Driver mutations of the adenoma-carcinoma sequence govern the intestinal epithelial global translational capacity.

Deregulated global mRNA translation is an emerging feature of cancer cells. Oncogenic transformation in colorectal cancer (CRC) is driven by mutations in APC, KRAS, SMAD4, and TP53, known as the adenoma-carcinoma sequence (ACS). Here we introduce each of these driver mutations into intestinal organoids to show that they are modulators of global translational capacity in intestinal epithelial cells. Increased global translation resulting from loss of Apc expression was potentiated by the presence of oncogenic KrasG12D Knockdown of Smad4 further enhanced global translation efficiency and was associated with a lower 4E-BP1-to-eIF4E ratio. Quadruple mutant cells with additional P53 loss displayed the highest global translational capacity, paralleled by high proliferation and growth rates, indicating that the proteome is heavily geared toward cell division. Transcriptional reprogramming facilitating global translation included elevated ribogenesis and activation of mTORC1 signaling. Accordingly, interfering with the mTORC1/4E-BP/eIF4E axis inhibited the growth potential endowed by accumulation of multiple drivers. In conclusion, the ACS is characterized by a strongly altered global translational landscape in epithelial cells, exposing a therapeutic potential for direct targeting of the translational apparatus.

Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning.

During the suckling-to-weaning transition, the intestinal epithelium matures, allowing digestion of solid food. Transplantation experiments with rodent fetal epithelium into subcutaneous tissue of adult animals suggest that this transition is intrinsically programmed and occurs in the absence of dietary or hormonal signals. Here, we show that organoids derived from mouse primary fetal intestinal epithelial cells express markers of late fetal and neonatal development. In a stable culture medium, these fetal epithelium-derived organoids lose all markers of neonatal epithelium and start expressing hallmarks of adult epithelium in a time frame that mirrors epithelial maturation in vivoIn vitro postnatal development of the fetal-derived organoids accelerates by dexamethasone, a drug used to accelerate intestinal maturation in vivo Together, our data show that organoids derived from fetal epithelium undergo suckling-to-weaning transition, that the speed of maturation can be modulated, and that fetal organoids can be used to model the molecular mechanisms of postnatal epithelial maturation.

Elevation of ST-segment in aVR is predictive of cardiogenic shock but not of multivessel disease in inferior myocardial infarction.

INTRODUCTION: Some studies suggest that ST elevation in aVR (aVR-STE) can predict the presence of left main or multivessel disease (MVD) and relates to prognosis. Our purpose was to analyze the relationship of aVR-STE to MVD disease or cardiogenic shock (CS) in patients with inferior myocardial infarction (inferior STEMI). METHODS: We analyzed two cohorts of consecutive patients admitted for inferior STEMI in the Coronary Unit of two university hospitals. ST elevation and ST depression in each derivation were compared between patients with and without MVD and with and without CS. RESULTS: We included 342 patients-19.6% women and 80.4% men-with a median age of 60 (52, 70); 18 patients (5.2%) had MVD, and 25 (7.3%) patients presented CS. There was no relationship between ST elevation or ST depression in either derivation and MVD. In contrast, CS was associated with aVR-STE, ST-segment depression in lead aVL, and the sum of ST-segment depression. aVR-STE of 0.25 mm had a sensitivity of 24.0% and a specificity of 95.9% for CS. After multivariate analysis including clinical variables, aVR-STE was independently associated with CS. CONCLUSIONS: In patients with inferior STEMI, ST-segment analysis was not useful in predicting multivessel disease. aVR-STE was an independent predictor of CS, with high specificity but low sensitivity.

Localization of the culprit artery in inferior myocardial infarction: Influence of the point of measurement of ST segment.

BACKGROUND: There are several approaches widely used in the localization of the responsible artery in inferior myocardial infarction. However, the existing papers show differences in the point where the ST segment is measured. The purpose of our investigation is to analyse the influence of the point at which elevation of the ST segment is measured on the results of these algorithms. METHODS: We analysed the 12­lead electrocardiograms of 90 consecutive patients with inferior myocardial infarction. The ST segment elevation or depression was measured at the J-point and at 80 ms, and three algorithms were applied to predict the culprit artery with both measurements. Sensitivity, specificity, the area under the curve, and the kappa index of agreement were analysed to compare each algorithm at the J-point and at 80 ms. RESULTS: The area under the curve was better at the J-point than at 80 ms in two algorithms (0.696 vs. 0.635, p < 0.043, and 0.754 vs. 0.661, p < 0.045) and did not change in one. Agreement between the J-point and 80 ms was suboptimal in all three algorithms (0.71, 0.65, and 0.58). CONCLUSIONS: The result of different algorithms to detect the culprit artery in inferior STEMI patients can change significantly depending on the point where ST elevation or depression is measured.

Polyvinyl pyrrolidone-coated silver nanoparticles in a human lung cancer cells: time- and dose-dependent influence over p53 and caspase-3 protein expression and epigenetic effects.

The present study was aimed at providing a better understanding of the influence of silver nanoparticles (AgNPs) on the p53 tumor suppressor protein. Cell line A549 was exposed to a range of concentrations of AgNPs, and a time course (up to 72 h) of cell viability was determined. We also determined the time course of gene and protein expression of p53, p21, murine double minute 2 (MDM2) and caspase-3. The expression of all of these proteins was also determined after daily exposure of the cells to 10 µg/mL of AgNPs for 7 days, or after discontinuous exposure by treating the cells every 3 days, for 15 or 30 days. Moreover, epigenetic changes in the acetylation of the histone H3 protein and in global DNA methylation patterns were determined after 72 h of exposure. Results showed that daily exposure to low doses of AgNPs, or a single exposure to high concentrations for 72 h, decreased gene and protein expression of p53, p21, MDM2 and caspase-3 in A549 cells. In contrast, a discontinuous exposure to low doses or a single exposure to low concentrations for 72 h increased the levels of the active forms of p53 and caspase-3, as well as the p21 and MDM2 protein levels. In addition, exposure to high concentrations of AgNPs for 72 h induced higher levels of global DNA methylation and global histone H3 deacetylation in A549 cells. These results provide new information on the toxic action of AgNPs.

A transcriptome-based model of central memory CD4 T cell death in HIV infection.

BACKGROUND: Human central memory CD4 T cells are characterized by their capacity of proliferation and differentiation into effector memory CD4 T cells. Homeostasis of central memory CD4 T cells is considered a key factor sustaining the asymptomatic stage of Human Immunodeficiency Virus type 1 (HIV-1) infection, while progression to acquired immunodeficiency syndrome is imputed to central memory CD4 T cells homeostatic failure. We investigated if central memory CD4 T cells from patients with HIV-1 infection have a gene expression profile impeding proliferation and survival, despite their activated state. METHODS: Using gene expression microarrays, we analyzed mRNA expression patterns in naive, central memory, and effector memory CD4 T cells from healthy controls, and naive and central memory CD4 T cells from patients with HIV-1 infection. Differentially expressed genes, defined by Log2 Fold Change (FC) ≥ |0.5| and Log (odds) > 0, were used in pathway enrichment analyses. RESULTS: Central memory CD4 T cells from patients and controls showed comparable expression of differentiation-related genes, ruling out an effector-like differentiation of central memory CD4 T cells in HIV infection. However, 210 genes were differentially expressed in central memory CD4 T cells from patients compared with those from controls. Expression of 75 of these genes was validated by semi quantitative RT-PCR, and independently reproduced enrichment results from this gene expression signature. The results of functional enrichment analysis indicated movement to cell cycle phases G1 and S (increased CCNE1, MKI67, IL12RB2, ADAM9, decreased FGF9, etc.), but also arrest in G2/M (increased CHK1, RBBP8, KIF11, etc.). Unexpectedly, the results also suggested decreased apoptosis (increased CSTA, NFKBIA, decreased RNASEL, etc.). Results also suggested increased IL-1ß, IFN-γ, TNF, and RANTES (CCR5) activity upstream of the central memory CD4 T cells signature, consistent with the demonstrated milieu in HIV infection. CONCLUSIONS: Our findings support a model where progressive loss of central memory CD4 T cells in chronic HIV-1 infection is driven by increased cell cycle entry followed by mitotic arrest, leading to a non-apoptotic death pathway without actual proliferation, possibly contributing to increased turnover.

Chromatographic NMR Spectroscopy with Hollow Silica Spheres.

The use of micrometric hollow silica spheres is described as a strategy to reduce magnetic field inhomogeneities in the context of NMR chromatography. When employed as a stationary phase, hollow silica microspheres allow the use of common solution-state NMR instruments to measure the diffusion coefficient perturbation induced by the interaction of the analytes with the silica surface.

Correction to 'Ancient DNA and the tropics: a rodent's tale'.

The present erratum is in regards to our article entitled 'Ancient DNA and the tropics: a rodent's tale'. We were made aware of problems with some of the ancient sequences submitted to GenBank and conducted a systematic review of all the files used in our study. We discovered that, unfortunately, an incorrect file was sent to GenBank and was also used in some of our downstream analyses. We immediately contacted GenBank, explained the situation and corrected the file. We have redone some analyses with the correct file and describe these changes below.

Ancient DNA and the tropics: a rodent's tale.

Most genetic studies of Holocene fauna have been performed with ancient samples from dry and cold regions, in which preservation of fossils is facilitated and molecular damage is reduced. Ancient DNA work from tropical regions has been precluded owing to factors that limit DNA preservation (e.g. temperature, hydrolytic damage). We analysed ancient DNA from rodent jawbones identified as Ototylomys phyllotis, found in Holocene and Late Pleistocene stratigraphic layers from Loltún, a humid tropical cave located in the Yucatan peninsula. We extracted DNA and amplified six short overlapping fragments of the cytochrome b gene, totalling 666 bp, which represents an unprecedented success considering tropical ancient DNA samples. We performed genetic, phylogenetic and divergence time analyses, combining sequences from ancient and modern O. phyllotis, in order to assess the ancestry of the Loltún samples. Results show that all ancient samples fall into a unique clade that diverged prior to the divergence of the modern O. phyllotis, supporting it as a distinct Pleistocene form of the Ototylomys genus. Hence, this rodent's tale suggests that the sister group to modern O. phyllotis arose during the Miocene-Pliocene, diversified during the Pleistocene and went extinct in the Holocene.

Delays in diagnosis and surgery of sarcoma patients during the COVID-19 outbreak in Spain.

Background and objectives: Social distancing and quarantine implanted during the COVID-19 outbreak could have delayed the accession of oncologic patients to hospitals and treatments. This study analysed the management of sarcoma patients during this period in five Spanish hospitals. Design and methods: Clinical data from adult sarcoma patients, soft tissue and bone sarcomas, managed during the COVID-19 outbreak, from 15 March to 14 September 2020 (Covid cohort), were retrospectively collected and time for diagnosis, surgery and active treatments were compared with sarcoma patients managed during the same pre-pandemic period in 2018 (Control cohort). Results: A total of 126 and 182 new sarcoma patients were enrolled in the Covid and Control cohorts, respectively, who were mainly diagnosed as soft tissue sarcomas (81.0% and 80.8%) and at localized stage (80.2% and 79.1%). A diagnostic delay was observed in the Covid cohort with a median time for the diagnosis of 102.5 days (range 6-355) versus 83 days (range 5-328) in the Control cohort (p = 0.034). Moreover, a delay in surgery was observed in cases with localized disease from the Covid cohort with a median time of 96.0 days (range 11-265) versus 54.5 days (range 2-331) in the Control cohort (p = 0.034). However, a lower delay for neoadjuvant radiotherapy was observed in the Covid cohort with a median time from the diagnosis to the neoadjuvant radiotherapy of 47 days (range 27-105) versus 91 days (range 27-294) in the Control cohort (p = 0.039). No significant differences for adjuvant radiotherapy, neoadjuvant/adjuvant chemotherapy and neoadjuvant/adjuvant palliative chemotherapy were observed between both cohorts. Neither progression-free survival (PFS) nor overall survival (OS) was significantly different. Conclusion: Delays in diagnosis and surgery were retrospectively observed in sarcoma patients during the COVID-19 outbreak in Spain, while the time for neoadjuvant radiotherapy was reduced. However, no impact on the PFS and OS was observed.

N-terminal pro-B-type natriuretic peptide and pulmonary echography are predictors of acute heart failure needing early mechanical ventilation in acute coronary syndrome.

AIM: The aim of this study was to determine the best clinical predictors of acute heart failure needing mechanical ventilation (MV) in the first 48 h of evolution of patients admitted because of acute coronary syndrome (ACS). METHODS: We analyzed a cohort of patients admitted for ACS between February 2017 and February 2018. A pulmonary ultrasound was performed on admission and was considered positive (PE+) when there were three or more B-lines in two quadrants or more of each hemithorax. It was compared with N-terminal pro-B-type natriuretic peptide (NT-proBNP), peak troponin T-us value GRACE (Global Registry of Acute Coronary Events), CRUSADE (Can Rapid risk stratification of Unstable angina patients Suppress ADverse outcomes with Early implementation of the American College of Cardiology and American Heart Association guidelines - Bleeding Score), CACS (Canada Acute Coronary Syndrome risk score), and HAMIOT (Heart Failure after Acute Myocardial Infarction with Optimal Treatment score) scores, shock index, ejection fraction, chest X-ray, and Killip class at admission as predictors of MV in the first 48 h of admission. RESULTS: A total of 119 patients were included: 54.6% with ST elevation and 45.4% without ST elevation. Twelve patients (10.1%) required MV in the first 48 h of evolution. The sensitivity of PE+ was 100% (73.5-100%), specificity 91.6% (84.6-96.1%), and area under the curve was 0.96 (0.93-0.96). The sensitivity of an NT-proBNP value more than 3647 was 88.9% (51.9-99.7%), specificity 92.1% (84.5-96.8%), and area under the curve was 0.905 (0.793-1). The κ statistic between both predictors was 0.579. All the other scores were significantly worse than PE + . CONCLUSION: Lung ultrasound and a high NT-proBNP (3647 ng/L in our series) on admission are the best predictors of acute heart failure needing MV in the first 48 h of ACS. The agreement between both tests was only moderate.

Pro-inflammatory T cells-derived cytokines enhance the maturation of the human fetal intestinal epithelial barrier.

Small intestine (SI) maturation during early life is pivotal in preventing the onset of gut diseases. In this study we interrogated the milestones of SI development by gene expression profiling and ingenuity pathway analyses. We identified a set of cytokines as main regulators of changes observed across different developmental stages. Upon cytokines stimulation, with IFNγ as the most contributing factor, human fetal organoids (HFOs) increase brush border gene expression and enzyme activity as well as trans-epithelial electrical resistance. Electron microscopy revealed developed brush border and loss of fetal cell characteristics in HFOs upon cytokine stimulation. We identified T cells as major source of IFNγ production in the fetal SI lamina propria. Co-culture of HFOs with T cells recapitulated the major effects of cytokine stimulation. Our findings underline pro-inflammatory cytokines derived from T cells as pivotal factors inducing functional SI maturation in vivo and capable of modulating the barrier maturation of HFOs in vitro.

Estradiol increases cell growth in human astrocytoma cell lines through ERα activation and its interaction with SRC-1 and SRC-3 coactivators.

Estradiol (E2) regulates several cellular functions through the interaction with estrogen receptor subtypes, ERα and ERß, which present different functional and regulation properties. ER subtypes have been identified in human astrocytomas, the most common and aggressive primary brain tumors. We studied the role of ER subtypes in cell growth of two human astrocytoma cell lines derived from tumors of different evolution grades: U373 and D54 (grades III and IV, respectively). E2 significantly increased the number of cells in both lines and the co-administration with an ER antagonist (ICI 182, 780) significantly blocked E2 effects. ERα was the predominant subtype in both cell lines. E2 and ICI 182, 780 down-regulated ERα expression. The number of U373 and D54 cells significantly increased after PPT (ERα agonist) treatment but not after DPN (ERß agonist) one. To determine the role of SRC-1 and SRC-3 coactivators in ERα induced cell growth, we silenced them with RNA interference. Coactivator silencing blocked the increase in cell number induced by PPT. The content of proteins involved in proliferation and metastasis was also determined after PPT treatment. Western blot analysis showed that in U373 cells the content of PR isoforms (PR-A and PR-B), EGFR, VEGF and cyclin D1 increased after PPT treatment while in D54 cells only the content of EGFR was increased. Our results demonstrate that E2 induces cell growth of human astrocytoma cell lines through ERα and its interaction with SRC-1 and SRC-3 and also suggest differential roles of ERα on cell growth depending on astrocytoma grade.

Practical eight-frame algorithms for fringe projection profilometry.

In this paper we present several eight-frame algorithms for their use in phase shifting profilometry and their application for the analysis of semi-fossilized materials. All algorithms are obtained from a set of two-frame algorithms and designed to compensate common errors such as phase shift detuning and bias errors.

Synthesis of vitamin D3 analogues with A-ring modifications to directly measure vitamin D levels in biological samples.

C-3-substituted 25-hydroxyvitamin D3 analogues were synthesized as tools to directly measure levels of vitamin D in biological samples. The strategy involves vinyloxycarbonylation of the 3ß-hydroxy group and formation of a carbamate bond with a hydroxyl or amino group at the end of the alkyl chain. Biotinylated conjugates of synthesized derivatives were generated to be linked with vitamin D binding protein (DBP). The spacer group present in the alkyl chain is important in the binding of antibodies to the analogue-DBP complex. When compared to 25-hydroxyvitamin D3-DBP, the binding of some antibodies to the analogue-DBP complex of the 25-hydroxyvitamin D3 derivative 10 that posses an 8-aminoctyl alkyl chain is significantly reduced, but this analogue displaced [26,27-(3)H]-25-hydroxyvitamin D3 from DBP. In contrast, the 8-hydroxyoctyl alkyl chain analogue 9 showed less displacement.

Utility of pulmonary echography with a handheld ultrasound device in patients with acute coronary syndrome.

The objectives of this study are to establish the usefulness of lung ultrasound with a handheld device to predict the risk of developing heart failure with the need for mechanical ventilation (MV) in acute coronary syndrome (ACS). This is a prospective study of consecutive patients admitted because of ACS-type myocardial infarction, without data of HF at admission in a tertiary hospital, between February 2017 and February 2018. Lung ultrasounds were performed with a handheld cardiologic device in the first 24 h, and defined as echo-positive (PE+) when exams revealed 3 or more B-lines in 2 or more bilateral quadrants. We related this finding to the need for MV during admission. We included 119 patients (65.1 ± 12.8 year; 75.6% male, 24.4% female; 87.4% in Killip class I, 12.6% in Killip class II). Pulmonary echography was positive (PE+) in 21 patients (17.6%). The sensitivity of PE+ to predict MV was 93.3%, the specificity 93.3%, and the area under the curve 0.93. In Cox regression analysis adjusted by CRUSADE score and Killip class, PE+ patients had a hazard ratio of 64.55 (CI 7.87; 529.25, p < 0.001) of needing MV. PE+ was associated with more frequent use of inotropes and mortality. Pulmonary ultrasonography with a handheld echocardiograph was predictive of severe heart failure and the need for mechanical ventilation in ACS with high specificity and sensitivity.

Significant Increase in Cytomegalovirus (CMV) Infection in Solid Organ Transplants Associated With Increased Use of Thymoglobulin as Induction Therapy?

BACKGROUND: Cytomegalovirus (CMV) infection remains one of the most common viral pathogens affecting solid organ transplants (SOT). In 10 years of following the outcome of transplants, we noticed an increased incidence of CMV infection, along with increased use of rabbit anti-thymocyte globulin (rATG). The study aims to assess the incidence of active CMV infection and disease, response to treatment, and recurrence in a cohort of SOT. Furthermore, we look for correlating the CMV incidence with the type of induction therapy: r-ATG or interleukin 2 receptor-blocking antibody (basiliximab). METHODS: This was a single-center, retrospective 10-year study in patients submitted to kidney, kidney-liver, and kidney-pancreas transplants who used a preemptive therapy protocol for CMV. RESULTS: Among the 476 enrolled transplant recipients, 306 (64.2 %) had at least one episode of CMV infection (replication), and 71/306 patients (23.2 %) presented CMV-related disease. The most frequent clinical conditions associated with CMV disease were gastrointestinal. Among the 476 transplant patients, 333 received immunosuppressive induction with rATG (69.9 %); 140 (29.4 %) received induction with interleukin 2 receptor-blocking antibody (basiliximab). The initial maintenance immunosuppressive therapy in the patients who presented CMV infection was primarily performed with prednisone, tacrolimus, and sodium mycophenolate (91.7 %). The induction with rATG increased from 35.2%-94.6% in 10 years. The incidence of CMV infection was 20.7 % in the first year of observation and gradually increased to 87.3 % in the last year. CONCLUSIONS: The data suggest that the increase in the use of rATG in recent years could be responsible for the very expressive increase in the incidence of CMV infection/disease.

The clonal origins of dysplasia from intestinal metaplasia in the human stomach.

BACKGROUND & AIMS: Studies of the clonal architecture of gastric glands with intestinal metaplasia are important in our understanding of the progression from metaplasia to dysplasia. It is not clear if dysplasias are derived from intestinal metaplasia or how dysplasias expand. We investigated whether cells within a metaplastic gland share a common origin, whether glands clonally expand by fission, and determine if such metaplastic glands are genetically related to the associated dysplasia. We also examined the clonal architecture of entire dysplastic lesions and the genetic changes associated with progression within dysplasia. METHODS: Cytochrome c oxidase-deficient (CCO⁻) metaplastic glands were identified using a dual enzyme histochemical assay. Clonality was assessed by laser capture of multiple cells throughout CCO⁻ glands and polymerase chain reaction sequencing of the entire mitochondrial DNA (mtDNA) genome. Nuclear DNA abnormalities in individual glands were identified by laser capture microdissection polymerase chain reaction sequencing for mutation hot spots and microsatellite loss of heterozygosity analysis. RESULTS: Metaplastic glands were derived from the same clone-all lineages shared a common mtDNA mutation. Mutated glands were found in patches that had developed through gland fission. Metaplastic and dysplastic glands can be genetically related, indicating the clonal origin of dysplasia from metaplasia. Entire dysplastic fields contained a founder mutation from which multiple, distinct subclones developed. CONCLUSIONS: There is evidence for a distinct clonal evolution from metaplasia to dysplasia in the human stomach. By field cancerization, a single clone can expand to form an entire dysplastic lesion. Over time, this field appears to become genetically diverse, indicating that gastric cancer can arise from a subclone of the founder mutation.

The extracellular matrix controls stem cell specification and crypt morphology in the developing and adult mouse gut.

The rapid renewal of the epithelial gut lining is fuelled by stem cells that reside at the base of intestinal crypts. The signal transduction pathways and morphogens that regulate intestinal stem cell self-renewal and differentiation have been extensively characterised. In contrast, although extracellular matrix (ECM) components form an integral part of the intestinal stem cell niche, their direct influence on the cellular composition is less well understood. We set out to systematically compare the effect of two ECM classes, the interstitial matrix and the basement membrane, on the intestinal epithelium. We found that both collagen I and laminin-containing cultures allow growth of small intestinal epithelial cells with all cell types present in both cultures, albeit at different ratios. The collagen cultures contained a subset of cells enriched in fetal-like markers. In contrast, laminin increased Lgr5+ stem cells and Paneth cells, and induced crypt-like morphology changes. The transition from a collagen culture to a laminin culture resembled gut development in vivo. The dramatic ECM remodelling was accompanied by a local expression of the laminin receptor ITGA6 in the crypt-forming epithelium. Importantly, deletion of laminin in the adult mouse resulted in a marked reduction of adult intestinal stem cells. Overall, our data support the hypothesis that the formation of intestinal crypts is induced by an increased laminin concentration in the ECM.