Molecular Pathogenesis and Peripheral Monitoring of Adult Fragile X-Associated Syndromes.

Abnormal trinucleotide expansions cause rare disorders that compromise quality of life and, in some cases, lifespan. In particular, the expansions of the CGG-repeats stretch at the 5'-UTR of the Fragile X Mental Retardation 1 (FMR1) gene have pleiotropic effects that lead to a variety of Fragile X-associated syndromes: the neurodevelopmental Fragile X syndrome (FXS) in children, the late-onset neurodegenerative disorder Fragile X-associated tremor-ataxia syndrome (FXTAS) that mainly affects adult men, the Fragile X-associated primary ovarian insufficiency (FXPOI) in adult women, and a variety of psychiatric and affective disorders that are under the term of Fragile X-associated neuropsychiatric disorders (FXAND). In this review, we will describe the pathological mechanisms of the adult "gain-of-function" syndromes that are mainly caused by the toxic actions of CGG RNA and FMRpolyG peptide. There have been intensive attempts to identify reliable peripheral biomarkers to assess disease progression and onset of specific pathological traits. Mitochondrial dysfunction, altered miRNA expression, endocrine system failure, and impairment of the GABAergic transmission are some of the affectations that are susceptible to be tracked using peripheral blood for monitoring of the motor, cognitive, psychiatric and reproductive impairment of the CGG-expansion carriers. We provided some illustrative examples from our own cohort. Understanding the association between molecular pathogenesis and biomarkers dynamics will improve effective prognosis and clinical management of CGG-expansion carriers.

Molecular characterization of Spanish patients with MECP2 duplication syndrome.

MECP2 duplication syndrome (MDS) is an X-linked neurodevelopmental disorder characterized by a severe to profound intellectual disability, early onset hypotonia and diverse psycho-motor and behavioural features. To date, fewer than 200 cases have been published. We report the clinical and molecular characterization of a Spanish MDS cohort that included 19 boys and 2 girls. Clinical suspicions were confirmed by array comparative genomic hybridization and multiplex ligation-dependent probe amplification (MLPA). Using, a custom in-house MLPA assay, we performed a thorough study of the minimal duplicated region, from which we concluded a complete duplication of both MECP2 and IRAK1 was necessary for a correct MDS diagnosis, as patients with partial MECP2 duplications lacked some typical clinical traits present in other MDS patients. In addition, the duplication location may be related to phenotypic severity. This observation may provide a new approach for genotype-phenotype correlations, and thus more personalized genetic counselling.

Isolation Methods of Peripheral Blood Mononuclear Cells in Spanish Biobanks: An Overview.

The Spanish Hematic Derivatives Group, consisting of 26 biobanks, was established in 2011. We describe here the viability results of our publically available standard operating procedure to freeze and thaw peripheral blood mononuclear cells (PBMCs). Our protocol maximizes PBMC viability while avoiding where possible interbiobank and intrabiobank assay variability.

Transcriptome profile of the early stages of breast cancer tumoral spheroids.

Oxygen or nutrient deprivation of early stage tumoral spheroids can be used to reliably mimic the initial growth of primary and metastatic cancer cells. However, cancer cell growth during the initial stages has not been fully explored using a genome-wide approach. Thus, in the present study, we investigated the transcriptome of breast cancer cells during the initial stages of tumoral growth using RNAseq in a model of Multicellular Tumor Spheroids (MTS). Network analyses showed that a metastatic signature was enriched as several adhesion molecules were deregulated, including EPCAM, E-cadherin, integrins and syndecans, which were further supported by an increase in cell migration. Interestingly, we also found that the cancer cells at this stage of growth exhibited a paradoxical hyperactivation of oxidative mitochondrial metabolism. In addition, we found a large number of regulated (long non coding RNA) lncRNAs, several of which were co-regulated with neighboring genes. The regulatory role of some of these lncRNAs on mRNA expression was demonstrated with gain of function assays. This is the first report of an early-stage MTS transcriptome, which not only reveals a complex expression landscape, but points toward an important contribution of long non-coding RNAs in the final phenotype of three-dimensional cellular models.

Three cases with L1 syndrome and two novel mutations in the L1CAM gene.

UNLABELLED: Mutations in the L1CAM gene have been identified in the following various X-linked neurological disorders: congenital hydrocephalus; mental retardation, aphasia, shuffling gait, and adducted thumbs (MASA) syndrome; spastic paraplegia; and agenesis of the corpus callosum. These conditions are currently considered different phenotypes of a single entity known as L1 syndrome. We present three families with L1 syndrome. Sequencing of the L1CAM gene allowed the identification of the following mutations involved: a known splicing mutation (c.3531-12G>A) and two novel ones: a missense mutation (c.1754A>C; p.Asp585Ala) and a nonsense mutation (c.3478C>T; p.Gln1160Stop). The number of affected males and carrier females identified in a relatively small population suggests that L1 syndrome may be under-diagnosed. CONCLUSION: L1 syndrome should be considered in the differential diagnosis of intellectual disability or mental retardation in children, especially when other signs such as hydrocephalus or adducted thumbs are present.

Physicochemical and functional characterization of the collagen-polyvinylpyrrolidone copolymer.

Collagen-polyvinylpyrrolidone (C-PVP) is a copolymer that is generated from the γ irradiation of a mixture of type I collagen and low-molecular-weight PVP. It is characterized by immunomodulatory, fibrolytic, and antifibrotic properties. Here, we used various physicochemical and biological strategies to characterize the structure, biochemical susceptibility, as well as its effects on metabolic activity in fibroblasts. C-PVP contained 16 times more PVP than collagen, but only 55.8% of PVP was bonded. Nevertheless, the remaining PVP exerted strong structural activity due to the existence of weak bonds that provided shielding in the NMR spectra. On SEM and AFM, freeze-dried C-PVP appeared as a film that uniformly covered the collagen fibers. Size analysis revealed the presence of abundant PVP molecules in the solution of the copolymer with a unique dimension related to macromolecular combinations. Calorimetric analysis showed that the copolymer in solution exhibited structural changes at 110 °C, whereas the lyophilized form showed such changes at temperatures below 50 °C. The copolymer presented a rheopectic behavior, with a predominant effect of the collagen. C-PVP had biological effects on the expression of integrin α2 and prolyl-hydroxylase but did not interact with cells through the collagen receptors because it did not inhibit or slow contraction.

Negative clinical outcomes of medication resulting in emergency department visits.

PURPOSE: The results of analyses of patients' health problems related to medication use have been highly variable due to various factors, such as different study methodology, diverse variables determined, fields of study. The aim of our study was to determine the prevalence and preventability of negative clinical outcomes of medication (NCOMs). METHODS: This was a cross-sectional study performed in the emergency departments (EDs) of nine Spanish hospitals during a 3-month period. A two-stage probabilistic sampling method was used , and a systematic appraisal tool was used to identify the NCOMs based on information gathered through patient interview and review of the medical records. Case evaluations were conducted in two phases by pharmacists and physicians. The prevalence and preventability of NCOM were calculated. A homogeneity test was performed to assess potential differences in the prevalence for each hospital. RESULTS: A total of 4,611 patients were included in the study. The overall prevalence of NCOMs was 35.7 % [95 % confidence interval (CI) 33.3-38.1]. These NCOMs could be divided into three categories: ineffectiveness (18.2 %; 95 % CI 16.2-20.1), necessity (14.9 %; 95 % CI 13.4-16.6), and lack of safety (2.4 %; 95 % CI 1.9-2.8). About 81 % (95 % CI 80.1-82.3) of the NCOMs could have been prevented. CONCLUSIONS: NCOMs provoked approximately one-third of visits to the EDs, and a high percentage of these were preventable. Implementation of strategies for patient safety and pharmaceutical care could help to prevent these problems and optimize the use of medications.

Mutational spectrum of the SPG4 (SPAST) and SPG3A (ATL1) genes in Spanish patients with hereditary spastic paraplegia.

BACKGROUND: Hereditary Spastic Paraplegias (HSP) are characterized by progressive spasticity and weakness of the lower limbs. At least 45 loci have been identified in families with autosomal dominant (AD), autosomal recessive (AR), or X-linked hereditary patterns. Mutations in the SPAST (SPG4) and ATL1 (SPG3A) genes would account for about 50% of the ADHSP cases. METHODS: We defined the SPAST and ATL1 mutational spectrum in a total of 370 unrelated HSP index cases from Spain (83% with a pure phenotype). RESULTS: We found 50 SPAST mutations (including two large deletions) in 54 patients and 7 ATL1 mutations in 11 patients. A total of 33 of the SPAST and 3 of the ATL1 were new mutations. A total of 141 (31%) were familial cases, and we found a higher frequency of mutation carriers among these compared to apparently sporadic cases (38% vs. 5%). Five of the SPAST mutations were predicted to affect the pre-mRNA splicing, and in 4 of them we demonstrated this effect at the cDNA level. In addition to large deletions, splicing, frameshifting, and missense mutations, we also found a nucleotide change in the stop codon that would result in a larger ORF. CONCLUSIONS: In a large cohort of Spanish patients with spastic paraplegia, SPAST and ATL1 mutations were found in 15% of the cases. These mutations were more frequent in familial cases (compared to sporadic), and were associated with heterogeneous clinical manifestations.

Analysis of HLA-E expression in human tumors.

Downregulation of MHC class Ia molecule expression is a widespread mechanism used by tumor cells to escape antitumor T-cell-mediated immune responses. However, it is not known why NK cells cannot lyse these MHC class-Ia-deficient tumor targets. Tumors must select additional routes of escape from NK cells. An attractive hypothesis is that the aberrant expression of nonclassical HLA class Ia molecules in tumors provides the required inhibitory signal to NK cells, rendering tumor cells resistant to NK lysis. To analyze the possible role of HLA-E molecules in providing tumor cells with an NK escape mechanism, we studied the cell surface expression of this HLA class Ib molecule in a variety of tumor cell lines with well-defined HLA class Ia alterations. Tests were done with the monoclonal antibody 3D12 recognizing cell surface HLA-E molecules. Our results indicate that HLA-E was mainly detected in leukemia-derived cell lines. In addition, HLA-E was detected in tumor cell lines of different origin. This expression was related with the availability of free beta(2)-microglobulin (beta(2)m) in the cytoplasm of tumor cells. Expression was detected in tumor cell lines showing an imbalance in heavy chain/beta(2)m expression, particularly in tumor cell lines with alterations in the expression of heavy-chain genes. Several lines of evidence favor these conclusions: (1) In the FM55 and NW145 melanoma tumor systems, the reduction in HLA class Ia expression paralleled the increased cell surface detection of HLA-E. (2) A cervical tumor (808) and a melanoma cell line (R22.2) expressing a single HLA-A1 allele also expressed HLA-E. (3) The addition of human beta(2)m to tumor cell lines that expressed the HLA-E(G) allele increased HLA-E cell surface expression. (4) There was no HLA-E cell surface expression in tumor cell lines with total loss of HLA class Ia expression, including cell lines with low transcription of HLA class I heavy chains or with beta(2)m mutations. Our findings suggest that the biological consequences of these cumulative genetic and molecular changes in tumor cells lead to the appearance of HLA-E in a limited number of tumor cell lines with peculiar phenotypic and genotypic characteristics, namely: HLA-class Ia downregulation, free beta(2)m and HLA-E(G) genotype. The aberrant HLA-E expression might be of particular biological relevance in those HLA tumor phenotypes that express a single HLA-A allele when NK inhibition is markedly reduced due to the downregulation of HLA-B and -C alleles.

[Evaluation and nutritional therapy in cirrhotic patients of the "Edgardo Rebagliati Martins" hospital]. / Evaluación y Terapia Nutricional en Pacientes Cirróticos del Hospital "Edgardo Rebagliati Martins"

A nutritional evaluation was carried out in 43 cirrhotic outpatients who came to the Digestive Disease Department of the Edgardo Rebagliati Martins Hospital for a checkup. The evaluation included a dietary composition analysis; nutritional status through the analysis of lean and fatty mass through anthropometric parameters (tricipital skin fold thickness, midarm muscle circumference, muscle arm circumference and creatinine excretion in the 24 h urine/height ratio), measuring of visceral proteins (albumin, transferrin) and bioelectric Impedance. Finally, a nutritional dietary intervention was made according to international standards for cirrhotic patients. The Child - Pugh score was: Child A: 16 patients (37.1%), Child B: 20 patients (46.5%) and Child C: 7 patients (16.4%). The dietary analysis showed the following: Hypoprotein diets in 42 patients (98%), hypocaloric diets in 38 patients (88%), low fat diets in 36 patients (83.7%) only one patient had a normal diet. Nutritional status was normal in 15 patients (35%), 24 patients had protein malnutrition (56%) and 4 patients had protein-calorie malnutrition (9.3%). There was an association between Child B and C and the malnutrition diagnosis was (p = 0.045). The nutritional intervention was carried out in 19 of the 43 patients (dietary therapy) during 15 months; the results were 17 hospital admissions in the group without nutritional intervention and 6 in the group that had received nutritional intervention: (p = 0.01). Malnutrition and inadequate diets are becoming a prevalent problem in our cirrhotic population. Nutritional support may provide an additional and valuable therapy to decrease patient morbidity.

Sobre los antecedentes, consecuencias, variedad y aplicación farmacológica de los nutrientes enterales en el Perú / About antecedents, consequences, variety and farmacologic aplication of enteral nutrition in Peru

En el contexto de la realidad tecnológica latinoamericana se presentan el problema del soporte nutricional artificial en el Perú comparando la amplísima difusión del uso de antibiótico versus la marcada ausencia del uso de nutrición artificial, siendo ello un factor en contra del paciente seriamente comprometido dada la positiva gravitación del uso de soporte nutricional enteral sobre la inmunidad de un paciente. La presentación de un estudio prospectivo del uso de 3 diferentes fórmulas de nutrición enteral en 4 pacientes tipo, permiten mostrar las consecuencias adecuadas del uso de la nutrición enteral y las razones por las cuales el soporte nutricional artificial debe ser de aplicación sujeta a regulaciones legales en el Perú.

Sobre el tratamiento quirúrgico de fístulas gastrointestinales postoperatorias / Surgical treatment of postoperatory gastrointestinal fistulas

Se reporta un protocolo de estudio prospectivo de 10 años sobre 28 pacientes operados, por fístulas enterocutáneas postoperatorias en una unidad referencial quirúrgica de soporte nutricional artificial (SNA), de un total de 284 pacientes con dicho tipo de fístulas. Sobrevivieron 24/28 (85.7 por ciento) pacientes. Requirieron SNA, 26/28 (92.9 por ciento) pacientes en sus variedades enteral y/o parenteral. Se destacan tres aspectos: 1ro, la importancia vital de un equipo interdisciplinario de soporte nutricional artificial para obtener replección nutricional adecuada con mínima morbimortalidad derivadas de un soporte nutricional farmacológico , 2do., la falla orgánica múltiple como causa principal de mortalidad si el tratamiento quirúrgico no es exitoso, y 3ero, el manejo protocolizado de estos pacientes para la toma de decisiones quirúrgicas. El SNA adecuado es de vital importancia para evitar o dismimuir la morbimortalidad derivada de las fístulas que complican un acto quirúrgico así como es la piedra angular en donde descansa la posibilidad de solucionar quirúrgicamente esta complicación.

[SURGICAL TREATMENT OF POSTOPERATORY GASTROINTESTINAL FISTULAS] / Sobre el Tratamiento Quirúrgico deFístulas Gastrointestinales Postoperatorias.

A prospective 10-year protocol is reported. Twenty-eight patients were operated from postoperatory intercutaneous fistulas by the same surgeon. They were treated at a Surgical Referral Unit of Artificial Nutritional Support (SNA), acronym in Spanish from 284 patients with similar fistulas. Survival was of 85.7 per cent (24/28). A 92.9 per cent of patients (26/28) required SNA either enteral and/or parenteral. Three major aspects should be considered: First, the vital importance on an Artificial Nutritional Support team in order to attain nutritional repletion with the lowest possible morbimortality derived from a Phamacological Nutritional Support. Second, should surgical treatment be unsuccessful, multiple organ failure is the main cause of mortality. Third, surgical decision making shoul be based on established protocols.An adequate SNA is of major importance to avoid or decrease morbimortality derived from postoperatory fistulas, and at the same time, it is the cornerstone to resolve this complication surgically.