First female with Allan-Herndon-Dudley syndrome and partial deletion of X-inactivation center.

Allan-Herndon-Dudley is an X-linked recessive syndrome caused by pathogenic variants in the SLC16A2 gene. Clinical manifestations are a consequence of impaired thyroid metabolism and aberrant transport of thyroid hormones to the brain. Carrier females are generally asymptomatic and may show subtle symptoms of the disease. We describe a female with a complete Allan-Herndon-Dudley phenotype, carrying a de novo 543-kb deletion of the X chromosome. The deletion encompasses exon 1 of the SLC16A2 gene and JPX and FTX genes; it is known that the latter two genes participate in the X-inactivation process upregulating XIST gene expression. Subsequent studies in the patient demonstrated the preferential expression of the X chromosome with the JPX and FTX deletion.

Surfing transcriptomic landscapes. A step beyond the annotation of chromosome 16 proteome.

The Spanish team of the Human Proteome Project (SpHPP) marked the annotation of Chr16 and data analysis as one of its priorities. Precise annotation of Chromosome 16 proteins according to C-HPP criteria is presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of DNA Elements (ENCODE) data sets were used to obtain further information relative to cell/tissue specific chromosome 16 coding gene expression patterns and to infer the presence of missing proteins. Twenty-four shotgun 2D-LC-MS/MS and gel/LC-MS/MS MIAPE compliant experiments, representing 41% coverage of chromosome 16 proteins, were performed. Furthermore, mapping of large-scale multicenter mass spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines into RNA-Seq data allowed further insights relative to correlation of chromosome 16 transcripts and proteins. Detection and quantification of chromosome 16 proteins in biological matrices by SRM procedures are also primary goals of the SpHPP. Two strategies were undertaken: one focused on known proteins, taking advantage of MS data already available, and the second, aimed at the detection of the missing proteins, is based on the expression of recombinant proteins to gather MS information and optimize SRM methods that will be used in real biological samples. SRM methods for 49 known proteins and for recombinant forms of 24 missing proteins are reported in this study.

SpadaHC: a database to improve the classification of variants in hereditary cancer genes in the Spanish population.

Accurate classification of genetic variants is crucial for clinical decision-making in hereditary cancer. In Spain, genetic diagnostic laboratories have traditionally approached this task independently due to the lack of a dedicated resource. Here we present SpadaHC, a web-based database for sharing variants in hereditary cancer genes in the Spanish population. SpadaHC is implemented using a three-tier architecture consisting of a relational database, a web tool and a bioinformatics pipeline. Contributing laboratories can share variant classifications and variants from individuals in Variant Calling Format (VCF) format. The platform supports open and restricted access, flexible dataset submissions, automatic pseudo-anonymization, VCF quality control, variant normalization and liftover between genome builds. Users can flexibly explore and search data, receive automatic discrepancy notifications and access SpadaHC population frequencies based on many criteria. In February 2024, SpadaHC included 18 laboratory members, storing 1.17 million variants from 4306 patients and 16 343 laboratory classifications. In the first analysis of the shared data, we identified 84 genetic variants with clinically relevant discrepancies in their classifications and addressed them through a three-phase resolution strategy. This work highlights the importance of data sharing to promote consistency in variant classifications among laboratories, so patients and family members can benefit from more accurate clinical management. Database URL: https://spadahc.ciberisciii.es/.

Embryonic sex induces differential expression of proteins in bovine uterine fluid.

The bovine endometrium recognizes early embryos and reacts differently depending on the developmental potential of the embryo. However, it is unknown whether the endometrium can distinguish embryonic sex. Our objective was to analyze sexual dimorphism in the uterus in response to male and female embryos. Differentially expressed (DE) proteins, different levels of hexoses, and other embryotrophic differences were analyzed in uterine fluid (UF). Proteomic analysis of day-8 UF recovered from heifers after the transfer of day-5 male or female embryos identified 23 DE proteins. Regulated proteasome/immunoproteasome protein subunits indicated differences in antigen processing between UF carrying male embryos (male-UF) or female embryos (female-UF). Several enzymes involved in glycolysis/gluconeogenesis and antioxidative/antistress responses were up-regulated in female-UF. Fructose concentration was increased in female-UF versus male-UF, while glucose levels were similar. In vitro cultures with molecules isolated from male-UF were found to improve male embryo development compared to female embryos cultured with molecules isolated from female-UF. We postulated that, in vivo, male embryos induce changes in the endometrium to help ensure their survival. In contrast, female embryos do not appear to induce these changes.

HSV-1 Cgal+ infection promotes quaking RNA binding protein production and induces nuclear-cytoplasmic shuttling of quaking I-5 isoform in human hepatoma cells.

Herpesvirus type 1 (HSV-1) based oncolytic vectors arise as a promising therapeutic alternative for neoplastic diseases including hepatocellular carcinoma. However, the mechanisms mediating the host cell response to such treatments are not completely known. It is well established that HSV-1 infection induces functional and structural alterations in the nucleus of the host cell. In the present work, we have used gel-based and shotgun proteomic strategies to elucidate the signaling pathways impaired in the nucleus of human hepatoma cells (Huh7) upon HSV-1 Cgal(+) infection. Both approaches allowed the identification of differential proteins suggesting impairment of cell functions involved in many aspects of host-virus interaction such as transcription regulation, mRNA processing, and mRNA splicing. Based on our proteomic data and additional functional studies, cellular protein quaking content (QKI) increases 4 hours postinfection (hpi), when viral immediate-early genes such as ICP4 and ICP27 could be also detected. Depletion of QKI expression by small interfering RNA results in reduction of viral immediate-early protein levels, subsequent decrease in early and late viral protein content, and a reduction in the viral yield indicating that QKI directly interferes with viral replication. In particular, HSV-1 Cgal(+) induces a transient increase in quaking I-5 isoform (QKI-5) levels, in parallel with an enhancement of p27(Kip1) protein content. Moreover, immunofluorescence microscopy showed an early nuclear redistribution of QKI-5, shuttling from the nucleus to the cytosol and colocalizing with nectin-1 in cell to cell contact regions at 16-24 hpi. This evidence sheds new light on mechanisms mediating hepatoma cell response to HSV-1 vectors highlighting QKI as a central molecular mediator.

Proteome of the early embryo-maternal dialogue in the cattle uterus.

We analyzed embryo-maternal interactions in the bovine uterus on day 8 of development. Proteomic profiles were obtained by two-dimensional difference gel electrophoresis from 8 paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus. Results were contrasted with UF obtained after artificial insemination. We detected 50 differential protein spots (t test, p < 0.05). Subsequent protein characterization by nano-LC-ESI-MS/MS enabled us to identify 38 proteins, obtaining for first time the earliest evidence of involvement of the down-regulated NFkB system in cattle as a pregnancy signature pathway. Embryos enhanced the embryotrophic ability of UF and decreased uterine protein, while blood progesterone was unaltered. Twinfilin, hepatoma-derived growth factor, and synaptotagmin-binding cytoplasmic RNA interacting protein have not previously been identified in the mammalian uterus. TNFα and IL-1B were localized to embryos by immunocytochemistry, and other proteins were validated by Western blot in UF. Glycosylated-TNFα, IL-1B, insulin, lactotransferrin, nonphosphorylated-peroxiredoxin, albumin, purine nucleoside phosphorylase, HSPA5, and NFkB were down-regulated, while phosphorylated-peroxiredoxin, annexin A4, and nonglycosylated-TNFα were up-regulated. The embryonic signaling agents involved could be TNFα and IL-1B, either alone or in a collective dialogue with other proteins. Such molecules might explain the immune privilege during early bovine development.

Identification of replication-competent HSV-1 Cgal+ strain signaling targets in human hepatoma cells by functional organelle proteomics.

In the present work, we have attempted a comprehensive analysis of cytosolic and microsomal proteomes to elucidate the signaling pathways impaired in human hepatoma (Huh7) cells upon herpes simplex virus type 1 (HSV-1; Cgal(+)) infection. Using a combination of differential in-gel electrophoresis and nano liquid chromatography/tandem mass spectrometry, 18 spots corresponding to 16 unique deregulated cellular proteins were unambiguously identified, which were involved in the regulation of essential processes such as apoptosis, mRNA processing, cellular structure and integrity, signal transduction, and endoplasmic-reticulum-associated degradation pathway. Based on our proteomic data and additional functional studies target proteins were identified indicating a late activation of apoptotic pathways in Huh7 cells upon HSV-1 Cgal(+) infection. Additionally to changes on RuvB-like 2 and Bif-1, down-regulation of Erlin-2 suggests stimulation of Ca(2+)-dependent apoptosis. Moreover, activation of the mitochondrial apoptotic pathway results from a time-dependent multi-factorial impairment as inferred from the stepwise characterization of constitutive pro- and anti-apoptotic factors. Activation of serine-threonine protein phosphatase 2A (PP2A) was also found in Huh7 cells upon HSV-1 Cgal(+) infection. In addition, PP2A activation paralleled dephosphorylation and inactivation of downstream mitogen-activated protein (MAP) kinase pathway (MEK(1/2), ERK(1/2)) critical to cell survival and activation of proapoptotic Bad by dephosphorylation of Ser-112. Taken together, our results provide novel molecular information that contributes to define in detail the apoptotic mechanisms triggered by HSV-1 Cgal(+) in the host cell and lead to the implication of PP2A in the transduction of cell death signals and cell survival pathway arrest.

Regulation of stathmin phosphorylation in mouse liver progenitor-29 cells during proteasome inhibition.

Proteasome inhibitors are potential therapeutic agents in the treatment of hepatocarcinoma and other liver diseases. The analysis of alternative protein phosphorylation states might contribute to elucidate the underlying mechanisms of proteasome inhibitor-induced apoptosis. We have investigated the response of mouse liver progenitor-29 (MLP-29) cells to MG132 using a combination of phosphoprotein affinity chromatography, DIGE, and nano LC-MS/MS. Thirteen unique deregulated phosphoproteins involved in chaperone activity, stress response, mRNA processing and cell cycle control were unambiguously identified. Alterations in NDRG1 and stathmin suggest new mechanisms associated to proteasome inhibitor-induced apoptosis in MLP-29 cells. Particularly, a transient modification of the phosphorylation state of Ser(16), Ser(25) and Ser(38), which are involved in the regulation of stathmin activity, was detected in three distinct isoforms upon proteasome inhibition. The parallel deregulation of calcium/calmodulin-activated protein kinase II, extracellular regulated kinase-1/2 and cyclin-dependent kinase-2, might explain the modified phosphorylation pattern of stathmin. Interestingly, stathmin phosphorylation profile was also modified in response to epoxomicin treatment, a more specific proteasome inhibitor. In summary, we report here data supporting that regulation of NDRG1 and stathmin by phosphorylation at specific Ser/Thr residues may participate in the cellular response induced by proteasome inhibitors.

Fragile X-associated tremor/ataxia syndrome: Regional decrease of mitochondrial DNA copy number relates to clinical manifestations.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that appears in at least one-third of adult carriers of a premutation (55-200 CGG repeats) in the fragile X mental retardation 1 (FMR1) gene. Several studies have shown that mitochondrial dysfunction may play a central role in aging and also in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease as well as in FXTAS. It has been recently proposed that mtDNA copy number, measured by the number of mitochondrial genomes per nuclear genome (diploid), could be a useful biomarker of mitochondrial dysfunction. In order to elucidate the role of mtDNA variation in the pathogenesis of FXTAS, mtDNA copy number was quantified by digital droplet Polymerase chain reaction. In human brain samples, mtDNA levels were measured in the cerebellar vermis, dentate nucleus, parietal and temporal cortex, thalamus, caudate nucleus and hippocampus from a female FXTAS patient, a FMR1 premutation male carrier without FXTAS and from three male controls. The mtDNA copy number was further analyzed using this technology in dermal fibroblasts primary cultures derived from three FXTAS patients and three controls as well as in cortex and cerebellum of a CGG knock in FXTAS mice model. Finally, qPCR was carried out in human blood samples. Results indicate reduced mtDNA copy number in the specific brain region associated with disease progression in FXTAS patients, providing new insights into the role of mitochondrial dysfunction in the pathogenesis of FXTAS.

Agave mapisaga aqueous extract shows in vitroand in vivoactivity on murine prostate cancer cells / El extracto acuoso del Agave mapisaga presenta actividad in vitro e in vivo sobre células de cáncer de próstata murino

Plants are a source of multiple antineoplastic treatments. However, the effect of many species used in traditional medicine has yet to be demonstrated. In this work, the taxonomic identification of Agave mapisaga was made and a high-performance liquid chromatography-mass spectrometry (HPLC-MS) study suggested the presence of the aglycone hecogenin, which is part of compounds such as agavoside C and cantalasaponin 4. The antineoplastic activity of an aqueous extract was tested in vitro and in vivo on PEC-Src epithelial murine prostate cancer cells. In vitro study revelead a significant chemosensivity at 0.125 mg/100 µL (p=0.0001). Also, in in vivo, using an isotransplantation model with 1x106 cells subcutaneously, it was observed that the group treated with 50 mg/kg presented a lower tumor implantation compared with the control without treatment (p=0.04).

Las plantas son fuente de múltiples tratamientos antineoplásicos. Sin embargo, aún falta demostrar el efecto de muchas especies usadas en la medicina tradicional. En este trabajo se realizó la identificación taxonómica del Agave mapisaga y un estudio de cromatografía líquida de alta definición­masas (HPLC-MS) que sugirió la presencia de la aglicona hecogenina, que forma parte de compuestos como el agavósido C y la cantalasaponina 4. Se probó la actividad antineoplásica de un extracto acuoso in vitro e in vivo sobre células de cáncer de próstata murino epitelial PEC-Src. En el estudio in vitro se observó una actividad citotóxica significativa a partir de 0.125 mg/100 µL (p=0.0001). Mientras que, en los experimentos in vivo, se isotransplantaron 1x106 células por vía subcutánea, se observó que el grupo tratado con 50 mg/kg presentó una menor implantación tumoral con respecto del testigo sin tratamiento (p=0.04).

A multicentric study to evaluate the use of relative retention times in targeted proteomics.

Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. BIOLOGICAL SIGNIFICANCE: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups.

[Cardiac troponin I in perioperative myocardial infarction after coronary artery bypass surgery]. / Troponina I cardíaca en el infarto de miocardio perioperatorio tras cirugía de revascularización coronaria.

BACKGROUND AND AIMS: Myocardial infarction after coronary artery bypass grafting is a serious complication and one of the most common causes of perioperative morbidity and mortality. The present study was designed to determine the relevance of serum cardiac troponin I as a specific diagnostic marker for perioperative myocardial infarction. METHODS: A cohort of 64 patients undergoing coronary artery bypass grafting was enrolled for prospective study. Postoperative blood samples were extracted and analyzed for total creatine kinase (CK), CKMB and cardiac troponin I activity. Perioperative infarction was defined as the development of new Q waves in the postoperative electrocardiogram together with congruent regional wall motion abnormalities in the echocardiogram and CK values greater than 400 IU/l with MB fraction greater than 40 IU/l. RESULTS: Perioperative infarction occurred in 12 patients. Higher cardiac troponin I values were observed in patients experiencing perioperative myocardial infarction than in those without infarction (p < 0.001). Cardiac troponin I values higher than 12 ng/ml 10 h after release of the aortic clamp best detected the presence of perioperative myocardial infarction, with an area under the characteristic receiver operating curve of 0.91 (95% CI, 0.82-0.97), a sensitivity of 90.9%, and a specificity of 88.5%. The mean stay in the intensive care unit was significantly longer for patients who suffered perioperative myocardial infarction (6.5 8.6 days) than for patients without perioperative infarction (4.7 7.5 days) (p < 0.005). CONCLUSIONS: Cardiac troponin I elevation appears to be an early, specific marker for the diagnosis of perioperative myocardial infarction after coronary artery bypass grafting.

Proteomic analysis of chemonaive pediatric osteosarcomas and corresponding normal bone reveals multiple altered molecular targets.

With a view to identify the proteins involved in transformation, metastasis or chemoresistance in pediatric osteosarcoma, we carried out a new experimental approach based on comparison of the proteomic profile of paired samples of osteosarcoma and normal bone tissues from the same patient. The proteomic profiles of five pairs of cell lines (normal vs tumoral) were obtained by two-dimensional difference gel electrophoresis. We detected 56 differential protein spots (t test, p < 0.05). Subsequent protein characterization by nano-LC-ESI-MS/MS enabled us to identify some of these proteins, 16 of which were chosen on the basis of the change of their relative abundance between osteosarcomas and paired normal bones and also because their involvement was supported by the genomic analysis. Two of the 16 proteins, Alpha-crystallin B chain (CRYAB) and ezrin (EZR1), were selected for further studies: an immunohistochemical analysis of a TMA (tissue microarray) and real-time PCR for a set of 14 osteosarcoma/normal-bone pairs. The results of this second tier of studies confirmed that there were significant increases in the amounts of CRYAB and ezrin, especially in advanced stages of the disease. Our overall conclusion is that proteomic profiling of paired samples of osteosarcoma and normal bone tissues from the same patient is a practicable and potentially powerful way of initiating and proceeding with a search for proteins and genes involved in pediatric osteosarcoma.

Identification of replication-competent HSV-1 Cgal+ strain targets in a mouse model of human hepatocarcinoma xenograft.

Recent studies based on animal models have shown the advantages and potential of oncolytic viral therapy using HSV-1 -based replication-competent vectors in the treatment of liver tumors, but little is known about the cellular targets that are modulated during viral infection. In the present work, we have studied the effects of intratumoral injections of HSV-1 Cgal(+) strain in a murine model of human hepatoma xenografts. Viral replication was assessed for more than 1month, leading to a significant reduction of tumor growth rate mediated, in part, by a cyclin B dependent cell proliferation arrest. Early events resulting in this effect were analyzed using a proteomic approach. Protein extracts from xenografted human hepatomas treated with saline or HSV-1 Cgal(+) strain during 24h were compared by 2-D DIGE and differential spots were identified by nanoLC-ESI-MS/MS. Alterations on glutathione S transferase 1 Omega, and ERp29 suggest novel HSV-1 Cgal(+) targets in solid liver tumors. Additionally, ERp29 showed a complex differential isoform pattern upon HSV-1 Cgal(+) infection, suggesting regulatory mechanisms based on post-translational modification events.

Molecular profiling of hepatocellular carcinoma in mice with a chronic deficiency of hepatic s-adenosylmethionine: relevance in human liver diseases.

S-adenosylmethionine arises as a central molecule in the preservation of liver homeostasis as a chronic hepatic deficiency results in spontaneous development of steatohepatitis and hepatocellular carcinoma. In the present work, we have attempted a comprehensive analysis of proteins associated with hepatocarcinogenesis in MAT1A knock out mice using a combination of two-dimensional electrophoresis and mass spectrometry, to then apply the resulting information to identify hallmarks of human HCC. Our results suggest the existence of individual-specific factors that might condition the development of preneoplastic lesions. Proteomic analysis allowed the identification of 151 differential proteins in MAT1A-/- mice tumors. Among all differential proteins, 27 changed in at least 50% of the analyzed tumors, and some of these alterations were already detected months before the development of HCC in the KO liver. The expression level of genes coding for 13 of these proteins was markedly decreased in human HCC. Interestingly, seven of these genes were also found to be down-regulated in a pretumoral condition such as cirrhosis, while depletion of only one marker was assessed in less severe liver disorders.