone slit lamp (DSL) in assessing anterior segment pathology in ophthalmology patients compared with conventional slit lamp (CSL).MethodsPatients were recruited from eye clinics at Hotel Dieu Hospital in Kingston, Ontario, Canada. Each patient was assessed by 2 examiners. Examiners consisted of ophthalmology residents and staff attendings. Each examiner assessed the anterior chamber (AC) depth, presence or absence of cells, and/or presence of flare of the patient first using the DSL, followed by CSL. Qualitative data were collected on the ability to assess corneal integrity, infiltrates, foreign bodies, epithelial defects, and conjunctival injection using the DSL.Results48 eyes of 42 participants were examined using the DSL and CSL. No significant within-examiner differences in AC depth or cell were detected. There was substantial agreement between the DSL and CSL when assessing AC cell and flare (κ = 72.6 and κ = 60.4, respectively) and moderate agreement when assessing AC depth (κ = 42.5). The DSL compared with CSL had a sensitivity and specificity of 98.3% (95% confidence interval [CI] 94-100) and 100% (95% CI 98.7-100), respectively, for detecting AC cell. The DSL had sensitivity and specificity of 100% (95% CI 97.5-100) and 88.2% (95% CI 80.2-96.1), respectively, for detecting AC flare.ConclusionsThere was substantial agreement between the DSL and CSL when assessing AC depth, cell, and flare. Sensitivity and specificity for assessing these findings ranged from 88.2% to 100%. This DSL provides excellent capability for examination of anterior segment pathology in live patients, performing similarly to a CSL.