Typing of Mycobacterium tuberculosis strains isolated in Community Health Centers of Rio de Janeiro City, Brazil

Fingerprinting of Mycobacterium tuberculosis strains from tuberculosis (TB) patients attended in Community Health Centers (CHCs) of Rio de Janeiro was performed to verify possible risk factors for TB transmission. A prospective community-based study was performed during the period of July 1996 to December 1996 by collecting sputum samples of 489 patients in 11 different CHCs in four different planning areas (APs) of the city. Bacteriological, clinical, and epidemiological information was collected and M. tuberculosis genotypes defined after restriction fragment length polymorphism (IS6110-RFLP) and double repetitive element (DRE) fingerprinting of RFLP-clustered cases. Risk factors for TB transmission were looked for using three levels of cluster stringency. Among 349 (71%) positive cultures obtained, IS6110-RFLP typing could be performed on strains from 153 different patients. When using identity of RFLP patterns as cluster definition, 49 (32%) of the strains belonged to a cluster and none of the clinical or epidemiologic characteristics was associated with higher clustering levels. However, higher clustering level was observed in the AP including the central region of the city when compared to others. This strongly suggests that more recent transmission occurs in that area and this may be related with higher incidence of TB and HIV in this region.     

Depositional environment and biofacies characterisation of the Triassic (Carnian to Rhaetian) carbonate succession of Punta Bassano (Marettimo Island,Sicily)

The aims of this study are to reconstruct the geological setting of the Punta Bassano series (Marettimo Island, Egadi Archipelago, western Sicily) and its palaeogeographic evolution. The reference section for the Upper Triassic of Marettimo shows an alternation of marl and limestone beds together with brecciated levels. The limestones are both homogeneous mudstones with evaporite pseudomorphs and laminated with fenestrae. Foraminiferal, palynomorph, and ostracod associations constrain the Punta Bassano sequence to the Carnian-Rhaetian interval. The Punta Bassano succession represents a shallow inner ramp, ranging from open-marine environment with good water circulation to lagoonal and peritidal protected environments. Freshwater input from rivers or groundwater on the carbonate ramp is indicated by the ostracod microfauna. The comparison of facies and microfauna with those from other sequences of the Mediterranean Upper Triassic (Pyrenees, Corsica, Sardinia, and Tunisia) allows us to confine the Punta Bassano sedimentation to the northern margin of the Tethys, between the Corsican and the Pyrenean depositional setting. These new results indicate that Marettimo Island, which is considered a single structural element being formed by four tectonic units, is a piece of the southern margin of the European Plate, displaced over a longer distance to become part of the other Egadi Islands, when the Corso-Sarde block made its rotation and successive collision with the North African Margin.     

A comparative infrared spectroscopic study of glycoside hydrolases from extremophilic archaea revealed different molecular mechanisms of adaptation to high temperatures

The identification of the determinants of protein thermal stabilization is often pursued by comparing enzymes from hyperthermophiles with their mesophilic counterparts while direct structural comparisons among proteins and enzymes from hyperthermophiles are rather uncommon. Here, oligomeric beta-glycosidases from the hyperthermophilic archaea Sulfolobus solfataricus (Ss beta-gly), Thermosphaera aggregans (Ta beta-gly), and Pyrococcus furiosus (Pf beta-gly), have been compared. Studies of FTIR spectroscopy and kinetics of thermal inactivation showed that the three enzymes had similar secondary structure composition, but Ss beta-gly and Ta beta-gly (temperatures of melting 98.1 and 98.4 degrees C, respectively) were less stable than Pf beta-gly, which maintained its secondary structure even at 99.5 degrees C. The thermal denaturation of Pf beta-gly, followed in the presence of SDS, suggested that this enzyme is stabilized by hydrophobic interactions. A detailed inspection of the 3D-structures of these enzymes supported the experimental results: Ss beta-gly and Ta beta-gly are stabilized by a combination of ion-pairs networks and intrasubunit S-S bridges while the increased stability of Pf beta-gly resides in a more compact protein core. The different strategies of protein stabilization give experimental support to recent theories on thermophilic adaptation and suggest that different stabilization strategies could have been adopted among archaea.     

A richness that cures

A study in Nature by Fischer et al. shows that environmental enrichment or increasing histone acetylation rescue the ability to form new memories and re-establish access to remote memories even in the presence of brain degeneration. Chromatin remodeling may be the final gate environmental enrichment opens to enhance plasticity and represents a promising target for therapeutical intervention in neurodegenerative diseases.     

Detection and typing of BKV,JCV, and SV40 by multiplex nested polymerase chain reaction

A multiplex nested polymerase chain reaction (PCR) method was developed for detecting and differentiating simultaneously the DNA of polyomaviruses JC, BK, and SV40 in a single tube. In the first amplification step the same set of primers was used to amplify a conserved DNA region of the large T antigen gene of JCV, BKV, and SV40. The second round was carried out using a set of primers designed to obtain products of different size for each related virus. Subsequently, the sensitivity of the multiplex nested PCR was maximized by optimizing parameters such as primer, magnesium, and dNTP concentrations. The sensitivity of the method ranged between 1 and 10 copies of the polyomavirus genome. The assay was then used for detecting polyomavirus DNA in urine, serum, and biopsy specimens from renal transplant recipients. Based on the results obtained, the multiplex nested PCR developed in our study represents a useful tool for supporting the diagnosis of polyomavirus infection and could be used for epidemiological purposes and to better define the role of polyomaviruses in human pathology.     

Epitope Recognition in the Human–Pig Comparison Model on Fixed and Embedded Material

The conditions and the specificity by which an antibody binds to its target protein in routinely fixed and embedded tissues are unknown. Direct methods, such as staining in a knock-out animal or in vitro peptide scanning of the epitope, are costly and impractical. We aimed to elucidate antibody specificity and binding conditions using tissue staining and public genomic and immunological databases by comparing human and pig—the farmed mammal evolutionarily closest to humans besides apes. We used a database of 146 anti-human antibodies and found that antibodies tolerate partially conserved amino acid substitutions but not changes in target accessibility, as defined by epitope prediction algorithms. Some epitopes are sensitive to fixation and embedding in a species-specific fashion. We also find that half of the antibodies stain porcine tissue epitopes that have 60% to 100% similarity to human tissue at the amino acid sequence level. The reason why the remaining antibodies fail to stain the tissues remains elusive. Because of its similarity with the human, pig tissue offers a convenient tissue for quality control in immunohistochemistry, within and across laboratories, and an interesting model to investigate antibody specificity.     

Involvement of the Na+/Ca2+ exchanger isoform 1 (NCX1) in Neuronal Growth Factor (NGF)-induced Neuronal Differentiation through Ca2+-dependent Akt Phosphorylation

NGF induces neuronal differentiation by modulating [Ca²⁺]_i. However, the role of the three isoforms of the main Ca²⁺-extruding system, the Na⁺/Ca²⁺ exchanger (NCX), in NGF-induced differentiation remains unexplored. We investigated whether NCX1, NCX2, and NCX3 isoforms could play a relevant role in neuronal differentiation through the modulation of [Ca²⁺]_i and the Akt pathway. NGF caused progressive neurite elongation; a significant increase of the well known marker of growth cones, GAP-43; and an enhancement of endoplasmic reticulum (ER) Ca²⁺ content and of Akt phosphorylation through an early activation of ERK1/2. Interestingly, during NGF-induced differentiation, the NCX1 protein level increased, NCX3 decreased, and NCX2 remained unaffected. At the same time, NCX total activity increased. Moreover, NCX1 colocalized and coimmunoprecipitated with GAP-43, and NCX1 silencing prevented NGF-induced effects on GAP-43 expression, Akt phosphorylation, and neurite outgrowth. On the other hand, the overexpression of its neuronal splicing isoform, NCX1.4, even in the absence of NGF, induced an increase in Akt phosphorylation and GAP-43 protein expression. Interestingly, tetrodotoxin-sensitive Na⁺ currents and 1,3-benzenedicarboxylic acid, 4,4′-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester-detected [Na⁺]_i significantly increased in cells overexpressing NCX1.4 as well as ER Ca²⁺ content. This latter effect was prevented by tetrodotoxin. Furthermore, either the [Ca²⁺]_i chelator(1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) (BAPTA-AM) or the PI3K inhibitor LY 294002 prevented Akt phosphorylation and GAP-43 protein expression rise in NCX1.4 overexpressing cells. Moreover, in primary cortical neurons, NCX1 silencing prevented Akt phosphorylation, GAP-43 and MAP2 overexpression, and neurite elongation. Collectively, these data show that NCX1 participates in neuronal differentiation through the modulation of ER Ca²⁺ content and PI3K signaling.     

Exceptional record of submarine cave communities from the Pleistocene of Sicily (Italy)

An Early Pleistocene benthic community, discovered inside the Rumena Cave in NW Sicily, Italy, was studied. Analysis of the community led to the recognition of several encrusting species – notably scleractinians, bryozoans, serpuloideans, cirripeds, foraminifera and brachiopods – and borings mostly referable to the ichnogenus Gastrochaenolites. All fossils detected are typical of the present‐day hard‐surface submarine cave biota, at both high taxonomic rank and species level. The biogenic crust, restricted to a few sectors of the cave but locally up to few centimetres thick, largely consist of scleractinians, mainly represented by dendrophylliids. Bryozoans and serpuloideans are also present with Hippaliosina depressa and Spiraserpula massiliensis, locally forming multi‐layered sheets and dense specimen aggregates, respectively. Basing mostly on the composition of the encrusting community and on morphological/morphometric features of some species, it has been hypothesized that at least part of the cave was blind when the crust formed, possibly at relatively shallow depth in a sheltered setting or, more probably, at higher depths, below the fair weather swell zone. Encrustations and borings on the rocky cave ceiling and on occasional speleothems broken surfaces document subsequent phases of cave submersion/colonization separated by emersion/erosion phases. The importance of the Early Pleistocene fossils of the Rumena Cave for the knowledge of submarine cave communities through time and for the understanding of sea‐level variations and the uplift of the area has been remarked.