Methylprednisolone Administration Following Spinal Cord Injury Reduces Aquaporin 4 Expression and Exacerbates Edema.

Spinal cord injury (SCI) is an incapacitating condition that affects motor, sensory, and autonomic functions. Since 1990, the only treatment administered in the acute phase of SCI has been methylprednisolone (MP), a synthetic corticosteroid that has anti-inflammatory effects; however, its efficacy remains controversial. Although MP has been thought to help in the resolution of edema, there are no scientific grounds to support this assertion. Aquaporin 4 (AQP4), the most abundant component of water channels in the CNS, participates in the formation and elimination of edema, but it is not clear whether the modulation of AQP4 expression by MP plays any role in the physiopathology of SCI. We studied the functional expression of AQP4 modulated by MP following SCI in an experimental model in rats along with the associated changes in the permeability of the blood-spinal cord barrier. We analyzed these effects in male and female rats and found that SCI increased AQP4 expression in the spinal cord white matter and that MP diminished such increase to baseline levels. Moreover, MP increased the extravasation of plasma components after SCI and enhanced tissue swelling and edema. Our results lend scientific support to the increasing motion to avoid MP treatment after SCI.

Unprocessed snRNAs Are a Prognostic Biomarker and Correlate with a Poorer Prognosis in Colorectal Cancer.

The human Integrator complex is a set of 15 subunits that mediates processing of small nuclear RNAs (snRNAs), and which later participates in splicing messenger RNAs (mRNAs). In addition, it controls the pause and release of RNA polymerase II (RNA pol II) at specific gene promoters in response to growth factors. Mutations in Integrator-complex subunit 6 (INTS6) are associated with different types of tumors. However, the INTS6 gene product does not have a significant prognostic value as a biomarker for tumor progression. Here we show that Integrator-complex deregulation is involved in 8.3% of the colorectal cancer cases diagnosed from the population screen carried out in La Rioja (Spain) from the years 2017 to 2019. Lack of Integrator-complex function, measured by an increased level of unprocessed snRNA, is a prognostic biomarker and correlates with a poorer prognosis in colorectal-cancer patients. The transcriptomic profile of all analyzed colorectal tumors shows a strong alteration of the metabolic state of tumor cells, which compromises standard energy production through mitochondrial respiration, known as the Warburg effect. Furthermore, there is a significant upregulation of genes involved in extracellular matrix organization and collagen rearrangement. This is consistent with tissue reorganization in a growing tumor forming a polyp. Crossing the molecular data generated in this study with the follow-up of patients from population screening indicates that population screening combined with early typing of tumors appears to be the most efficient way to increase patient survival.

Extracellular vesicles from neural progenitor cells promote functional recovery after stroke in mice with pharmacological inhibition of neurogenesis.

Neural progenitor cells (NPCs) of the subventricular zone proliferate in response to ischemic stroke in the adult mouse brain. Newly generated cells have been considered to influence recovery following a stroke. However, the mechanism underlying such protection is a matter of active study since it has been thought that proliferating NPCs mediate their protective effects by secreting soluble factors that promote recovery rather than neuronal replacement in the ischemic penumbra. We tested the hypothesis that this mechanism is mediated by the secretion of multimolecular complexes in extracellular vesicles (EVs). We found that the molecular influence of oxygen and glucose-deprived (OGD) NPCs-derived EVs is very limited in improving overt neurological alterations caused by stroke compared to our recently reported astrocyte-derived EVs. However, when we inhibited the ischemia-triggered proliferation of NPCs with the chronic administration of the DNA synthesis inhibitor Ara-C, the effect of NPC-derived EVs became evident, suggesting that the endogenous protection exerted by the proliferation of NPC is mainly carried out through a mechanism that involves the intercellular communication mediated by EVs. We analyzed the proteomic content of NPC-derived EVs cargo with label-free relative abundance mass spectrometry and identified several molecular mediators of neuronal recovery within these vesicles. Our findings indicate that NPC-derived EVs are protective against the ischemic cascade activated by stroke and, thus, hold significant therapeutic potential.

Preparation and ex vivo investigation of an injectable microparticulate formulation for gastrointestinal mucosa polyp resection.

Endoscopic submucosal dissection (ESD) and endoscopic submucosal resection (EMR) are non-invasive endoscopic techniques. They allow an early excised gastrointestinal (GI) mucosal precancerous lessions. For their application is necessary to use a submucosal injection that lifts the area to excise. The main objective of this study was the preparation of a microparticulate-based fluid for injection in the GI submucosa. Alginate microparticles (MPs) were developed by the solvent displacement technique and characterized by particle size, surface electrical properties, swelling, degradation, rheology, adhesion, leakage, syringeablity and stability. Furthermore, their potential to form a submucosal cushion was assayed in porcine stomach mucosa and porcine colon mucosa. Results showed MPs sizes below 160 µm, negative surface charge around -50 mV at pH = 6, high rates of swelling and good adhesion. The microparticulate-based fluid exhibited pseudoplastic behavior following the Ostwald-de Waele rheological model. A brief force is sufficient for its injection through a syringe. Finally, formulations were able to provide a submucosa elevation of 1.70 cm for more than 90 min and 120 min in the porcine stomach and colon, respectively.

Gln3-Gcn4 hybrid transcriptional activator determines catabolic and biosynthetic gene expression in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is able to sense the availability and quality of nitrogen sources and the intrinsic variation of amino acid disponibility for protein synthesis. When this yeast is provided with secondary nitrogen sources, transcription of genes encoding enzymes involved in their catabolism is elicited through the action of Gln3, which constitutes the main activator of the Nitrogen Catabolite Repression network (NCR). Activation of genes encoding enzymes involved in the amino acid biosynthetic pathways is achieved through the action of the GCN4-encoded transcriptional modulator whose transcriptional activation is induced at the translational level by limitation for any amino acid. Thus the role of each one of these activators had been secluded to either catabolic or biosynthetic pathways. However, some observations have suggested that under peculiar physiological conditions, Gln3 and Gcn4 could act simultaneously in order to contemporaneously increase expression of both sets of genes. This paper addresses the question of whether Gln3 and Gcn4 cooperatively determine expression of their target genes. Results presented herein show that induced expression of catabolic and biosynthetic genes when cells are grown under nitrogen derepressive conditions and amino acid deprivation is dependent on the concurrent action of Gln3 and Gcn4, which form part of a unique transcriptional complex. We propose that the combination of Gln3 and Gcn4 results in the constitution of a hybrid modulator which elicits a novel transcriptional response, not evoked when these modulators act in a non-combinatorial fashion.

Hap2-3-5-Gln3 determine transcriptional activation of GDH1 and ASN1 under repressive nitrogen conditions in the yeast Saccharomyces cerevisiae.

The transcriptional activation response relies on a repertoire of transcriptional activators, which decipher regulatory information through their specific binding to cognate sequences, and their capacity to selectively recruit the components that constitute a given transcriptional complex. We have addressed the possibility of achieving novel transcriptional responses by the construction of a new transcriptional regulator--the Hap2-3-5-Gln3 hybrid modulator--harbouring the HAP complex polypeptides that constitute the DNA-binding domain (Hap2-3-5) and the Gln3 activation domain, which usually act in an uncombined fashion. The results presented in this paper show that transcriptional activation of GDH1 and ASN1 under repressive nitrogen conditions is achieved through the action of the novel Hap2-3-5-Gln3 transcriptional regulator. We propose that the combination of the Hap DNA-binding and Gln3 activation domains results in a hybrid modulator that elicits a novel transcriptional response not evoked when these modulators act independently.

Towards a collaborative structure of interpreter-mediated medical consultations: Complementing functions between healthcare interpreters and providers.

In today's multilingual and multicultural societies, healthcare interpreters are increasingly needed to mitigate communication barriers in language-discordant, intercultural medical consultations. To orient these interactions, existing guidelines, best practices and recommendations shed light on the behaviour and responsibilities of interpreters and healthcare providers involved. These documents, however, mainly treat both professionals as individuals that take care of separate, unrelated dimensions of consultations, thus failing to address how they can work collaboratively. This seems to be particularly relevant if we consider that prescriptive documents advocate for an invisible interpreter rather than an active participant, consequently ignoring the positive functions interpreters are playing when they step out of their prescribed roles. In this context, this paper sets out to explore potential collaboration between both professional groups to improve communication as a whole. Drawing on Goffman's production format (1981), we examined excerpts from real interpreter-mediated medical consultations that took place at a public hospital in Madrid (Spain) over a period of five months (February-June 2017). Data analysis reveals that interpreters enact an author role as main participants of consultations and serve several functions in medical encounters, consequently sharing some of the responsibilities which are conventionally seen as doctors'. This may reveal potential areas of interest for interprofessional collaboration. In addition to interpreting, participants performed other clinical functions, thus accounting for complementary functions of that performed by healthcare providers. Interpreters act as clinical and therapeutic allies, patient empowerers and metalinguistic negotiators. In light of our findings, the next step is to design a new model for the interpreter-mediated medical consultations that integrates both perspectives in a collaborative, non-excluding proposal.

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy.

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

Novel Polymeric Formulation for Removal of Gastrointestinal Polyps by Digestive Endoscopy.

Endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) are two techniques used in the resection of gastrointestinal mucosal polyps. The aim of this work is the development and evaluation of an innovative polymeric solution containing sodium carboxymethylcellulose and hyaluronic acid. For this purpose, several mixtures of these two main components, as well as other components such as fructose, citric acid, and zinc, are evaluated in terms of physicochemical and microbiological properties, rheological behavior, extensibility, syringeability, and stability at different storage conditions. Furthermore, the potential production of mucosal elevation and duration is also studied by an ex vivo model using porcine stomach and colon. Results show that the developed polymeric solutions possess optimal values of pH, from 4.58 to 6.63, for their use in the gastrointestinal tract. The formulations exhibit both Newtonian and pseudoplastic behaviors with different viscosity values as a function of their composition. All formulations exhibit high stability properties and no bacterial or fungal growth is detected. MCS01 and MCS05 are the polymeric solutions with the best syringeability results. In this line, MCS05 is the formulation that provides the highest, 2.20 ± 0.18 cm and 1.40 ± 0.11 cm, and longest-lasting, for more than 120 min, elevation effect on porcine submucosal stomach and colon tissues, respectively. Thus, it can be concluded that polymeric solution MCS05 might be considered as a promising tool for use in human EMR and ESD.

Efficacy and Safety of a Novel Submucosal Injection Solution for Resection of Gastrointestinal Lesions.

Endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) are minimally invasive and efficient techniques for the removal of gastrointestinal (GI) mucosal polyps. In both techniques, submucosal injection solutions are necessary for complete effectiveness and safety during the intervention to be obtained. The main objective of this study was to evaluate the efficacy and safety of a new sterile submucosal injection solution for EMR/ESD used within a clinical protocol in patients with intestinal polyps. We carried out a prospective study between 2016 and 2017 with patients who attended the Endoscopy Consultation-Digestive Department of Primary Hospital. Patients were selected for EMR/ESD after the application of clinical protocols. Thirty-six patients were selected (≥ 66 years with comorbidities and risk factors). Lesions were located mainly in the colon. Our solution presented an intestinal lift ≥ 60 min in EMR/ESD and a high expansion of tissue, optimum viscosity, and subsequent complete resorption. The genes S100A9 and TP53 presented an expression increase in the distal regions. TP53 and PCNA were the only genes whose expression was increased in polyp specimens vs. the surrounding tissue at the mRNA level. In EMR/ESD, our solution presented a prolonged effect at the intestinal level during all times of the intervention. Thus, our solution seems be an effective and safe alternative in cases of flat lesions in both techniques.

Telomere shortening is associated with poor prognosis and telomerase activity correlates with DNA repair impairment in non-small cell lung cancer.

BACKGROUND AND PURPOSE: Telomere function and DNA damage response pathways are frequently inactivated in cancer. Moreover, some telomere-binding proteins have been implicated in DNA repair. The purpose of this work consists of evaluating the prognostic impact of telomere dysfunction and its relationship with DNA repair systems in non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: We analysed 83 NSCLCs and their corresponding control samples obtained from patients submitted to surgery. Telomere function was evaluated by determining telomerase activity and telomere length. DNA repair expression assays were established by using cDNA arrays containing 96 DNA-repair genes and by Real Time Quantitative PCR. RESULTS: Our data indicated that telomere attrition was significantly associated with poor clinical outcome of patients (P=0.02), being this parameter a significant prognostic factor independent of tumour stage (P=0.012; relative risk=1.887; 95% CI: 1.147-3.102). DNA-repair gene expression studies showed down regulation of DCLRE1C and GTF2H1 and a clear FLJ10858 up regulation in tumour tissues, as compared to controls. In addition, a number of genes related to DNA-repair were significantly down regulated in tumours that reactivated telomerase (DCLRE1C, GTF2H1, PARP-3, MLH1, and TRF2). CONCLUSIONS: Telomere shortening emerged as a poor clinical evolution parameter in NSCLC. Moreover, results from this work suggest a relationship between the loss of several DNA repair genes and telomerase activity, which may be of relevance in the pathogenesis of non-small lung cancer.

[Leprosy: control of household contacts in the municipality of Londrina-PR for a ten-year period]. / Hanseníase: o controle dos contatos no município de Londrina-PR em um período de dez anos.

The purpose of this descriptive study is to analyze variables related to leprosy patients' household contacts who received treatment in Londrina-PR-Brazil for a ten-year period. The data analysis was based on the health service's records and from a system of infectious disease. Out of 1055 leprosy's patients, it was recorded 3394 contacts with an average of 3,2. The most exposed individuals were those aged up to 40 (71,5%); son/daughter (40.6%) and husband/wife (17.8%). Of the1731 contacts (51.0%) examined, 183 showed some signs of the disease: there were 16 confirmed cases, 47 were excluded and 120 did not finish the clinical investigation. Most of the contacts (51.6%) were exposed to the multibacillary forms and 12.8% proved they were vaccinated with two doses of BCG. It is possible to conclude that the follow-up of the contacts was not adequate.

Biological and clinical significance of MMP-2, MMP-9, TIMP-1 and TIMP-2 in non-small cell lung cancer.

Our main aim consists of investigating the clinical usefulness of gelatinases and their tissue inhibitors in non-small cell lung cancer (NSCLC). Thus, we have analysed in 111 NSCLCs, levels and activity of MMP-2, MMP-9, TIMP-1 and TIMP-2, by Enzymoimmunoassay and Gelatine zymography, respectively. Our data revealed higher MMP-2 net activity in the NSCLC population analyzed in this study, this parameter showing a significant association with the TNM stage of tumours (P=0.002). Moreover, MMP-9 levels were significantly associated with poor clinical evolution of patients (P=0.02). Also, disease-free survival time was higher for patients whose tumours showed TIMP-1 increased levels (P=0.04). Of interest, Cox multivariate analysis revealed that TIMP-1 levels can be considered as an independent prognostic factor in NSCLC. Relative Risk (RR) to tumour relapse was more than two times lower for patients showing high TIMP-1 levels (RR=0.420, P=0.041). Therefore, according to our results, we conclude that MMP-9 and TIMP-1 levels of synthesis could be useful for the selection of patients with potentially unfavourable clinical evolution in order to establish adjuvant therapy protocols. Among these parameters, TIMP-1 level evaluation emerges as the main factor to predict the clinical outcome of patients.

Expression of MMP-9 and TIMP-1 as prognostic markers in gastric carcinoma.

BACKGROUND/AIMS: The aim of the present work is to clarify the role of metalloproteinase-9 and its inhibitor in the evolution of gastric cancer after surgical resection. METHODOLOGY: We have studied 44 gastric cancer patients submitted to surgery. There were 13 proximal tumors, 16 located in the middle third and 15 in the distal one. Overall survival was 26% at 6 years. Metalloproteinase-9 and tissue inhibitor of metalloproteinase concentrations were investigated by means of ELISA in frozen samples of tumoral and normal gastric mucosa. RESULTS: Mean concentration of metalloproteinase-9 in tumoral tissue was 42 ng/mg of total protein, and this value was 6.9 times greater than the mean concentration in non-tumoral tissue. Cancer tissue also expressed higher levels of TIMP-1, 7.25 versus 4.39 ng/mg of protein. Higher levels of metalloproteinase expression in tumoral tissue, greater [metalloproteinase in tumor]/[metalloproteinase in non-tumor] ratio and greater [metalloproteinase]/[inhibitor] ratio in tumor cells, were all of them statistically related to a worse prognosis when T1 and T2 tumors were considered. CONCLUSIONS: The expression of metalloproteinase-9 or its inhibitor is related to a more aggressive phenotype of gastric cancer.

Impairment of stromelysin-1 transcriptional activity by promoter mutations in high microsatellite instability colorectal tumors.

Colorectal tumorigenesis is characterized by the sequential inactivation of a series of tumor suppressor genes (microsatellite-stable tumors) and genetic or epigenetic alterations in mismatch repair genes in nonpoliposic hereditary tumours and 13% to 15% of sporadic colorectal cancer [high microsatellite instability (MSI-H) tumors]. We hypothesized a molecular mechanism for MSI-H colorectal tumors related to matrix metalloproteinase 3 (MMP-3) promoter mutations, down-regulation of MMP-3 expression, and impairment of MMP-9 activation. We have now analyzed the 2.2-kb full MMP-3 promoter to assess the mutation distribution. The mutations found are restricted to the polymorphic region that includes the zinc-binding protein (ZBP-89) binding element. To show that these alterations were the cause of the low expression of this gene, we have generated three constructs with different MMP-3 promoters (wild type and two mutants) and we have expressed them in SW480 human colorectal cells. The basal transcriptional activity of wild-type MMP-3 promoter was much higher than the mutants activity. In addition, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transcriptional activity of wild-type MMP-3 promoter was 10-fold higher than the mutants activity. Dexamethasone inhibited the basal transcriptional activity of wild-type MMP-3 promoter and of the two mutants found in the MSI-H subgroup of colorectal tumors. Significantly, dexamethasone almost completely blunted the TPA-induced effect on wild-type MMP-3 promoter transcriptional activity and on the mutants, even below their basal activity. Our data show that mutations found in the polymorphic region of the MMP-3 promoter from MSI-H colorectal tumors impair its basal and induced transcriptional activity, which may contribute to their better clinical outcome.

The influence of crossbite in early development of mandibular bone asymmetries in paediatric patients.

BACKGROUND: All authors agree that posterior crossbite is a malocclusion that affects mandibular growth and may lead to skeletal asymmetry but there are few data on which age these modifications are easily quantifiable. MATERIAL AND METHODS: For this study, the researchers used x-ray records of 217 children between 6 and 9 years of age, in the mixed dentition stage and with unilateral posterior crossbite. All the horizontal variables were traced and evaluated by the principal researcher, using the tpsDig version 2 computer program. Subsequently, a descriptive and statistical analysis was carried out, using the SPSS 17.0 for Windows program. RESULTS AND DISCUSSION: After analysing the vertical mandibular traces on the x-rays, the researchers found, in all cases, quantifiable differences between the crossbite side and the non-crossbite side. The differences between horizontal variables were statistically significant (p<0.005) for the entire sample (H3-H4), in the group of boys (H3-H4) and in the 7-year old age group (H1-H2 and H3-H4). Differences were observed in the size of the horizontal measures between the crossbite side and the non-crossbite side. Some of these differences were significant as a function of the sex and age of the study sample. Key words:Crossbite, Mandibular asymmetry, Panoramic.

Características de los pacientes con dolor musculoesquelético moderado a intenso, tratados con comprimidos bucodispersables de paracetamol 325 mg/tramadol HCI 37,5 mg (Paxiflas(R)) respecto a otras formas orales de la misma combinación. Estudio PROPAX / Characteristics of patients suffering mild to severe pain treated with orodispersible tablets of paracetamol 325 mg/tramadol HCL 37,5mg (Paxiflas(R)) in comparison with other oral formulations containing the same combination. PROPAX study

OBJETIVO: Comparar el perfil del paciente con dolor musculoesquelético moderado a intenso en tratamiento con los comprimidos bucodispersables, Paxiflas® [tramadol HCl (37,5 mg) y paracetamol (325 mg)] respecto a otras combinaciones de tramadol HCl (37,5 mg) y paracetamol (325 mg). Secundariamente, se comparó la adherencia, satisfacción y preferencia entre grupos. MATERIALES Y MÉTODOS: El estudio Propax es un estudio postautorización, observacional, transversal, retrospectivo y multicéntrico. La variable principal fue el perfil sociodemográfico y clínico del paciente. La satisfacción de los pacientes en tratamiento se midió mediante el cuestionario genérico SATMED-Q; la adherencia, con el cuestionario Morisky-Green; y la preferencia, mediante una batería de preguntas con respuesta tipo Likert. RESULTADOS: Se evaluaron 835 pacientes. No se observaron diferencias estadísticamente significativas en el perfil clínico y sociodemográfico de los pacientes entre ambos grupos de tratamiento, pero sí entre los pacientes en tratamiento con Paxiflas®, en relación con la evaluación de la satisfacción, tanto en la puntuación total (p = 0,002) como en las dimensiones: la interferencia de los efectos secundarios de la medicación en las actividades cotidianas (p = 0,006), la comodidad de uso (p < 0,001) y la opinión general respecto a la medicación y su estado de salud (p = 0,010). Además, la preferencia de los pacientes por el tratamiento con Paxiflas® fue estadísticamente significativas (p < 0,001) en comparación con otras combinaciones orales de tramadol HCI (37,5 mg) y paracetamol (325 mg), incluyendo la percepción de rapidez en el alivio del dolor, comodidad de la medicación, sabor y sensación agradable, conveniencia del tamaño del comprimido, y elección final del tratamiento. CONCLUSIONES: Entre los distintos grupos de tratamiento no se encontraron diferencias estadísticamente significativas en el perfil de los pacientes, ni en el grado de adherencia a la medicación. Los comprimidos bucodispersables Paxiflas® han demostrado un mayor grado de satisfacción y preferencia por parte de los pacientes con dolor musculoesquelético agudo y crónico moderado e intenso frente a otras formas orales

OBJECTIVE: To compare profiles of patients suffering mild to severe pain treated with orodispersible formulation of paracetamol 325 mg/tramadol HCL 37,5 mg (Paxiflas®) in comparison with other oral formulations of paracetamol 325 mg/tramadol HCL 37,5mg. In addition, to compare adherence, satisfaction and preference between groups. MATERIALS AND METHODS: Propax is a postautorization, observational, cross-sectional retrospective and multicentre study. The primary variable was the clinic and sociodemographic profile of the patient. Patient satisfaction was measured by STAMED generic questionnaire; adherence was measured by Morisky-Green questionnaire and preference was measured using a battery of questions with Likert-like answer. RESULTS: A number 835 patients were evaluated. Clinical and sociodemographic statistically significant differences were not observed between both groups of patients. However, there were statistically significant differences among Paxiflas® group of patients in satisfaction assessment, in both total score (p = 0,002) and dimensions: side effects interferences in everyday life (p = 0,006), convenience and ease of use (p < 0,001) and overall opinion of medication and health condition (p = 0,010). In addition, patient preference of Paxiflas® over other oral combinations of tramadol HCI (37.5 mg) and paracetamol (325 mg) was statistically significant (p < 0,001), including perception of pain relief speed, medication convenience, taste and likeable sensation, tablet size convenience and final election of treatment. CONCLUSIONS: Statistically significant differences were not observed in neither patient profile nor adherence. Paxiflas® orodispersible tablets demonstrated to provide greater satisfaction and preference among patients suffering acute and mild chronic musculoskeletal pain than other oral formulations

Differential impact of p16 inactivation by promoter methylation in non-small cell lung and colorectal cancer: clinical implications.

Inactivation of p16 has been reported as one of the more frequent events in human carcinogenesis. In order to contribute to the knowledge of the impact of p16 silencing by promoter methylation, we have investigated p16 expression and inactivation of p16 by methylation in two of the major types of human cancer, non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). p16 expression was evaluated by Western blot, and p16 promoter methylation by a methylation-specific PCR procedure (MSP). Clinical correlations were established using the chi-square test, and distributions of disease-free survival (DFS) were estimated with the Kaplan-Meier method. Analyses for p16 revealed that 61.22% (60 of 98) of NSCLCs, and 32.9% (26 of 79) of CRCs here considered, lacked p16 expression. Moreover, 36.7% (22/60) of the non-small cell lung tumours without p16 expression showed p16 promoter methylation, detecting a significant correlation between p16 methylation and the histological subtype of squamous cell carcinomas (SCC) (P=0.04). With respect to CRCs, p16 promoter methylation was observed in 26.9% of tumours that lacked p16 expression (7/26), all tumours studied showing partial methylation. Survival studies demonstrated a clear correlation between p16 negative expression and poor prognosis in NSCLC patients. Moreover, we found a trend toward poor clinical evolution in the group of patients with tumours showing total p16 methylation, in NSCLC, without statistically significant differences in CRC. In conclusion, our results indicate that p16 alterations constitute a major molecular abnormality in NSCLC with a considerable prognosis impact, promoter methylation being an important mechanism involved in p16 silencing. In CRC, however, p16 promoter methylation could be considered as a less definitive molecular factor without prognostic implication, since partial methylation constitutes a prevalent mechanism.

Targeted gene delivery by new folate-polycationic amphiphilic cyclodextrin-DNA nanocomplexes in vitro and in vivo.

AIM: Development and evaluation of a new targeted gene delivery system by first preforming self-assembled nanocomplexes from a polycationic amphiphilic cyclodextrin (paCD) and pDNA and then decorating the surface of the nanoparticles with folic acid (FA). EXPERIMENTAL SECTION: The cyclodextrin derivative (T2) is a tetradecacationic structure incorporating 14 primary amino groups and 7 thioureido groups at the primary face of a cyclomaltoheptaose (ß-CD) core and 14 hexanoyl chains at the secondary face. RESULTS AND CONCLUSIONS: T2 complexed and protected pDNA (luciferase-encoding plasmid DNA, pCMVLuc) and efficiently mediated transfection in vitro and in vivo with no associated toxicity. The combination of folic acid with CDplexes afforded ternary nanocomplexes (Fol-CDplexes) that enhanced significantly the transfection activity of pCMVLuc in human cervix adenocarcinoma HeLa cells, especially when formulated with 1 µg FA/µg DNA. The observed transfection enhancement was associated to specific folate receptor (FR)-mediated internalization of Fol-CDplexes, as corroborated by employing a receptor-deficient cell line (HepG2) and an excess of free folic acid. The in vivo studies, including luciferase reporter gene expression and biodistribution, indicated that 24h after intravenous administration of the T2-pDNA nanocomplexes, transfection takes part mainly in the liver and partially in the lung. Interestingly, the corresponding Fol-CDplexes lead to an increase in the transfection activity in the lung and the liver compared to non-targeted CDplexes. Folate-CDplexes developed in this study have improved transfection efficiency and although various methods have been used for the preparation of ligand-DNA-complexes, covalent binding is usually needed and insoluble aggregates are formed unless the concentration of the components is minimized. However, the complexes developed by first time in this work were prepared by simple mixing. The synthetic nature of this formulation provides the potential of flexibility in terms of composition and the capability of inexpensive and large-scale production of the complexes. These nanovectors may be an adequate alternative to viral vectors for gene therapy in the future.

Paralogous ALT1 and ALT2 retention and diversification have generated catalytically active and inactive aminotransferases in Saccharomyces cerevisiae.

BACKGROUND: Gene duplication and the subsequent divergence of paralogous pairs play a central role in the evolution of novel gene functions. S. cerevisiae possesses two paralogous genes (ALT1/ALT2) which presumably encode alanine aminotransferases. It has been previously shown that Alt1 encodes an alanine aminotransferase, involved in alanine metabolism; however the physiological role of Alt2 is not known. Here we investigate whether ALT2 encodes an active alanine aminotransferase. PRINCIPAL FINDINGS: Our results show that although ALT1 and ALT2 encode 65% identical proteins, only Alt1 displays alanine aminotransferase activity; in contrast ALT2 encodes a catalytically inert protein. ALT1 and ALT2 expression is modulated by Nrg1 and by the intracellular alanine pool. ALT1 is alanine-induced showing a regulatory profile of a gene encoding an enzyme involved in amino acid catabolism, in agreement with the fact that Alt1 is the sole pathway for alanine catabolism present in S. cerevisiae. Conversely, ALT2 expression is alanine-repressed, indicating a role in alanine biosynthesis, although the encoded-protein has no alanine aminotransferase enzymatic activity. In the ancestral-like yeast L. kluyveri, the alanine aminotransferase activity was higher in the presence of alanine than in the presence of ammonium, suggesting that as for ALT1, LkALT1 expression could be alanine-induced. ALT2 retention poses the questions of whether the encoded protein plays a particular function, and if this function was present in the ancestral gene. It could be hypotesized that ALT2 diverged after duplication, through neo-functionalization or that ALT2 function was present in the ancestral gene, with a yet undiscovered function. CONCLUSIONS: ALT1 and ALT2 divergence has resulted in delegation of alanine aminotransferase activity to Alt1. These genes display opposed regulatory profiles: ALT1 is alanine-induced, while ALT2 is alanine repressed. Both genes are negatively regulated by the Nrg1 repressor. Presented results indicate that alanine could act as ALT2 Nrg1-co-repressor.