Wilson disease: revision of diagnostic criteria in a clinical series with great genetic homogeneity.

BACKGROUND: Wilson disease is an autosomal recessive disorder of copper metabolism caused by mutations in the ATP7B gene. An early diagnosis is crucial to prevent evolution of the disease, as implantation of early therapeutic measures fully prevents its symptoms. As population genetics data predict a higher than initially expected prevalence, it was important to define the basic diagnostic tools to approach population screening. METHODS: A highly genetically homogeneous cohort of 70 patients, belonging to 50 unrelated families, has been selected as a framework to analyze all their clinical, biochemical and genetic characteristics, to define the disease in our population, with an estimated prevalence of 1 in 12,369, and determine the most useful features that reach diagnostic value. RESULTS: Serum ceruloplasmin below 11.5 mg/dL and cupremia below 60 µg/mL, were the best analytical predictors of the disease in asymptomatic individuals, while cupruria or hepatic copper determination were less powerful. Genetic analysis reached a conclusive diagnosis in all 65 patients available for complete testing. Of them, 48 were carriers of at least one p.Leu708Pro mutant allele, with 24 homozygotes. Nine patients carried a promoter deletion mutation, revealing that extended sequencing beyond the ATP7B gene-coding region is essential. All mutations caused hepatic damage since early ages, increasing its severity as diagnosis was delayed, and neurological symptoms appear. CONCLUSION: Serum ceruloplasmin determination followed by genetic screening would reduce costs and favor the prioritization of non-invasive procedures to reach a definitive diagnosis, even for asymptomatic cases.

Suicidal risk, hopelessness and depression in patients with schizophrenia and internalized stigma.

BACKGROUND: Internalized stigma has negative consequences on subjective and objective aspects of the recovery in people diagnosed with mental disorders. Despite its relevance, it has been poorly studied in patients with schizophrenia. METHODS: A sample of 71 outpatients with a diagnosis of schizophrenia who attended Psychosocial Rehabilitation Centers of Gran Canaria, Spain, were evaluated. We assessed the prevalence of internalized stigma and its possible association with sociodemographic, clinical, psychological and psychopathological variables, as well as suicidal behaviour and suicidal ideation. RESULTS: 21.1% of the patients had internalized stigma. Internalized stigma was associated with higher prevalence of suicidal ideation during the last year, higher number of suicide attempts, higher current suicidal risk, worse self-compassion, higher self-esteem, higher scores on depression, higher prevalence of depression and higher hopelessness. After multivariate analysis, hopelessness and the existence of depression were independently associated with internalized stigma, although depression showed trend towards significance. CONCLUSIONS: The association between internalized stigma and higher hopelessness, depression and higher suicidal risk suggests the necessity to systematically assess internalized stigma in patients with schizophrenia, and to intervene to reduce it.

Riesgo suicida, desesperanza y depresión en pacientes con esquizofrenia y autoestigma / Suicidal risk, hopelessness and depression in patients with schizophrenia and internalized stigma

Introducción. El autoestigma tiene efectos negativos en aspectos subjetivos y objetivos de la recuperación en las personas con trastornos mentales. A pesar de su importancia, ha sido poco estudiado en la esquizofrenia. Metodología. Evaluamos el autoestigma en 71 pacientes diagnosticados de esquizofrenia, en Centros de Día de Rehabilitación Psicosocial de Gran Canaria. Se evaluó su prevalencia y su posible asociación con variables sociodemográficas, clínicas generales, psicopatológicas y aspectos psicológicos, así como la conducta suicida e ideación suicida. Resultados. El 21,1% de los pacientes presentaron autoestigma. El autoestigma se asoció a mayor prevalencia de ideación suicida en el último año, mayor número total de tentativas suicidas, mayor riesgo suicida actual, peor autocompasión, mayor autoestima y peores puntuaciones en depresión, mayor prevalencia de depresión y mayor desesperanza. En el análisis multivariante, la desesperanza y la existencia de depresión se asociaron de manera independiente con el autoestigma, si bien la depresión lo hizo con tendencia a la significación. Conclusiones. La asociación entre autoestigma y mayor desesperanza, depresión y riesgo suicida sugiere la necesidad de evaluar sistemáticamente el autoestigma en los pacientes con esquizofrenia, e intervenir para su reducción

Background. Internalized stigma has negative consequences on subjective and objective aspects of the recovery in people diagnosed with mental disorders. Despite its relevance, it has been poorly studied in patients with schizophrenia. Methods. A sample of 71 outpatients with a diagnosis of schizophrenia who attended Psychosocial Rehabilitation Centers of Gran Canaria, Spain, were evaluated. We assessed the prevalence of internalized stigma and its possible association with sociodemographic, clinical, psychological and psychopathological variables, as well as suicidal behavior and suicidal ideation. Results. 21.1% of the patients had internalized stigma. Internalized stigma was associated with higher prevalence of suicidal ideation during the last year, higher number of suicide attempts, higher current suicidal risk, worse self-compassion, higher self-esteem, higher scores on depression, higher prevalence of depression and higher hopelessness. After multivariate analysis, hopelessness and the existence of depression were independently associated with internalized stigma, although depression showed trend towards significance. Conclusions. The association between internalized stigma and higher hopelessness, depression and higher suicidal risk suggests the necessity to systematically assess internalized stigma in patients with schizophrenia, and to intervene to reduce it

Development of a POC test for TB based on multiple immunodominant epitopes of M. tuberculosis specific cell-wall proteins.

The need for an accurate, rapid, simple and affordable point-of-care (POC) test for Tuberculosis (TB) that can be implemented in microscopy centers and other peripheral health-care settings in the TB-endemic countries remains unmet. This manuscript describes preliminary results of a new prototype rapid lateral flow TB test based on detection of antibodies to immunodominant epitopes (peptides) derived from carefully selected, highly immunogenic M. tuberculosis cell-wall proteins. Peptide selection was initially based on recognition by antibodies in sera from TB patients but not in PPD-/PPD+/BCG-vaccinated individuals from TB-endemic settings. The peptides were conjugated to BSA; the purified peptide-BSA conjugates striped onto nitrocellulose membrane and adsorbed onto colloidal gold particles to devise the prototype test, and evaluated for reactivity with sera from 3 PPD-, 29 PPD+, 15 PPD-unknown healthy subjects, 10 patients with non-TB lung disease and 124 smear-positive TB patients. The assay parameters were adjusted to determine positive/negative status within 15 minutes via visual or instrumented assessment. There was minimal or no reactivity of sera from non-TB subjects with the striped BSA-peptides demonstrating the lack of anti-peptide antibodies in subjects with latent TB and/or BCG vaccination. Sera from most TB patients demonstrated reactivity with one or more peptides. The sensitivity of antibody detection ranged from 28-85% with the 9 BSA-peptides. Three peptides were further evaluated with sera from 400 subjects, including additional PPD-/PPD+/PPD-unknown healthy contacts, close hospital contacts and household contacts of untreated TB patients, patients with non-TB lung disease, and HIV+TB- patients. Combination of the 3 peptides provided sensitivity and specificity>90%. While the final fully optimized lateral flow POC test for TB is under development, these preliminary results demonstrate that an antibody-detection based rapid POC lateral flow test based on select combinations of immunodominant M. tb-specific epitopes may potentially replace microscopy for TB diagnosis in TB-endemic settings.

Development of a generic microfluidic device for simultaneous detection of antibodies and nucleic acids in oral fluids.

A prototype dual-path microfluidic device (Rheonix CARD) capable of performing simultaneously screening (antigen or antibody) and confirmatory (nucleic acid) detection of pathogens is described. The device fully integrates sample processing, antigen or antibody detection, and nucleic acid amplification and detection, demonstrating rapid and inexpensive "sample-to-result" diagnosis with performance comparable to benchtop analysis. For the chip design, a modular approach was followed allowing the optimization of individual steps in the sample processing process. This modular design provides great versatility accommodating different disease targets independently of the production method. In the detection module, a lateral flow (LF) protocol utilizing upconverting phosphor (UCP) reporters was employed. The nucleic acid (NA) module incorporates a generic microtube containing dry reagents. Lateral flow strips and PCR primers determine the target or disease that is diagnosed. Diagnosis of HIV infection was used as a model to investigate the simultaneous detection of both human antibodies against the virus and viral RNA. The serological result is available in less than 30 min, and the confirmation by RNA amplification takes another 60 min. This approach combines a core serological portable diagnostic with a nucleic acid-based confirmatory test.

Development of an ultrasensitive immunochromatography test to detect nicotine metabolites in oral fluids.

Passive exposure to tobacco smoke causes a variety of illnesses ranging from allergic responses to cancer. Assessment of exposure to second-hand tobacco smoke (SHS), particularly among vulnerable populations enables intervention and prevention of future disease. A minimally invasive oral fluids-based onsite test to detect such exposure would create a valuable tool for researchers and clinicians. Here we describe the development of a test that uses an inexpensive reader that utilizes a CMOS image sensor to reliably quantify a reporter signal and determine nicotine exposure. The rapid lateral flow test consists of a nitrocellulose strip with a control line containing goat anti-rabbit IgG, used as an internal standard, and a test line containing BSA-cotinine conjugate. To run the test, diluted sample containing antibodies against cotinine, the major metabolite of nicotine, is mixed with protein A-gold nanoparticles and placed on the sample pad. As the sample runs up to the nitrocellulose pad, antibodies in the running buffer bind to available cotinine. If cotinine is absent, the antibodies will bind to the BSA-cotinine derivative immobilized on the test line, resulting in an intense purple-red band. The concentration of cotinine equivalents in the sample can be estimated from interpretation of the test line. In this article we describe the effect of different cotinine derivatives, oral fluid pretreatment, and application and running buffers on assay sensitivity. The test can reliably detect as little as 2 ng mL(-1) cotinine equivalents. The assay is sensitive, simple, rapid, inexpensive, and easily implementable in point-of-care facilities to detect second-hand smoke exposure.

Designing an inducer-feeding schedule to enhance production of recombinant protein in Escherichia coli by microbial reaction engineering.

Metabolic constraints during the production of recombinant protein in Escherichia coli impede the efficient utilization of resources by the cells, thus reducing their production potential. In order to minimize these adverse effects, we have proposed to segregate the cell population into two groups: the first one formed by non-induced cells, growing at a high specific growth rate and rapidly contributing cells to the system, and the second one formed by fully induced cells, growing slowly but using the cell machinery to express the target protein. An adequate balance between these two populations should maximize the protein expression in a given system. This segregation is accomplished experimentally by taking advantage of the "all or none" phenomenon, in which at subsaturated inducer conditions the cells are either fully induced or fully uninduced. Based on this two-population theory, a mathematical model was developed in which a parameter alpha was defined as the fraction of the fully induced cells in the total population. In this study three different induction strategies were investigated and their effect on the protein production was established. It was found that the linear increase of this fraction, achieving maximum induction (alpha = 1) only at the end of the fermentation and with a slope m = 0.15 gave the best results. Finally these results were validated experimentally with the finding that they closely match the mathematical simulation with a 26% increase in protein production with respect to the conventional induction approach described.

A study of the movements of the human temporomandibular joint complex in the cadaver.

The objective of this study was to define the movements in the sagittal plane of the articular disk relative to the temporal bone and the mandibular condyle respectively, on jaw-opening in the absence of loads. Four temporomandibular joints from two human cadavers were used as materials in the study. Ten steel needles were placed in the lateral plane. Lateral photographs were taken of the articular area in different mandibular positions. OXY reference systems were adapted to the temporal and condylar points. The movements of the disk relative to the temporal bone are statistically different from those relative to the condyle. These findings are consistent with the new concept that the mandible joins the skull through two Temporomandibular Joint Complexes (TMJCs), one on each side and each one made up of a reciprocally fitting temporodiskal joint and a disko-condyle of the condylar type.