CRISPR-Cas9 Multiplex Editing of the α-Amylase/Trypsin Inhibitor Genes to Reduce Allergen Proteins in Durum Wheat

Wheat and its derived foods are widespread, representing one of the main food sources globally. During the last decades, the incidence of disorders related to wheat has become a global issue for the human population, probably linked to the spread of wheat-derived foods. It has been ascertained that structural and metabolic proteins, like \u3b1-amylase/trypsin inhibitors (ATI), are involved in the onset of wheat allergies (bakers' asthma) and probably Non-Coeliac Wheat Sensitivity (NCWS). The ATI are a group of exogenous protease inhibitors, which are encoded by a multigene family dispersed over several chromosomes in durum and bread wheat. WTAI-CM3 and WTAI-CM16 subunits are considered among the main proteins involved in the onset of bakers' asthma and probably NCWS. A CRISPR-Cas9 multiplexing strategy was used to edit the ATI subunits WTAI-CM3 and WTAI-CM16 in the grain of the Italian durum wheat cultivar Svevo with the aim to produce wheat lines with reduced amount of potential allergens involved in adverse reactions. Using a marker gene-free approach, whereby plants are regenerated without selection agents, homozygous mutant plants without the presence of CRISPR vectors were obtained directly from T0 generation. This study demonstrates the capability of CRISPR technology to knock out immunogenic proteins in a reduced time compared to conventional breeding programmes. The editing of the two target genes was confirmed either at molecular (sequencing and gene expression study) or biochemical (immunologic test) level. Noteworthy, as a pleiotropic effect, is the activation of the ATI 0.28 pseudogene in the edited lines

Melanoma, ancestralidad y variantes MC1R en la población mestizada uruguaya

Malignant melanoma (MM) is the most dangerous type of skin cancer and the main cause of death produced by skin diseases. In Uruguay, the incidence rate is 3.8/100,000, one of the highest in Latin America. We analyzed the contribution of ancestry and MC1R as a candidate gene for sporadic melanoma in Uruguay. Our objective wasto investigate the possible associations between ancestry and the MC1R gene with sporadic melanoma in the Uruguayan population. To that end, one hundred patients with sporadic MM and 107 controls were recruited. Phenotypic factors and lifestyle were evaluated as risk factors. At the same time, we analyzed fiveancestry informative markers, the MC1R variants (R151, R160 and D294H) and five tag-SNPs. Phototype, atypical nevi, sunburns and recreational exposure were the main risk factors for MM in the Uruguayan population. We confirmed 16q as a candidate region for MM. R151C, and R160W showed an important association with risk of melanoma (OR= 3.85, P= 1 x 10-2; OR= 10.15, P= 7 x 10-3, respectively). Furthermore, three novel MC1R haplotypes from the promoter region were detected,and the two most common haplotypes for the coding region were different to the ones found in Europeans through HapMap. However, MC1R coding region haplotypes revealed a highly similar frequency to that of the Spanish population. Our results showed that the chromosomal 16q region confers susceptibility to MM risk in the Uruguayan population. In addition, the admixed genome structure of the MC1R region could be part of the explanation of melanoma etiology.El melanoma maligno (MM) es uno de los más peligrosos, y la principal causa de muerte producida por tumores de piel. En el Uruguay, la tasa de incidencia es de 3,8/100.000, una de las más altas de América Latina. En este trabajo analizamos la ancestría y el gen candidato MC1R entre los pacientes con MM del Uruguay. Nuestro objetivo fue investigar la posible asociación entre ancestría y el gen MC1R en pacientes con melanoma esporádico en la población uruguaya. Con tal finalidad, se reclutaron 100 pacientes con MM esporádico y 107 controles. Se evaluó el riesgo de factores fenotípicos y de estilo de vida. Además se analizaron cinco marcadores informativos de ancesdencia, variantes del gen MC1R (R151, R160 y D294H) y cinco tagSNPs. El fototipo, los nevos atípicos, quemaduras solares y la exposición recreativa fueron los principales factores de riesgo para MM en la población uruguaya. La región cromosómica 16q es candidata para MM, mientras que R151C y R160W mostraron una importante asociación con el riesgo para MM (OR= 3,85, P= 1 x 10-2; OR= 10,15, P= 7 x 10-3, respectivamente). Por otra parte, se detectaron tres nuevos haplotipos en la región promotora y los dos haplotipos más frecuentes en la región codificante son diferentes a los encontrados en la población europea. Sin embargo, los haplotipos de la región codificante presentan una frecuencia muy similar a las encontradas en la población española. Los resultados muestran que la región cromosómica 16q confiere susceptibilidad al riesgo de MM en la población uruguaya. Por otra parte, la estructura genómica mestizada de la región del MC1R podría explicar la etiología del melanoma

Qui meurt après une néphrectomie pour cancer ? Étude des facteurs de risque de décès, des causes de décès et des réunions de morbi-mortalité (étude UroCCR-33)

BACKGROUND AND METHODS: Nephrectomy is the treatment for renal cell cancer from T1-4 tumors but remains at risk. To determine the thirty-day mortality rate after nephrectomy for cancer and to identify causes and risk factors of death in order to find clinical applications. From 2014 to 2017, we performed a retrospective multicentric analysis of prospectively collected data study involving the French network for research on kidney cancer (UroCCR). All patients who died after nephrectomy for cancer during the first thirty days were identified. Patients\u27 characteristics, causes of death and morbidity and mortality reviews reports were analyzed for each death. RESULTS AND LIMITATIONS: In total, 2578 patients underwent nephrectomy and 35 deaths occurred. The thirty-day mortality rate was 1.4%. In univariate analysis, symptoms at diagnosis (P=0.006, OR=2.56 IC (1.3-5.03)), c stage superior to cT1 (P<0.0001, OR=6.13 IC (2.8-13.2)), cT stage superior to cT2 (P<0.0001, OR=8.8 IC (4.39-17.8)), nodal invasion (P<0.0001, OR=4.6 IC (1.9-10.7)), distant metastasis (P=0.001, OR=4.01 IC (1.7-8.9)), open surgery (P<0.0001, OR=0.272 IC (0.13-0.54)) and radical nephrectomy (P=0.007, OR=2.737 IC (1.3-5.7)) were risk factors of thirty-day mortality. In a multivariable model, only cT stage superior to T2 (P=0.015, OR=3.55 IC (1.27-10.01)) was a risk factor of thirty-day mortality. The main cause of postoperative death was pulmonary (n=15; 43%). The second cause was postoperative digestive sepsis for 7 patients (20%). Only 2 morbidity and mortality reviews had been done for the 35 deaths. Limitations are related to the thirty-day mortality criteria and descriptive study design. CONCLUSIONS: Symptomatic patients, stage cTNM and type and techniques of surgery are determinants of thirty-day mortality after nephrectomy for cancer. The first cause of postoperative death is pulmonary. Morbidity and mortality reviews should be considered to better understand causes of death and to reduce early mortality after nephrectomy for cancer. LEVEL OF EVIDENCE: 4

Tight junctions: from simple barriers to multifunctional molecular gates

Epithelia and endothelia separate different tissue compartments and protect multicellular organisms from the outside world. This requires the formation of tight junctions, selective gates that control paracellular diffusion of ions and solutes. Tight junctions also form the border between the apical and basolateral plasma-membrane domains and are linked to the machinery that controls apicobasal polarization. Additionally, signalling networks that guide diverse cell behaviours and functions are connected to tight junctions, transmitting information to and from the cytoskeleton, nucleus and different cell adhesion complexes. Recent advances have broadened our understanding of the molecular architecture and cellular functions of tight junctions

Neurofuzzy modeling to determine recurrence risk following radical cystectomy for nonmetastatic urothelial carcinoma of the bladder

Purpose: Bladder cancer recurrence occurs in 40% of patients following radical cystectomy (RC) and pelvic lymphadenectomy (PLND). Although recurrence can be reduced with adjuvant chemotherapy, the toxicity and low response rates of this treatment restrict its use to patients at highest risk.We developed a neurofuzzymodel (NFM) to predict disease recurrence following RC and PLNDin patients who are not usually administered adjuvant chemotherapy. Experimental Design: The study comprised 1,034 patients treated with RC and PLND for bladder urothelial carcinoma. Four hundred twenty-five patients were excluded due to lymph node metastases and/or administration of chemotherapy. For the remaining 609 patients, we obtained complete clinicopathologic data relating to their tumor.We trained, tested, and validated two NFMs that predicted risk (Classifier) and timing (Predictor) of post-RC recurrence.We measured the accuracy of our model at various postoperative time points. Results: Cancer recurrence occurred in 172 (28%) patients. With a median follow-up of 72.7 months, our Classifier NFMidentified recurrence with an accuracy of 0.84 (concordance index 0.92, sensitivity 0.81, and specificity 0.85) and an excellent calibration.Thiswas better than two predictive nomograms (0.72 and 0.74 accuracies). The Predictor NFMs identified the timing of tumor recurrencewith a median error of 8.15 months. Conclusions:We have developed an accurate and well-calibrated model to identify disease recurrence following RC and PLND in patients with nonmetastatic bladder urothelial carcinoma. It seems superior to other available predictivemethods and could be used to identify patientswho would potentially benefit from adjuvant chemotherapy

Intérêt de la résection complémentaire (ou « second look ») pour les carcinomes urothéliaux de vessie classés pTa haut grade

International audienc

Prostate cancer proteomics: The urgent need for clinically validated biomarkers

Prostate cancer (PCa) is the most common cancer diagnosis and the second most common cause of cancer-related deaths in men. Currently, serum prostate-specific antigen (PSA) is the only biomarker widely used in the diagnosis and management of patients with PCa. However, PSA lacks diagnostic sensitivity and specificity, leading to false-negative and false-positive test results. PSA cannot distinguish indolent from aggressive disease, leading to many patients being over-treated with associated side-effects. Furthermore, PSA is unable to identify which tumors are likely to become unresponsive to treatment at an early stage. Thus, there is an urgent need for clinically validated biomarkers which will improve the diagnosis and management of PCa. Given the heterogeneity of PCa it is likely that a panel of biomarkers will be required. In the quest for PCa biomarkers, a wide range of samples including urine, serum, tissues, and cell lines have been studied using proteomic approaches such as 2-DE, SELDI-TOF, SILAC, ICAT, iTRAQ, and MALDI-IMS. The value of these technologies, and other emerging platforms such as selected reaction monitoring (SRM) and multiple reaction monitoring (MRM), are discussed in the context of biomarker discovery, validation and addressing the "bottle-necks" that exist prior to clinical translation. © 2009 Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim