Caracterización de la pérdida del gen NOMO1 en cáncer colorrectal

[ES] A nivel global, el cáncer colorrectal es el tercer tumor más frecuente y la segunda causa de muerte por cáncer, generando más de 900.000 fallecimientos cada año. El CCR se considera una enfermedad del adulto mayor donde la edad es el factor más influyente para su aparición, en consonancia con la mayoría de las enfermedades neoplásicas. Sin embargo, aunque la incidencia del CCR está en aumento debido al envejecimiento poblacional, la mortalidad por este tipo de tumor ha ido disminuyendo. Esto se debe a la implementación y mejora de los programas de cribado para el diagnóstico precoz a los que se someten las personas mayores de 50 años, así como a la aparición de estrategias terapéuticas más eficaces. En cambio, la incidencia del cáncer colorrectal en pacientes menores de 50 años (EOCRC) ha aumentado considerablemente en las últimas décadas, representando en el momento actual el 10% del total de casos de CCR. Así, el EOCRC esporádico (75-80% de todos los casos de EOCRC) presenta importantes limitaciones, tanto para su diagnóstico precoz como para la identificación de biomarcadores específicos que permitan diagnosticar, pronosticar y diseñar planes de tratamiento eficientes. Este subgrupo de pacientes se caracteriza por un diagnóstico de la enfermedad en estadio avanzado, peor pronóstico y por presentar fenotipos más agresivos del tumor. Sin embargo, a diferencia del CCR de aparición tardía, las bases genéticas y los mecanismos de carcinogénesis implicados en el EOCRC son, hoy en día, una incógnita por resolver. Un estudio preliminar de nuestro grupo, con el objetivo de identificar diferencias moleculares entre el CCR de aparición temprana y tardía, identificó una deleción recurrente en la región cromosómica 16p13.12-p13.11 en pacientes con EOCRC esporádico. Dentro de esta región se localiza el gen NOMO1, delecionado en homocigosis de forma somática en más del 80% de los casos de EOCRC, independientemente del estado de la región 16p156. Además, la pérdida en heterocigosis del gen NOMO1 también fue identificada en lesiones benignas precursoras de la enfermedad (pólipos). Con todo esto, la deleción del gen NOMO1 se posiciona como una alteración recurrente en EOCRC, aunque se desconocen los mecanismos moleculares alterados en tumores con este perfil genómico. Por ello, en este trabajo de tesis doctoral se hipotetiza que la pérdida del gen NOMO1 podría ser un evento precoz en el desarrollo del EOCRC, por lo que podría utilizarse como un biomarcador de malignización. Vistos estos antecedentes, se propusieron los siguientes objetivos con el fin de identificar los mecanismos moleculares que podrían estar alterados en aquellos tumores con inactivación de NOMO1: 1. Caracterizar funcionalmente la pérdida de NOMO1 en cáncer colorrectal de aparición temprana (EOCRC). 1.1 Generar distintas líneas celulares knockout (KO) para NOMO1 mediante el sistema de edición génica CRISPR/cas9. 1.2 Caracterizar el fenotipo de las líneas celulares NOMO1-KO. 1.3 Identificar posibles rutas de señalización alteradas tras la inactivación de NOMO1 mediante el análisis del perfil transcriptómico y proteómico de las líneas celulares carentes de este gen. 2. Determinar si la pérdida de Nomo1 es una mutación driver o passenger en EOCRC. 2.1 Generar un modelo murino condicional para Nomo1 mediante la herramienta de edición génica CRISPR/cas9. 2.2 Realizar un análisis anatomopatológico de los modelos murinos con inactivación de Nomo1. 3. Analizar la sensibilidad al tratamiento con 5-fluorouracilo, irinotecán, oxaliplatino y cisplatino de las líneas celulares carentes de NOMO1. 4. Estudiar la longitud telomérica y analizar su implicación en EOCRC

Recurrent NOMO1 gene deletion is a potential clinical marker in early-onset colorectal cancer and is involved in the regulation of cell migration

The incidence of early-onset colorectal cancer (EOCRC; age younger than 50 years) has been progressively increasing over the last decades globally, with causes unexplained. A distinct molecular feature of EOCRC is that compared with cases of late-onset colorectal cancer, in EOCRC cases, there is a higher incidence of Nodal Modulator 1 (NOMO1) somatic deletions. However, the mechanisms of NOMO1 in early-onset colorectal carcinogenesis are currently unknown. In this study, we show that in 30% of EOCRCs with heterozygous deletion of NOMO1, there were pathogenic mutations in this gene, suggesting that NOMO1 can be inactivated by deletion or mutation in EOCRC. To study the role of NOMO1 in EOCRC, CRISPR/cas9 technology was employed to generate NOMO1 knockout HCT-116 (EOCRC) and HS-5 (bone marrow) cell lines. NOMO1 loss in these cell lines did not perturb Nodal pathway signaling nor cell proliferation. Expression microarrays, RNA sequencing, and protein expression analysis by LC–IMS/MS showed that NOMO1 inactivation deregulates other signaling pathways independent of the Nodal pathway, such as epithelial–mesenchymal transition and cell migration. Significantly, NOMO1 loss increased the migration capacity of CRC cells. Additionally, a gut-specific conditional NOMO1 KO mouse model revealed no subsequent tumor development in mice. Overall, these findings suggest that NOMO1 could play a secondary role in early-onset colorectal carcinogenesis because its loss increases the migration capacity of CRC cells. Therefore, further study is warranted to explore other signalling pathways deregulated by NOMO1 loss that may play a significant role in the pathogenesis of the disease.This study was supported by the health research program of the Instituto de Salud Carlos III (Spanish Ministry of Economy and Competitiveness, PI20/01569 and PI20/0974), co-funded by FEDER funds, and Mutua Madrileña Foundation (FMM20/001). A.M.-M was supported by a predoctoral research grant from the Dr. Moraza Fundation (FMoraza18/001). P.G.V and N.G.-U were supported by a predoctoral research grant from the Consejería de Educación—Junta de Castilla y León. A.N.H. was supported by the National Institutes of Health K12 HD043483 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development

The First Stars: Mass Growth Under Protostellar Feedback

We perform three-dimensional cosmological simulations to examine the growth of metal-free, Population III (Pop III) stars under radiative feedback. We begin our simulation at z=100 and trace the evolution of gas and dark matter until the formation of the first minihalo. We then follow the collapse of the gas within the minihalo up to densities of n = 10^12 cm^-3, at which point we replace the high-density particles with a sink particle to represent the growing protostar. We model the effect of Lyman-Werner (LW) radiation emitted by the protostar, and employ a ray-tracing scheme to follow the growth of the surrounding H II region over the next 5000 yr. We find that a disk assembles around the first protostar, and that radiative feedback will not prevent further fragmentation of the disk to form multiple Pop III stars. Ionization of neutral hydrogen and photodissociation of H_2 by LW radiation leads to heating of the dense gas to several thousand Kelvin, and this warm region expands outward at the gas sound speed. Once the extent of this warm region becomes equivalent to the size of the disk, the disk mass declines while the accretion rate onto the protostars is reduced by an order of magnitude. This occurs when the largest sink has grown to ~ 20 M_sol while the second sink has grown to 7 M_sol, and we estimate the main sink will approach an asymptotic value of ~ 30 M_sol by the time it reaches the main sequence. Our simulation thus indicates that the most likely outcome is a massive Pop III binary. However, we simulate only one minihalo, and the statistical variation between minihaloes may be substantial. If Pop III stars were typically unable to grow to more than a few tens of solar masses, this would have important consequences for the occurence of pair-instability supernovae in the early Universe as well as the Pop III chemical signature in the oldest stars observable today.Comment: 21 pages, 11 figures, to appear in MNRA

The first stars: formation of binaries and small multiple systems

We investigate the formation of metal-free, Population III (Pop III), stars within a minihalo at z ~ 20 with a smoothed particle hydrodynamics (SPH) simulation, starting from cosmological initial conditions. Employing a hierarchical, zoom-in procedure, we achieve sufficient numerical resolution to follow the collapsing gas in the center of the minihalo up to number densities of 10^12 cm^-3. This allows us to study the protostellar accretion onto the initial hydrostatic core, which we represent as a growing sink particle, in improved physical detail. The accretion process, and in particular its termination, governs the final masses that were reached by the first stars. The primordial initial mass function (IMF), in turn, played an important role in determining to what extent the first stars drove early cosmic evolution. We continue our simulation for 5000 yr after the first sink particle has formed. During this time period, a disk-like configuration is assembled around the first protostar. The disk is gravitationally unstable, develops a pronounced spiral structure, and fragments into several other protostellar seeds. At the end of the simulation, a small multiple system has formed, dominated by a binary with masses ~ 40 M_Sun and ~ 10 M_Sun. If Pop III stars were to form typically in binaries or small multiples, the standard model of primordial star formation, where single, isolated stars are predicted to form in minihaloes, would have to be modified. This would have crucial consequences for the observational signature of the first stars, such as their nucleosynthetic pattern, and the gravitational-wave emission from possible Pop III black-hole binaries.Comment: Accepted to MNRAS. New section with new figure added. 18 pages, 13 figures. Supplementary material and high resolution version at http://www.as.utexas.edu/~minerva

Cosmological Hydrodynamics with Adaptive Mesh Refinement: a new high resolution code called RAMSES

A new N-body and hydrodynamical code, called RAMSES, is presented. It has been designed to study structure formation in the universe with high spatial resolution. The code is based on Adaptive Mesh Refinement (AMR) technique, with a tree based data structure allowing recursive grid refinements on a cell-by-cell basis. The N-body solver is very similar to the one developed for the ART code (Kravtsov et al. 97), with minor differences in the exact implementation. The hydrodynamical solver is based on a second-order Godunov method, a modern shock-capturing scheme known to compute accurately the thermal history of the fluid component. The accuracy of the code is carefully estimated using various test cases, from pure gas dynamical tests to cosmological ones. The specific refinement strategy used in cosmological simulations is described, and potential spurious effects associated to shock waves propagation in the resulting AMR grid are discussed and found to be negligible. Results obtained in a large N-body and hydrodynamical simulation of structure formation in a low density LCDM universe are finally reported, with 256^3 particles and 4.1 10^7 cells in the AMR grid, reaching a formal resolution of 8192^3. A convergence analysis of different quantities, such as dark matter density power spectrum, gas pressure power spectrum and individual haloes temperature profiles, shows that numerical results are converging down to the actual resolution limit of the code, and are well reproduced by recent analytical predictions in the framework of the halo model.Comment: 21 pages and 13 low resolution JPEG images. Accepted for publication in A&

Rotation Speed of the First Stars

We estimate the rotation speed of Population III (Pop III) stars within a minihalo at z ~ 20 using a smoothed particle hydrodynamics (SPH) simulation, beginning from cosmological initial conditions. We follow the evolution of the primordial gas up to densities of 10^12 cm^-3. Representing the growing hydrostatic cores with accreting sink particles, we measure the velocities and angular momenta of all particles that fall onto these protostellar regions. This allows us to record the angular momentum of the sinks and estimate the rotational velocity of the Pop III stars expected to form within them. The rotation rate has important implications for the evolution of the star, the fate encountered at the end of its life, and the potential for triggering a gamma-ray burst (GRB). We find that there is sufficient angular momentum to yield rapidly rotating stars (> 1000 km s^-1, or near break-up speeds). This indicates that Pop III stars likely experienced strong rotational mixing, impacting their structure and nucleosynthetic yields. A subset of them was also likely to result in hypernova explosions, and possibly GRBs.Comment: 14 pages, 7 figures, accepted for publication in MNRA

The Cold Big-Bang Cosmology as a Counter-example to Several Anthropic Arguments

A general Friedmann big-bang cosmology can be specified by fixing a half-dozen cosmological parameters such as the photon-to-baryon ratio Eta, the cosmological constant Lambda, the curvature scale R, and the amplitude Q of (assumed scale-invariant) primordial density fluctuations. There is currently no established theory as to why these parameters take the particular values we deduce from observations. This has led to proposed `anthropic' explanations for the observed value of each parameter, as the only value capable of generating a universe that can host intelligent life. In this paper, I explicitly show that the requirement that the universe generates sun-like stars with planets does not fix these parameters, by developing a class of cosmologies (based on the classical `cold big-bang' model) in which some or all of the cosmological parameters differ by orders of magnitude from the values they assume in the standard hot big-bang cosmology, without precluding in any obvious way the existence of intelligent life. I also give a careful discussion of the structure and context of anthropic arguments in cosmology, and point out some implications of the cold big-bang model's existence for anthropic arguments concerning specific parameters.Comment: 13 PRD-style pages, 2 postscript figures. Reference 26 corrected. Accepted to Phys. Rev.

A dualistic model of primary anal canal adenocarcinoma with distinct cellular origins, etiologies, inflammatory microenvironments and mutational signatures: implications for personalised medicine.

Primary adenocarcinoma of the anal canal is a rare and aggressive gastrointestinal disease with unclear pathogenesis. Because of its rarity, no clear clinical practice guideline has been defined and a targeted therapeutic armamentarium has yet to be developed. The present article aimed at addressing this information gap by in-depth characterising the anal glandular neoplasms at the histologic, immunologic, genomic and epidemiologic levels. In this multi-institutional study, we first examined the histological features displayed by each collected tumour (n = 74) and analysed their etiological relationship with human papillomavirus (HPV) infection. The intratumoural immune cell subsets (CD4, CD8, Foxp3), the expression of immune checkpoints (PD-1, PD-L1), the defect in mismatch repair proteins and the mutation analysis of multiple clinically relevant genes in the gastrointestinal cancer setting were also determined. Finally, the prognostic significance of each clinicopathological variable was assessed. Phenotypic analysis revealed two region-specific subtypes of anal canal adenocarcinoma. The significant differences in the HPV status, density of tumour-infiltrating lymphocytes, expression of immune checkpoints and mutational profile of several targetable genes further supported the separation of these latter neoplasms into two distinct entities. Importantly, anal gland/transitional-type cancers, which poorly respond to standard treatments, displayed less mutations in downstream effectors of the EGFR signalling pathway (i.e., KRAS and NRAS) and demonstrated a significantly higher expression of the immune inhibitory ligand-receptor pair PD-1/PD-L1 compared to their counterparts arising from the colorectal mucosa. Taken together, the findings reported in the present article reveal, for the first time, that glandular neoplasms of the anal canal arise by HPV-dependent or independent pathways. These etiological differences leads to both individual immune profiles and mutational landscapes that can be targeted for therapeutic benefits

Mechanical behaviour and formation process of silkworm silk gut

High performance silk fibers were produced directly from the silk glands of silkworms ("Bombyx mori") following an alternative route to natural spinning. This route is based on a traditional procedure that consists of soaking the silk glands in a vinegar solution and stretching them by hand leading to the so called silkworm guts. Here we present, to the authors’ best knowledge, the first comprehensive study on the formation, properties and microstructure of silkworm gut fibers. Comparison of the tensile properties and microstructural organization of the silkworm guts with those of naturally spun fibers allows gain of a deeper insight into the mechanisms that lead to the formation of the fiber, as well as the relationship between the microstructure and properties of these materials. In this regard, it is proved that an acidic environment and subsequent application of tensile stress in the range of 1000 kPa are sufficient conditions for the formation of a silk fiber