Short term evolution of a highly transmissible methicillin-resistant Staphylococcus aureus clone (ST228) in a tertiary care hospital.

Staphylococcus aureus is recognized as one of the major human pathogens and is by far one of the most common nosocomial organisms. The genetic basis for the emergence of highly epidemic strains remains mysterious. Studying the microevolution of the different clones of S. aureus is essential for identifying the forces driving pathogen emergence and spread. The aim of the present study was to determine the genetic changes characterizing a lineage belonging to the South German clone (ST228) that spread over ten years in a tertiary care hospital in Switzerland. For this reason, we compared the whole genome of eight isolates recovered between 2001 and 2008 at the Lausanne hospital. The genetic comparison of these isolates revealed that their genomes are extremely closely related. Yet, a few more important genetic changes, such as the replacement of a plasmid, the loss of large fragments of DNA, or the insertion of transposases, were observed. These transfers of mobile genetic elements shaped the evolution of the ST228 lineage that spread within the Lausanne hospital. Nevertheless, although the strains analyzed differed in their dynamics, we have not been able to link a particular genetic element with spreading success. Finally, the present study showed that new sequencing technologies improve considerably the quality and quantity of information obtained for a single strain; but this information is still difficult to interpret and important investments are required for the technology to become accessible for routine investigations.

Identification of Mycobacteria by Thin Layer Chromatographic Analysis of Mycolic Acids and Conventional Biochemical Method: Four Years of Experience

Mycolic acids analysis by thin-layer chromatography (TLC) has been employed by several laboratories worldwide as a method for fast identification of mycobacteria. This method was introduced in Brazil by our laboratory in 1992 as a routine identification technique. Up to the present, 861 strains isolated were identified by mycolic acids TLC and by standard biochemical tests; 61% out of these strains came as clinical samples, 4% isolated from frogs and 35% as environmental samples. Mycobacterium tuberculosis strains identified by classical methods were confirmed by their mycolic acids contents (I, III and IV). The method allowed earlier differentiation of M. avium complex - MAC (mycolic acids I, IV and VI) from M. simiae (acids I, II and IV), both with similar biochemical properties. The method also permitted to distinguish M. fortuitum (acids I and V) from M. chelonae (acids I and II) , and to detect mixed mycobacterial infections cases as M. tuberculosis with MAC and M. fortuitum with MAC. Concluding, four years experience shows that mycolic acids TLC is an easy, reliable, fast and inexpensive method, an important tool to put together conventional mycobacteria identification methods.

An approximate solution method for boundary layer flow of a power law fluid over a flat plate

The work in this paper deals with the development of momentum and thermal boundary layers when a power law fluid flows over a flat plate. At the plate we impose either constant temperature, constant flux or a Newton cooling condition. The problem is analysed using similarity solutions, integral momentum and energy equations and an approximation technique which is a form of the Heat Balance Integral Method. The fluid properties are assumed to be independent of temperature, hence the momentum equation uncouples from the thermal problem. We first derive the similarity equations for the velocity and present exact solutions for the case where the power law index n = 2. The similarity solutions are used to validate the new approximation method. This new technique is then applied to the thermal boundary layer, where a similarity solution can only be obtained for the case n = 1.

Intracortical connectivity of layer VI pyramidal cells in the barrel cortex of mice

ABSTRACT : The whisker-to-barrel pathway of rodents is formed by a series of somatotopic projections from the mystacial whisker follicles to the layer IV of the primary somatosensory cortex such that each follicle corresponds to a cluster of cortical neurons called barrel. Barrels are present in layer IV but form part of functional columns that comprise the entire depth of the somatosensory cortex. Interestingly, the cortex of the barrelless mouse strain (BRL) is organized such a manner that thalamocortical afferents do not remodel their projections in layer IV and barrels fail to appear. Nevertheless, functionally, a columnar organization persists, indicating that functional columns are not only provided by thalamocortical projections and layer IV cells. Since in the visual cortex of cats, layer VI cells contribute to the response properties of layer IV neurons, we wonder whether layer VI pyramidal cells could contribute to the columnar organization of the primary somatosensory cortex of mice. To address -this question, we morphologically analyzed the distribution of intracortical axon collaterals of layer VI neurons after in-vivo juxtacellular injections of biocytin in the C2 barrel column. Injected hemispheres were tangentially serial cut and intracortical collaterals of individual layer VI neurons were reconstructed at the light microscopic level. The position of axonal boutons was recorded to evaluate the distribution of presumed synaptic contacts. In normal (NOR) mice, cluster analysis shows that layer VI pyramidal cells can be classified in four statistically different clusters of neurons. Moreover, we assume that two classes are formed by cortico-cortical neurons and two classes are formed by cortico-thalamic neurons. Looking at the direction of the main axon in the white matter, we noticed that its orientation correlates perfectly with the type of neuron: cortico-cortical neurons send main axon medially whereas cortico-thalamic neurons send main axon laterally. Performing the same study in the BRL strain, we showed that the BRL mutation affects layer VI pyramidal cells tangentially and radially: the effects of the mutation are illustrated by a significant decrease of the index of colurnnarization and a significant decrease of percentage of boutons in granular and supragranular layers comparing to NOR neurons. In spite of these differences, the same four classes of layer VI neurons have been found in BRL mice. Using a tangential analysis of the boutons distribution, we showed that putative synapses are distributed mainly in the C2 barrel column. This was observed for each layer, type of neuron, cluster or strain, indicating that layer VI pyramidal cells could participate to the functional columnar organization of the barrel cortex. To determine post-synaptic partners of layer VI neurons in layer IV, we conducted an ultrastructural analysis of layer VI-to-IV contacts. We showed that synapses principally occur on spines and spiny dendritic shafts, supposed to belong to excitatory neurons. We furthermore showed that pre-synaptic elements are significantly different between en passant and terminaux contacts, which support hypothesis that terminaux boutons should show longer duration of facilitation than en passant boutons. RÉSUMÉ : Le «whisker-to-barrel pathway» des rongeurs est caractérisé par une série de projections somatotopiques depuis les follicules des moustaches ('whiskers') jusqu'à la couche IV de l'aire somatosensorielle primaire, de telle façon que chaque follicule corresponde à un groupe de neurones corticaux appelés tonneaux (`barrels'). Les tonneaux sont seulement présents en couche IV mais font partie de colonnes fonctionnelles qui s'étendent sur toute la profondeur du cortex somatosensoriel. Chez les souris mutantes barrelless (BRL), le cortex somatosensoriel est organisé de façon telle que lés afférences thalamocorticales ne remodellent pas leurs projections en couche IV et que les tonneaux n'apparaissent pas. Fonctionnellement, pourtant, une organisation en colonnes persiste, ce qui indique que les colonnes fonctionnelles ne sont pas uniquement produites par les projections thalamocorticales et par les cellules de la couche IV. Puisque les cellules de la couche VI contribuent à influencer les réponses des cellules de la couche IV dans le cortex visuel du chat, nous nous sommes demandé si ces cellules ne pourraient pas aussi contribuer à l'organisation en colonnes du cortex somatosensoriel primaire de la souris. Pour répondre à cette question, nous avons analysé de façon morphologique la distribution intracorticale des collatéraux axonaux de neurones de la couche VI. Suite à des injections juxtacellulaires de biocytine in-vivo dans la colonne C2, les hémisphères cérébraux ont été tangentiellement coupés en série et les collatéraux intracorticaux des neurones de la couche VI ont été reconstruits en microscopie optique. La position des boutons axonaux a aussi été enregistrée pour évaluer la distribution des contacts synpptiques potentiels. Chez les souris NOR, une analyse multivariée montre que les cellules pyramidales de la couche VI sont distribuées en quatre classes. Deux de ces classes sont probablement formées de neurons cortico-corticaux, alors que les deux autres sont probablement formées de neurones corticothalamiques. En observant la direction de l'axone principal dans la matière blanche, nous avons noté que son orientation est parfaitement corrélée avec le type supposé de neurone : les neurones corticocorticaux envoient leurs axones principaux médiallement, alors que les neurons cortico-thalamiques envoient leurs axones principaux latéralement. En menant la même étude chez les souris BRL, nous avons montré que la mutation affecte les cellules pyramidales de la couche VI de façon tangentielle, mais aussi radiaire : les effets de 1a mutation se traduisent par une diminution significative de l'index de « columnarization » et de la connectivité en couches granulaire et supragranulaire. Malgré ces différences, les quatre mêmes classes de neurones ont été retrouvées. En utilisant une analyse tangentielle de la distribution des boutons, nous avons montré que les synapses potentielles sont distribuées principalement dans la colonne C2. Cette observation a été faite dans chaque couche, chaque type de neurones, chaque classe de neurones et chaque souche de souris, indicant que les cellules de la couche VI participent certainement à l'organisation en colonne du cortex somatosensoriel. Pour déterminer les partenaires post-synaptiques des cellules de la couche VI en couche IV, nous avons conduit une analyse ultrastructurelle de ces contacts. Nous avons montré que les synapses interviennent principalement sur les épines et sur les dendrites supposés appartenir à des cellules excitatrices. Nous avons aussi montré que les éléments pré-synaptiques de ces synapses sont significativement differents selon le type de bouton, en passant ou terminal, ce qui supporte l'hypothèse que les boutons terminaux seraient capables d'une plus longue facilitation.

Sawhorse-type diruthenium tetracarbonyl complexes containing porphyrin-derived ligands as highly selective photosensitizers for female reproductive cancer cells.

Diruthenium tetracarbonyl complexes of the type [Ru2(CO)4(l2-g2-O2CR)2L2] containing a Ru-Ru backbone with four equatorial carbonyl ligands, two carboxylato bridges, and two axial two-electron ligands in a sawhorse-like geometry have been synthesized with porphyrin-derived substituents in the axial ligands [1: R is CH3, L is 5-(4-pyridyl)-10,15,20-triphenyl-21,23H-porphyrin], in the bridging carboxylato ligands [2: RCO2H is 5-(4-carboxyphenyl)-10,15,20-triphenyl-21,23H-porphyrin, L is PPh3; 3: RCO2H is 5-(4-carboxyphenyl)-10,15,20-triphenyl-21,23H-porphyrin, L is 1,3,5-triaza-7-phosphatricyclo [3.3.1.1]decane], or in both positions [4: RCO2H is 5-(4-carboxyphenyl)-10,15,20-triphenyl-21,23H-porphyrin, L is 5-(4-pyridyl)-10,15,20-triphenyl-21,23H-porphyrin]. Compounds 1-3 were assessed on different types of human cancer cells and normal cells. Their uptake by cells was quantified by fluorescence and checked by fluorescence microscopy. These compounds were taken up by human HeLa cervix and A2780 and Ovcar ovarian carcinoma cells but not by normal cells and other cancer cell lines (A549 pulmonary, Me300 melanoma, PC3 and LnCap prostate, KB head and neck, MDAMB231 and MCF7 breast, or HT29 colon cancer cells). The compounds demonstrated no cytotoxicity in the absence of laser irradiation but exhibited good phototoxicities in HeLa and A2780 cells when exposed to laser light at 652 nm, displaying an LD50 between 1.5 and 6.5 J/cm2 in these two cell lines and more than 15 J/cm2 for the others. Thus, these types of porphyric compound present specificity for cancer cell lines of the female reproductive system and not for normal cells; thus being promising new organometallic photosensitizers.

Forensic examination of ink by high-performance thin layer chromatography - The United States Secret Service Digital Ink Library

Forensic examinations of ink have been performed since the beginning of the 20th century. Since the 1960s, the International Ink Library, maintained by the United States Secret Service, has supported those analyses. Until 2009, the search and identification of inks were essentially performed manually. This paper describes the results of a project designed to improve ink samples' analytical and search processes. The project focused on the development of improved standardization procedures to ensure the best possible reproducibility between analyses run on different HPTLC plates. The successful implementation of this new calibration method enabled the development of mathematical algorithms and of a software package to complement the existing ink library.

Angiotensinergic innervation of the kidney: Localization and relationship with catecholaminergic postganglionic and sensory nerve fibers.

We describe an angiotensin (Ang) II-containing innervation of the kidney. Cryosections of rat, pig and human kidneys were investigated for the presence of Ang II-containing nerve fibers using a mouse monoclonal antibody against Ang II (4B3). Co-staining was performed with antibodies against synaptophysin, tyrosine 3-hydroxylase, and dopamine beta-hydroxylase to detect catecholaminergic efferent fibers and against calcitonin gene-related peptide to detect sensory fibers. Tagged secondary antibodies and confocal light or laser scanning microscopy were used for immunofluorescence detection. Ang II-containing nerve fibers were densely present in the renal pelvis, the subepithelial layer of the urothelium, the arterial nervous plexus, and the peritubular interstitium of the cortex and outer medulla. They were infrequent in central veins and the renal capsule and absent within glomeruli and the renal papilla. Ang II-positive fibers represented phenotypic subgroups of catecholaminergic postganglionic or sensory fibers with different morphology and intrarenal distribution compared to their Ang II-negative counterparts. The Ang II-positive postganglionic fibers were thicker, produced typically fusiform varicosities and preferentially innervated the outer medulla and periglomerular arterioles. Ang II-negative sensory fibers were highly varicose, prevailing in the pelvis and scarce in the renal periphery compared to the rarely varicose Ang II-positive fibers. Neurons within renal microganglia displayed angiotensinergic, cate-cholaminergic, or combined phenotypes. Our results suggest that autonomic fibers may be an independent source of intrarenal Ang II acting as a neuropeptide co-transmitter or neuromodulator. The angiotensinergic renal innervation may play a distinct role in the neuronal control of renal sodium reabsorption, vasomotion and renin secretion.

A genome-wide association study of resistance to HIV infection in highly exposed uninfected individuals with hemophilia A.

Human genetic variation contributes to differences in susceptibility to HIV-1 infection. To search for novel host resistance factors, we performed a genome-wide association study (GWAS) in hemophilia patients highly exposed to potentially contaminated factor VIII infusions. Individuals with hemophilia A and a documented history of factor VIII infusions before the introduction of viral inactivation procedures (1979-1984) were recruited from 36 hemophilia treatment centers (HTCs), and their genome-wide genetic variants were compared with those from matched HIV-infected individuals. Homozygous carriers of known CCR5 resistance mutations were excluded. Single nucleotide polymorphisms (SNPs) and inferred copy number variants (CNVs) were tested using logistic regression. In addition, we performed a pathway enrichment analysis, a heritability analysis, and a search for epistatic interactions with CCR5 Δ32 heterozygosity. A total of 560 HIV-uninfected cases were recruited: 36 (6.4%) were homozygous for CCR5 Δ32 or m303. After quality control and SNP imputation, we tested 1 081 435 SNPs and 3686 CNVs for association with HIV-1 serostatus in 431 cases and 765 HIV-infected controls. No SNP or CNV reached genome-wide significance. The additional analyses did not reveal any strong genetic effect. Highly exposed, yet uninfected hemophiliacs form an ideal study group to investigate host resistance factors. Using a genome-wide approach, we did not detect any significant associations between SNPs and HIV-1 susceptibility, indicating that common genetic variants of major effect are unlikely to explain the observed resistance phenotype in this population.

Non-tuberculous mycobacteria I: one year clinical isolates identification in Tertiary Hospital Aids Reference Center, Rio de Janeiro, Brazil, in pre highly active antiretroviral therapy era

The aim of this study was to determine the prevalence of non-tuberculous mycobacteria (NTM) isolates at University Hospital, Reference Center for Aids in Rio de Janeiro, Brazil, during one year. We used standard biochemical tests for species identification and IS1245 PCR amplification was applied as a Mycobacterium avium specific identification marker. Four hundred and four specimens from 233 patients yielded acid-fast bacilli growth. M. tuberculosis was identified in 85% of the patients and NTM in 15%. NTM disseminated infection was a common event correlated with human immunodeficiency virus (HIV) infected patients and only in HIV negative patients the source of NTM was non sterile site. M. avium complex (MAC) was biochemically identified in 57.8% (49/83) of NTM isolates, most of them from sterile sites (75.5%), and in 94% (46/49) the IS 1245 marker specific for M. avium was present. Twenty NTM strains showed a MAC biochemical pattern with the exception of a urease-positive (99% of MAC are urease-negative), however IS1245 was detected in 96% of the strains leading to their identification as M. avium. In this group differences in NTM source was not significant. The second most frequently isolated NTM was identified as M. scrofulaceum (7.2%), followed by M. terrae (3.6%), M. gordonae (2.4%), M. chelonae (1.2%), M. fortuitum (1.2%) and one strain which could not be identified. All were IS1245 negative except for one strain identified as M. scrofulaceum. It is interesting to note that non-sterile sites were the major source of these isolates (92.8%). Our finding indicated that M. avium is still the major atypical species among in the MAC isolates recovered from Brazilian Aids patients without highty active antiretroviral therapy schema. Some discrepancies were seen between the identification methods and further investigations must be done to better characterize NTM isolates using other phenotypic and genotypic methods.

Obtenció de capes primes d'alúmina mitjançant "Atomic Layer Deposition"

En aquest projecte s'ha estudiat la posada a punt d’un equip comercial ALD per a l’obtenció de capes primes d'alúmina a escala nanomètrica utilitzant vapor d’aigua i TMA com a precursors. Per tal de comprovar a bondat de les receptes experimentals aportades pel fabricant així com comprovar alguns aspectes de la teoria ALD s’han realitzat una sèrie de mostres variant els diferents paràmetres experimentals, principalment la temperatura de deposició, el nombre de cicles, la durada del cicle i el tipus de substrat. Per a la determinació dels gruixos nanomètrics de les capes i per tant dels ritmes de creixement s’ha utilitzat la el·lipsometria, una de les poques tècniques no destructives capaç de mesurar amb gran precisió gruixos de capes o interfases de pocs àngstroms o nanòmetres. En una primera etapa s'han utilitzat els valors experimentals donats pel fabricant del sistema ALD per determinar el ritme de creixement en funció de la temperatura de dipòsit i del numero de cicles, en ambdós casos sobre diversos substrats. S'ha demostrat que el ritme de creixement augmenta lleugerament en augmentar la temperatura de dipòsit, tot i que amb una variació petita, de l'ordre del 12% en variar 70ºC la temperatura de deposició. Així mateix s'ha demostrat la linealitat del gruix amb el número de cicles, tot i que no s’observa una proporcionalitat exacta. En una segona etapa s'han optimitzat els paràmetres experimentals, bàsicament els temps de purga entre pols i pols per tal de reduir considerablement les durades dels experiments realitzats a relativament baixes temperatures. En aquest cas s’ha comprovat que es mantenien els ritmes de creixement amb una diferencia del 3,6%, 4,8% i 5,5% en optimitzar el cicles en 6,65h, 8,31h, o 8,33h, respectivament. A més, per una d'aquestes condicions s’ha demostrat que es mantenia l’alta conformitat de les capes d’alúmina. A més, s'ha realitzat un estudi de l'homogeneïtat del gruix de les capes en tota la zona de dipòsit del reactor ALD. S’ha demostrat que la variació en gruix de les capes dipositades a 120ºC és com a màxim del 6,2% en una superfície de 110 cm2. Confirmant l’excepcional control de gruixos de la tècnica ALD.

Oviposition response of the mosquito, Culex quinquefasciatus to the secondary metabolite(s) of the fungus, Trichoderma viride

Secondary metabolites produced by Trichoderma viride, a deuteromycetes fungus, under submerged culture condition were formulated and evaluated for oviposition attractancy against gravid females of Culex quinquefasciatus mosquito. At a concentration of 10 µg ml-1 the formulation showed remarkable attractancy with an oviposition active index (OAI) of +0.52. When the oviposition attractancy of the formulation was compared with a known oviposition attractant, p-cresol, both at 10 µg ml-1, the former was found to be more attractive to result in 70% egg laying than the later with 30% egg laying. Thin layer chromatography fractions of the secondary metabolites showed that a fraction with Rf value of 0.88 was highly active as oviposition attractant with an OAI of +0.65. Further work on identification of the active principle(s) of the microbial formulation might lead to an oviposition attractant useful in mosquito vector management.

Highly diverse TCRα chain repertoire of pre-immune CD8(+) T cells reveals new insights in gene recombination.

Although the T-cell receptor αδ (TCRαδ) locus harbours large libraries of variable (TRAV) and junctional (TRAJ) gene segments, according to previous studies the TCRα chain repertoire is of limited diversity due to restrictions imposed by sequential coordinate TRAV-TRAJ recombinations. By sequencing tens of millions of TCRα chain transcripts from naive mouse CD8(+) T cells, we observed a hugely diverse repertoire, comprising nearly all possible TRAV-TRAJ combinations. Our findings are not compatible with sequential coordinate gene recombination, but rather with a model in which contraction and DNA looping in the TCRαδ locus provide equal access to TRAV and TRAJ gene segments, similarly to that demonstrated for IgH gene recombination. Generation of the observed highly diverse TCRα chain repertoire necessitates deletion of failed attempts by thymic-positive selection and is essential for the formation of highly diverse TCRαβ repertoires, capable of providing good protective immunity.

DIP-STR: Highly Sensitive Markers for the Analysis of Unbalanced Genomic Mixtures.

Samples containing highly unbalanced DNA mixtures from two individuals commonly occur both in forensic mixed stains and in peripheral blood DNA microchimerism induced by pregnancy or following organ transplant. Because of PCR amplification bias, the genetic identification of a DNA that contributes trace amounts to a mixed sample represents a tremendous challenge. This means that standard genetic markers, namely microsatellites, also referred as short tandem repeats (STR), and single-nucleotide polymorphism (SNP) have limited power in addressing common questions of forensic and medical genetics. To address this issue, we developed a molecular marker, named DIP-STR that relies on pairing deletion-insertion polymorphisms (DIP) with STR. This novel analytical approach allows for the unambiguous genotyping of a minor component in the presence of a major component, where DIP-STR genotypes of the minor were successfully procured at ratios up to 1:1,000. The compound nature of this marker generates a high level of polymorphism that is suitable for identity testing. Here, we demonstrate the power of the DIP-STR approach on an initial set of nine markers surveyed in a Swiss population. Finally, we discuss the limitations and potential applications of our new system including preliminary tests on clinical samples and estimates of their performance on simulated DNA mixtures.

Oral Candida flora from Brazilian human immunodeficiency virus-infected patients in the highly active antiretroviral therapy era

One of the main opportunistic fungal infections amongst immunocompromised individuals is oral candidosis, which has been found in up to 90% of human immunodeficiency virus (HIV)-infected patients. This study employed yeasts isolated from the saliva and oral cavities of 114 HIV-infected patients living in Campinas, São Paulo. Of the isolates, 57.8% were identified as Candida albicans and 42.1% as non-C. albicans. The latter isolates were subsequently identified as C. krusei (7.5%), C. lusitaniae (5.2%), C. tropicalis (4.6%), C. parapsilosis (4.6%), C. glabrata (2.8%), C. kefyr (1.7%), C. guilliermondii (1.7%), C. intermedia (1.1%), C. norvegensis (0.5%), and Rhodotorula rubra (1.7%). Susceptibility of the isolates to amphotericin B, fluconazole, miconazole, and itraconazole was also determined by a microdilution method adopted by the National Committee for Clinical Laboratory Standards. The isolates demonstrated various susceptibilities to the antifungal agents. In particular 29 C. albicans and 13 non-C. albicans isolates showed low susceptibility to FLCZ (> 64 µg/ml). This study revealed huge diversity of Candida species, in particular the increasing emergence of non-C. albicans associated with the oral flora of HIV-infected patients.