Hypoxia-mediated repression of pyruvate carboxylase drives immunosuppression.

BACKGROUND: Metabolic plasticity mediates breast cancer survival, growth, and immune evasion during metastasis. However, how tumor cell metabolism is influenced by and feeds back to regulate breast cancer progression are not fully understood. We identify hypoxia-mediated suppression of pyruvate carboxylase (PC), and subsequent induction of lactate production, as a metabolic regulator of immunosuppression. METHODS: We used qPCR, immunoblot, and reporter assays to characterize repression of PC in hypoxic primary tumors. Steady state metabolomics were used to identify changes in metabolite pools upon PC depletion. In vivo tumor growth and metastasis assays were used to evaluate the impact of PC manipulation and pharmacologic inhibition of lactate transporters. Immunohistochemistry, flow cytometry, and global gene expression analyzes of tumor tissue were employed to characterize the impact of PC depletion on tumor immunity. RESULTS: PC is essential for metastatic colonization of the lungs. In contrast, depletion of PC in tumor cells promotes primary tumor growth. This effect was only observed in immune competent animals, supporting the hypothesis that repression of PC can suppress anti-tumor immunity. Exploring key differences between the pulmonary and mammary environments, we demonstrate that hypoxia potently downregulated PC. In the absence of PC, tumor cells produce more lactate and undergo less oxidative phosphorylation. Inhibition of lactate metabolism was sufficient to restore T cell populations to PC-depleted mammary tumors. CONCLUSIONS: We present a dimorphic role for PC in primary mammary tumors vs. pulmonary metastases. These findings highlight a key contextual role for PC-directed lactate production as a metabolic nexus connecting hypoxia and antitumor immunity.

Low-Cost, High-Pressure-Synthesized Oxygen-Entrapping Materials to Improve Treatment of Solid Tumors.

Tumor hypoxia drives resistance to many cancer therapies, including radiotherapy and chemotherapy. Methods that increase tumor oxygen pressures, such as hyperbaric oxygen therapy and microbubble infusion, are utilized to improve the responses to current standard-of-care therapies. However, key obstacles remain, in particular delivery of oxygen at the appropriate dose and with optimal pharmacokinetics. Toward overcoming these hurdles, gas-entrapping materials (GeMs) that are capable of tunable oxygen release are formulated. It is shown that injection or implantation of these materials into tumors can mitigate tumor hypoxia by delivering oxygen locally and that these GeMs enhance responsiveness to radiation and chemotherapy in multiple tumor types. This paper also demonstrates, by comparing an oxygen (O2 )-GeM to a sham GeM, that the former generates an antitumorigenic and immunogenic tumor microenvironment in malignant peripheral nerve sheath tumors. Collectively the results indicate that the use of O2 -GeMs is promising as an adjunctive strategy for the treatment of solid tumors.

Video games as a method of training basic laparoscopic skills. / Videojuegos como método de entrenamiento para las habilidades quirúrgicas laparoscópicas básicas.

INTRODUCTION: The use of video games has been proposed as an alternative to shorten the learning curve of basic laparoscopic skills. However, it is not yet clar how useful this practice is. METHODS: We searched in Epistemonikos, the largest database of systematic reviews in health, which is maintained by screening multiple information sources, including MEDLINE, EMBASE, Cochrane, among others. We extracted data from the systematic reviews, reanalyzed data of primary studies, conducted a meta-analysis and generated a summary of findings table using the GRADE approach. RESULTS AND CONCLUSIONS: We identified three systematic reviews including eight primary studies, of which four were randomized trials. We concluded video games training could help shorten the learning curve of basic laparoscopic visuospatial skills measured in a virtual platform, but the certainty of the available evidence is low.

INTRODUCCIÓN: El uso de videojuegos ha sido propuesto como alternativa para acortar la curva de aprendizaje de las habilidades laparoscópicas básicas. Sin embargo, aún no está clara su real utilidad. MÉTODOS: Realizamos una búsqueda en Epistemonikos, la mayor base de datos de revisiones sistemáticas en salud, la cual es mantenida mediante el cribado de múltiples fuentes de información, incluyendo MEDLINE, EMBASE, Cochrane, entre otras. Extrajimos los datos desde las revisiones identificadas, analizamos los datos de los estudios primarios, realizamos un metanálisis y preparamos una tabla de resumen de los resultados utilizando el método GRADE. RESULTADOS Y CONCLUSIONES: Se identificaron tres revisiones sistemáticas que en conjunto incluyeron ocho estudios primarios, de los cuales cuatro son ensayos aleatorizados. Se concluyó que el entrenamiento mediante el uso de videojuegos podría ayudar a acortar la curva de aprendizaje de habilidades visuoespaciales laparoscópicas básicas medido en una plataforma virtual, sin embargo la certeza de la evidencia disponible es baja.

Validation of a self-reported work disability questionnaire for ulcerative colitis.

Ulcerative colitis (UC) may severely limit patients' capacity to work. Recently, we validated a work disability questionnaire (WDQ) for Crohn disease. As UC shares clinical characteristics with Crohn disease, we hypothesized that the questionnaire might also be useful for UC. The study was aimed to validate the WDQ for use in UC.Consecutive patients with UC (n = 142, 67 women; age 48 ±â€Š1) completed the UC-WDQ and the inflammatory bowel disease questionnaire-9 (IBDQ-9), and EuroQoL-5D quality-of-life questionnaires. Validation of the UC-WDQ included an assessment of its construct validity, including: discriminant validity, convergent validity, and reproducibility (test-retest). We also calculated the intraclass correlation and the Cronbach alpha.The UC-WDQ is a valid and reliable tool for measuring work disability in patients with UC.

Development and Validation of the Short Crohn's Disease Work Disability Questionnaire.

BACKGROUND: The aim was to develop and validate a self-reported short Crohn's disease work disability questionnaire (sCDWDQ). METHODS: (1) Development of a shortened questionnaire-Patients' responses to the validation process (n = 108) of a previously developed, 16-item Spanish Crohn's disease work disability questionnaire (CDWDQ) were analyzed using the Rasch model for multiple response items. After this process, a 9-item sCDWDQ was obtained. (2) Validation phase-The validation assessed the questionnaire's convergent validity, discriminant validity, test-retest reproducibility, and internal consistency. Spearman rank correlation, t test, intra-class correlation and Cronbach's alpha were used for the analysis. RESULTS: One hundred fifty-one patients were included in the validation phase. (1) Convergent validity was confirmed by correlations between the sCDWDQ and clinical activity (r = 0.66, P < 0.01), the short inflammatory bowel disease questionnaire IBDQ-9 (r = 0.74, P < 0.001), Euroqol-5D (r = 0.63, P < 0.01), the EuroQol-5D visual analog scale (r = 0.54, P < 0.01), and overall work impairment (r = 0.66, P < 0.01); (2) Discriminant validity-sCDWDQ scores were higher in patients with active disease (20.1 ± 6.3 versus 13.0 ± 3.8 inactive, P < 0.001), in those requiring previous sick leave (19.6 ± 6.9 versus no sick leave 14.2 ± 4.8, P < 0.01) and in those requiring hospitalization (20.0 ± 7.3 [n = 29] versus no hospitalization 14.1 ± 7.3 [n = 90], P < 0.01); (3) Internal consistency was also good (Cronbach's alpha = 0.92); and (4) Reproducibility-sCDWDQ measures obtained 2 weeks apart showed an excellent intraclass correlation coefficient of 0.92 (95% confidence interval, 0.90-0.94). CONCLUSIONS: The self-reported sCDWDQ appears to be a simple, valid, and reliable tool for measuring work disability in Crohn's disease.

Subtype-specific genomic alterations define new targets for soft-tissue sarcoma therapy.

Soft-tissue sarcomas, which result in approximately 10,700 diagnoses and 3,800 deaths per year in the United States, show remarkable histologic diversity, with more than 50 recognized subtypes. However, knowledge of their genomic alterations is limited. We describe an integrative analysis of DNA sequence, copy number and mRNA expression in 207 samples encompassing seven major subtypes. Frequently mutated genes included TP53 (17% of pleomorphic liposarcomas), NF1 (10.5% of myxofibrosarcomas and 8% of pleomorphic liposarcomas) and PIK3CA (18% of myxoid/round-cell liposarcomas, or MRCs). PIK3CA mutations in MRCs were associated with Akt activation and poor clinical outcomes. In myxofibrosarcomas and pleomorphic liposarcomas, we found both point mutations and genomic deletions affecting the tumor suppressor NF1. Finally, we found that short hairpin RNA (shRNA)-based knockdown of several genes amplified in dedifferentiated liposarcoma, including CDK4 and YEATS4, decreased cell proliferation. Our study yields a detailed map of molecular alterations across diverse sarcoma subtypes and suggests potential subtype-specific targets for therapy.

Targeted quantitation of overexpressed and endogenous cystic fibrosis transmembrane conductance regulator using multiple reaction monitoring tandem mass spectrometry and oxygen stable isotope dilution.

Cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ion channel in the apical plasma membrane of epithelial cells. Mutations in the gene coding for CFTR cause cystic fibrosis (CF). A major cellular dysfunction is insufficient apical plasma membrane expression of the protein. Its correction is important for developing new CF therapeutics and treatments, which requires a sensitive and precise method for quantifying apical plasma membrane CFTR. We report the first method of liquid chromatography-tandem mass spectrometry for quantifying endogenous and overexpressed CFTR in HT29 and BHK cells. For low level of endogenous CFTR from HT29, the target protein in the cell lysate was enriched by immunoprecipitation using anti-CFTR antibody MAB3484 or M3A7. For overexpressed CFTR from BHK, the cell lysate prepared by differential detergent fractionation or surface biotinylation was used directly without immunoprecipitation. Proteins in the enriched CFTR preparations or cell lysates were digested with proteases, and a surrogate marker peptide designated as CFTR01 (NSILTETLHR) was successfully quantified using the method of multiple reaction monitoring and stable isotope dilution with an (18)O-labeled reference peptide (CFTR01-(18)O(4)) as the internal standard. CFTR quantified in this work ranged from a few tens of picograms to low nanograms per million of cells.

Averagine-scaling analysis and fragment ion mass defect labeling in peptide mass spectrometry.

A method termed as the averagine-scaling analysis (ASA) is proposed for predictive design and selection of chemical reagents for modifying peptides, as well as for facile mass spectral analysis of peptide fragment ions with increased mass defects. The ASA method scales mass spectral data using the mass of the hypothetical averagine residue as reference. The scaling analysis is used in conjunction with a strategy of fragment ion mass defect labeling (FIMDL) for effectively using the broad, unoccupied mass zones in the low m/ z region of mass spectra. The FIMDL approach involves the solution modification of peptide termini with chemical reagents of large mass defects and the gas-phase generation of peptide terminal fragment ions that carry the FIMDL groups. The scaling analysis reveals that iodine has the highest FIMDL efficiency among halogens. Iodine-containing reagents, 4-iodophenylisocyanate and 4-iodophenylisothiocyanate, are used to label primary amines on peptides to demonstrate the scaling analysis. The ASA method successfully distinguishes peptide fragment ions with and without an FIMDL group and specifically and efficiently reduces the data complexity of peptide tandem mass spectra. The combination of ASA with FIMDL extends the instrument suitability for the mass defect analysis from mass spectrometers of ultrahigh mass resolution and accuracy to those of medium ones. This combination is expected to have a profound impact on peptide tandem mass spectrometry.

Tandem parallel fragmentation of peptides for mass spectrometry.

Parallel fragmentations of peptides in the source region and in the collision cell of tandem mass spectrometers are sequentially combined to develop parallel collision-induced-dissociation mass spectrometry (p2CID MS). Compared to MS/MS spectra, the p2CID mass spectra show increased signal intensities (2-400-fold) and number of sequence ions. This improvement is attributed to the fact that p2CID MS virtually samples all the ions generated by electrospray ionization, including intact and fragment ions of different charge states from a peptide. We implement the method using a quadrupole time-of-flight tandem mass spectrometer. The instrument is operated in TOF-MS mode that allows the ions from source region broadband-passing the first mass analyzer to enter the collision cell. Cone voltage and collision energy are investigated to optimize the outcome of the two parallel CID processes. In the in-source parallel CID, elevated cone voltage produces singly charged intact peptide ions and large fragment ions, as well as decreases the charge-state distribution of peptide ions mainly to double and single charges. The in-collision-cell parallel CID is optimized to dissociate the ions from the source region to produce small and medium fragment ions. The method of p2CID MS is especially useful for sequencing of large peptides with labile amide bonds and peptides with C-terminal arginine. It has unique potential for de novo sequencing of peptides and proteome analysis, especially for affinity-enriched subproteomes.

Paladar Hendido, Tratamiento quirúrgico, Injerto óseo combinado con plasma rico en plaquetas

Hace unos años en las décadas 40 y 50 del siglo pasado se realizaron estudios, debido a que se observó hipodesarrollo del tercio medio de la cara como consecuencia del tratamiento quirúrgico en épocas tempranas de la vida del paciente con paladar fisurado. Cirujanos odontólogos comenzaron a utilizar el injerto óseo para conseguir la integridad anatómica del arco alveolar y así facilitar su desarrollo, la erupción dentaria y las funciones intermaxilares. Estas intervenciones son muy cruentas por la toma del transplante en zonas como la tibia, costilla e iliom. En la actualidad existen técnicas menos cruentas y agresivas como la que utiliza el plasma rico en plaquetas mezclado con hueso autógeno medular. Se hace el reporte de un caso.

Some years ago in the 40 and 50 decades of last century were carried out studies, because little development of the half third of the face was observed as consequence of the surgical treatment in early times of the patient’s life with cleft palate. Surgeons dentists began to use bone graft to get the anatomical integrity of the alveolar arch and this way to facilitate their development, the eruption would jag and the intermaxillary’s functions. These interventions are very bloody for the taking of the transplant in areas like the lukewarm one, rib and ilium. At the present time less bloody and aggressive techniques exist as the one that uses the platelet rich plasma in blended platelet with medullary autogenous bone. The repot of a case is made.

Anos a nas décadas 40 e 50 dos estudos do último século foram feitos, porque o hipo-desarrolho de terceiros meios da cara foi observado em conseqüência do tratamento cirúrgico em épocas adiantadas da vida do paciente com paladar fisurado. Os cirurgião dentistas começou a usar o enxerto ósseo poder a integridade anatômica do arco alveolar e facilitar assim seu desenvolvimento, o erupção dental e os intermaxilares das funções. Estes intervenção são muito sangrentos fazer exame do transplante nas zonas como o tibia, reforço e iliom. No tempo atual existem mais técnicas menos sangrentas e agressivas como qual usa o plasma rico nos plaquetas misturado com o osso medular autógeno. O relatório acontece com um caso.