Critical role of the transcriptional repressor neuron-restrictive silencer factor in the specific control of connexin36 in insulin-producing cell lines.

Connexin36 (Cx36) is specifically expressed in neurons and in pancreatic beta-cells. Cx36 functions as a critical regulator of insulin secretion and content in beta-cells. In order to identify the molecular mechanisms that control the beta-cell expression of Cx36, we initiated the characterization of the human 5' regulatory region of the CX36 gene. A 2043-bp fragment of the human CX36 promoter was identified from a human BAC library and fused to a luciferase reporter gene. This promoter region was sufficient to confer specific expression to the reporter gene in insulin-secreting cell lines. Within this 5' regulatory region, a putative neuron-restrictive silencer element conserved between rodent and human species was recognized and binds the neuron-restrictive silencing factor (NRSF/REST). This factor is not expressed in insulin-secreting cells and neurons; it functions as a potent repressor through the recruitment of histone deacetylase to the promoter of neuronal genes. The NRSF-mediated repression of Cx36 in HeLa cells was abolished by trichostatin A, confirming the functional importance of histone deacetylase activity. Ectopic expression, by viral gene transfer, of NRSF/REST in different insulin-secreting beta-cell lines induced a marked reduction in Cx36 mRNA and protein content. Moreover, mutations in the Cx36 neuron-restrictive silencer element relieved the low transcriptional activity of the human CX36 promoter observed in HeLa cells and in INS-1 cells expressing NRSF/REST. The data showed that cx36 gene expression in insulin-producing beta-cell lines is strictly controlled by the transcriptional repressor NRSF/REST indicating that Cx36 participates to the neuronal phenotype of the pancreatic beta-cells.

CD8beta endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes.

The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8beta chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8alphabeta, but not CD8alphaalpha or soluble CD8alphabeta, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8beta endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8beta constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2K(d), and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8beta, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56(lck). In addition, the cytoplasmic portion of CD8beta mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8alphabeta partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56(lck) in rafts, which in turn phosphorylates CD3 and initiates T cell activation.

Host genetic factors in American cutaneous leishmaniasis: a critical appraisal of studies conducted in an endemic area of Brazil

American cutaneous leishmaniasis (ACL) is a vector-transmitted infectious disease with an estimated 1.5 million new cases per year. In Brazil, ACL represents a significant public health problem, with approximately 30,000 new reported cases annually, representing an incidence of 18.5 cases per 100,000 inhabitants. Corte de Pedra is in a region endemic for ACL in the state of Bahia (BA), northeastern Brazil, with 500-1,300 patients treated annually. Over the last decade, population and family-based candidate gene studies were conducted in Corte de Pedra, founded on previous knowledge from studies on mice and humans. Notwithstanding limitations related to sample size and power, these studies contribute important genetic biomarkers that identify novel pathways of disease pathogenesis and possible new therapeutic targets. The present paper is a narrative review about ACL immunogenetics in BA, highlighting in particular the interacting roles of the wound healing gene FLI1 with interleukin-6 and genes SMAD2 and SMAD3 of the transforming growth factor beta signalling pathway. This research highlights the need for well-powered genetic and functional studies on Leishmania braziliensis infection as essential to define and validate the role of host genes in determining resistance/susceptibility regarding this disease.

Trabecular bone score (TBS) the new parameter of 2D texture analysis for the evaluation of 3D bone micro architecture status

X-ray is a technology that is used for numerous applications in the medical field. The process of X-ray projection gives a 2-dimension (2D) grey-level texture from a 3- dimension (3D) object. Until now no clear demonstration or correlation has positioned the 2D texture analysis as a valid indirect evaluation of the 3D microarchitecture. TBS is a new texture parameter based on the measure of the experimental variogram. TBS evaluates the variation between 2D image grey-levels. The aim of this study was to evaluate existing correlations between 3D bone microarchitecture parameters - evaluated from μCT reconstructions - and the TBS value, calculated on 2D projected images. 30 dried human cadaveric vertebrae were acquired on a micro-scanner (eXplorer Locus, GE) at isotropic resolution of 93 μm. 3D vertebral body models were used. The following 3D microarchitecture parameters were used: Bone volume fraction (BV/TV), Trabecular thickness (TbTh), trabecular space (TbSp), trabecular number (TbN) and connectivity density (ConnD). 3D/2D projections has been done by taking into account the Beer-Lambert Law at X-ray energy of 50, 100, 150 KeV. TBS was assessed on 2D projected images. Correlations between TBS and the 3D microarchitecture parameters were evaluated using a linear regression analysis. Paired T-test is used to assess the X-ray energy effects on TBS. Multiple linear regressions (backward) were used to evaluate relationships between TBS and 3D microarchitecture parameters using a bootstrap process. BV/TV of the sample ranged from 18.5 to 37.6% with an average value at 28.8%. Correlations' analysis showedthat TBSwere strongly correlatedwith ConnD(0.856≤r≤0.862; p<0.001),with TbN (0.805≤r≤0.810; p<0.001) and negatively with TbSp (−0.714≤r≤−0.726; p<0.001), regardless X-ray energy. Results show that lower TBS values are related to "degraded" microarchitecture, with low ConnD, low TbN and a high TbSp. The opposite is also true. X-ray energy has no effect onTBS neither on the correlations betweenTBS and the 3Dmicroarchitecture parameters. In this study, we demonstrated that TBS was significantly correlated with 3D microarchitecture parameters ConnD and TbN, and negatively with TbSp, no matter what X-ray energy has been used. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.

Getting ready for malaria elimination: a check list of critical issues to consider

In recent years, a renewed interest in malaria elimination and eradication has emerged and seems to be rooting in the minds of the scientific community, public health specialists, funding bodies, policy makers and politicians. Malaria eradication will certainly benefit from improved and innovative tools; notwithstanding novel knowledge in fields ranging from basic science to mathematical modelling and health systems research. However, the elimination of malaria also encompasses a broad range of essential aspects that countries and other actors need to consider when thinking of embarking on such an adventure, including the implementation of innovative strategies, the ability to incorporate the most up-to-date evidence into policy, the integration of malaria into the broader health agenda, the strengthening of surveillance and health systems, capacity building, funding, advocacy and, very importantly, research. While in some cases this enthusiasm is clearly justified, some countries are still a long way from realistically advancing towards elimination. This paper attempts to provide guidance on all the necessary issues that should be considered when initiating a malaria elimination program.

Intraocular Straylight And Contrast Sensitivity Two Months After Lasik: Intraocular Straylight Measurement As A New Parameter In Visual Quality Assessment

PROPÒSIT: Estudiar l'efecte de la cirurgia LASIK en la llum dispersa i la sensibilitat al contrast. MÈTODES: Vint-i-vuit pacients van ser tractats amb LASIK. La qualitat visual es va avaluar abans de l'operació i dos mesos després. RESULTATS: La mitjana de llum dispersa i la sensibilitat al contrast abans de l'operació no va canviar en dos mesos després. Només un ull tenia un marcat augment en la llum dispersa. Nou ulls van presentar una lleugera disminució en la sensibilitat al contrast. S'han trobat dues complicacions. CONCLUSIÓ: Després de LASIK la majoria dels pacients (80%) no van tenir complicacions i van mantenir la seva qualitat visual. Uns pocs pacients (16%) van tenir una mica de qualitat visual disminuïda. Molt pocs (4%) van tenir complicacions clíniques amb disminució en la qualitat visual.

Studies of genotoxicity and mutagenicity of nitroimidazoles: demystifying this critical relationship with the nitro group

Nitroimidazoles exhibit high microbicidal activity, but mutagenic, genotoxic and cytotoxic properties have been attributed to the presence of the nitro group. However, we synthesised nitroimidazoles with activity against the trypomastigotes of Trypanosoma cruzi, but that were not genotoxic. Herein, nitroimidazoles (11-19) bearing different substituent groups were investigated for their potential induction of genotoxicity (comet assay) and mutagenicity (Salmonella/Microsome assay) and the correlations of these effects with their trypanocidal effect and with megazol were investigated. The compounds were designed to analyse the role played by the position of the nitro group in the imidazole nucleus (C-4 or C-5) and the presence of oxidisable groups at N-1 as an anion receptor group and the role of a methyl group at C-2. Nitroimidazoles bearing NO2 at C-4 and CH3 at C-2 were not genotoxic compared to those bearing NO2 at C-5. However, when there was a CH3 at C-2, the position of the NO2 group had no influence on the genotoxic activity. Fluorinated compounds exhibited higher genotoxicity regardless of the presence of CH3 at C-2 or NO2 at C-4 or C-5. However, in compounds 11 (2-CH3; 4-NO2; N-CH2OHCH2Cl) and 12 (2-CH3; 4-NO2; N-CH2OHCH2F), the fluorine atom had no influence on genotoxicity. This study contributes to the future search for new and safer prototypes and provide.

Role of Sam68 in post-transcriptional gene regulation.

The STAR family of proteins links signaling pathways to various aspects of post-transcriptional regulation and processing of RNAs. Sam68 belongs to this class of heteronuclear ribonucleoprotein particle K (hnRNP K) homology (KH) single domain-containing family of RNA-binding proteins that also contains some domains predicted to bind critical components in signal transduction pathways. In response to phosphorylation and other post-transcriptional modifications, Sam68 has been shown to have the ability to link signal transduction pathways to downstream effects regulating RNA metabolism, including transcription, alternative splicing or RNA transport. In addition to its function as a docking protein in some signaling pathways, this prototypic STAR protein has been identified to have a nuclear localization and to take part in the formation of both nuclear and cytosolic multi-molecular complexes such as Sam68 nuclear bodies and stress granules. Coupling with other proteins and RNA targets, Sam68 may play a role in the regulation of differential expression and mRNA processing and translation according to internal and external signals, thus mediating important physiological functions, such as cell death, proliferation or cell differentiation.

Angiographic demonstration of neoangiogenesis after intra-arterial infusion of autologous bone marrow mononuclear cells in diabetic patients with critical limb ischemia.

Critical limb ischemia in diabetic patients is associated with high rates of morbidity and mortality. Suboptimal responses to the available medical and surgical treatments are common in these patients, who also demonstrate limited vascular homeostasis. Neovasculogenesis induced by stem cell therapy could be a useful approach for these patients. Neovasculogenesis and clinical improvement were compared at baseline and at 3 and 12 months after autologous bone marrow-derived mononuclear cell (BMMNC) transplantation in diabetic patients with peripheral artery disease. We conducted a prospective study to evaluate the safety and efficacy of intra-arterial administration of autologous BMMNCs (100-400 × 10(6) cells) in 20 diabetic patients with severe below-the-knee arterial ischemia. Although the time course of clinical effects differed among patients, after 12 months of follow-up all patients presented a notable improvement in the Rutherford-Becker classification, the University of Texas diabetic wound scales, and the Ankle-Brachial Index in the target limb. The clinical outcome was consistent with neovasculogenesis, which was assessed at 3 months by digital subtraction angiography and quantified by MetaMorph software. Unfortunately, local cell therapy in the target limb had no beneficial effect on the high mortality rate in these patients. In diabetic patients with critical limb ischemia, intra-arterial perfusion of BMMNCs is a safe procedure that generates a significant increase in the vascular network in ischemic areas and promotes remarkable clinical improvement.

Activity-dependent regulation of energy metabolism by astrocytes: an update.

Astrocytes play a critical role in the regulation of brain metabolic responses to activity. One detailed mechanism proposed to describe the role of astrocytes in some of these responses has come to be known as the astrocyte-neuron lactate shuttle hypothesis (ANLSH). Although controversial, the original concept of a coupling mechanism between neuronal activity and glucose utilization that involves an activation of aerobic glycolysis in astrocytes and lactate consumption by neurons provides a heuristically valid framework for experimental studies. In this context, it is necessary to provide a survey of recent developments and data pertaining to this model. Thus, here, we review very recent experimental evidence as well as theoretical arguments strongly supporting the original model and in some cases extending it. Aspects revisited include the existence of glutamate-induced glycolysis in astrocytes in vitro, ex vivo, and in vivo, lactate as a preferential oxidative substrate for neurons, and the notion of net lactate transfer between astrocytes and neurons in vivo. Inclusion of a role for glycogen in the ANLSH is discussed in the light of a possible extension of the astrocyte-neuron lactate shuttle (ANLS) concept rather than as a competing hypothesis. New perspectives offered by the application of this concept include a better understanding of the basis of signals used in functional brain imaging, a role for neuron-glia metabolic interactions in glucose sensing and diabetes, as well as novel strategies to develop therapies against neurodegenerative diseases based upon improving astrocyte-neuron coupled energetics.

Critical ruptures in a bundle of slowly relaxing fibers

We study the damage enhanced creep rupture of disordered materials by means of a fiber bundle model. Broken fibers undergo a slow stress relaxation modeled by a Maxwell element whose stress exponent m can vary in a broad range. Under global load sharing we show that due to the strength disorder of fibers, the lifetime ʧ of the bundle has sample-to-sample fluctuations characterized by a log-normal distribution independent of the type of disorder. We determine the Monkman-Grant relation of the model and establish a relation between the rupture life tʄ and the characteristic time tm of the intermediate creep regime of the bundle where the minimum strain rate is reached, making possible reliable estimates of ʧ from short term measurements. Approaching macroscopic failure, the deformation rate has a finite time power law singularity whose exponent is a decreasing function of m. On the microlevel the distribution of waiting times is found to have a power law behavior with m-dependent exponents different below and above the critical load of the bundle. Approaching the critical load from above, the cutoff value of the distributions has a power law divergence whose exponent coincides with the stress exponent of Maxwell elements

Mean intrinsic tensile properties of stone groundwood fibers from softwood

Stone groundwood (SGW) is a fibrous matter commonly prepared in a high yield process, and mainly used for papermaking applications. In this work, the use of SGW fibers is explored as reinforcing element of polypropylene (PP) composites. Due to its chemical and superficial features, the use of coupling agents is needed for a good adhesion and stress transfer across the fiber-matrix interface. The intrinsic strength of the reinforcement is a key parameter to predict the mechanical properties of the composite and to perform an interface analysis. The main objective of the present work was the determination of the intrinsic tensile strength of stone groundwood fibers. Coupled and non-coupled PP composites from stone groundwood fibers were prepared. The influence of the surface morphology and the quality at interface on the final properties of the composite was analyzed and compared to that of fiberglass PP composites. The intrinsic tensile properties of stone groundwood fibers, as well as the fiber orientation factor and the interfacial shear strength of the current composites were determined

Unraveling the complex metabolic nature of astrocytes.

Since the initial description of astrocytes by neuroanatomists of the nineteenth century, a critical metabolic role for these cells has been suggested in the central nervous system. Nonetheless, it took several technological and conceptual advances over many years before we could start to understand how they fulfill such a role. One of the important and early recognized metabolic function of astrocytes concerns the reuptake and recycling of the neurotransmitter glutamate. But the description of this initial property will be followed by several others including an implication in the supply of energetic substrates to neurons. Indeed, despite the fact that like most eukaryotic non-proliferative cells, astrocytes rely on oxidative metabolism for energy production, they exhibit a prominent aerobic glycolysis capacity. Moreover, this unusual metabolic feature was found to be modulated by glutamatergic activity constituting the initial step of the neurometabolic coupling mechanism. Several approaches, including biochemical measurements in cultured cells, genetic screening, dynamic cell imaging, nuclear magnetic resonance spectroscopy and mathematical modeling, have provided further insights into the intrinsic characteristics giving rise to these key features of astrocytes. This review will provide an account of the different results obtained over several decades that contributed to unravel the complex metabolic nature of astrocytes that make this cell type unique.

A critical lineage-nonspecific role for pTalpha in mediating allelic and isotypic exclusion in TCRbeta-transgenic mice.

Although it is well established that early expression of TCRbeta transgenes in the thymus leads to efficient inhibition of both endogenous TCRbeta and TCRgamma rearrangement (also known as allelic and "isotypic" exclusion, respectively) the role of pTalpha in these processes remains controversial. Here, we have systematically re-evaluated this issue using three independent strains of TCRbeta-transgenic mice that differ widely in transgene expression levels, and a sensitive intracellular staining assay that detects endogenous TCRVbeta expression in individual immature thymocytes. In the absence of pTalpha, both allelic and isotypic exclusion were reversed in all three TCRbeta-transgenic strains, clearly demonstrating a general requirement for pre-TCR signaling in the inhibition of endogenous TCRbeta and TCRgamma rearrangement. Both allelic and isotypic exclusion were pTalpha dose dependent when transgenic TCRbeta levels were subphysiological. Moreover, pTalpha-dependent allelic and isotypic exclusion occurred in both alphabeta and gammadelta T cell lineages, indicating that pre-TCR signaling can potentially be functional in gammadelta precursors. Finally, levels of endogenous RAG1 and RAG2 were not down-regulated in TCRbeta-transgenic immature thymocytes undergoing allelic or isotypic exclusion. Collectively, our data reveal a critical but lineage-nonspecific role for pTalpha in mediating both allelic and isotypic exclusion in TCRbeta-transgenic mice.