Publications by authors named "Alberto Muñoz"

221 Publications

Organoids and Colorectal Cancer.

Cancers (Basel) 2021 May 28;13(11). Epub 2021 May 28.

Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid (UAM), 28029 Madrid, Spain.

Organoids were first established as a three-dimensional cell culture system from mouse small intestine. Subsequent development has made organoids a key system to study many human physiological and pathological processes that affect a variety of tissues and organs. In particular, organoids are becoming very useful tools to dissect colorectal cancer (CRC) by allowing the circumvention of classical problems and limitations, such as the impossibility of long-term culture of normal intestinal epithelial cells and the lack of good animal models for CRC. In this review, we describe the features and current knowledge of intestinal organoids and how they are largely contributing to our better understanding of intestinal cell biology and CRC genetics. Moreover, recent data show that organoids are appropriate systems for antitumoral drug testing and for the personalized treatment of CRC patients.
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http://dx.doi.org/10.3390/cancers13112657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8197877PMC
May 2021

Tankyrases as modulators of pro-tumoral functions: molecular insights and therapeutic opportunities.

J Exp Clin Cancer Res 2021 Apr 28;40(1):144. Epub 2021 Apr 28.

Instituto de Parasitología y Biomedicina López Neyra, CSIC, CIBERONC, 18016, Granada, Spain.

Tankyrase 1 (TNKS1) and tankyrase 2 (TNKS2) are two homologous proteins that are gaining increasing importance due to their implication in multiple pathways and diseases such as cancer. TNKS1/2 interact with a large variety of substrates through the ankyrin (ANK) domain, which recognizes a sequence present in all the substrates of tankyrase, called Tankyrase Binding Motif (TBM). One of the main functions of tankyrases is the regulation of protein stability through the process of PARylation-dependent ubiquitination (PARdU). Nonetheless, there are other functions less studied that are also essential in order to understand the role of tankyrases in many pathways. In this review, we concentrate in different tankyrase substrates and we analyze in depth the biological consequences derived of their interaction with TNKS1/2. We also examine the concept of both canonical and non-canonical TBMs and finally, we focus on the information about the role of TNKS1/2 in different tumor context, along with the benefits and limitations of the current TNKS inhibitors targeting the catalytic PARP domain and the novel strategies to develop inhibitors against the ankyrin domain. Available data indicates the need for further deepening in the knowledge of tankyrases to elucidate and improve the current view of the role of these PARP family members and get inhibitors with a better therapeutic and safety profile.
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http://dx.doi.org/10.1186/s13046-021-01950-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080362PMC
April 2021

Assessing the impact of COVID-19 on liver cancer management (CERO-19).

JHEP Rep 2021 Jun 23;3(3):100260. Epub 2021 Feb 23.

GI/Liver Unit, Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.

Background & Aims: The coronavirus disease 2019 (COVID-19) pandemic has posed unprecedented challenges to healthcare systems and it may have heavily impacted patients with liver cancer (LC). Herein, we evaluated whether the schedule of LC screening or procedures has been interrupted or delayed because of the COVID-19 pandemic.

Methods: An international survey evaluated the impact of the COVID-19 pandemic on clinical practice and clinical trials from March 2020 to June 2020, as the first phase of a multicentre, international, and observational project. The focus was on patients with hepatocellular carcinoma or intrahepatic cholangiocarcinoma, cared for around the world during the first COVID-19 pandemic wave.

Results: Ninety-one centres expressed interest to participate and 76 were included in the analysis, from Europe, South America, North America, Asia, and Africa (73.7%, 17.1%, 5.3%, 2.6%, and 1.3% per continent, respectively). Eighty-seven percent of the centres modified their clinical practice: 40.8% the diagnostic procedures, 80.9% the screening programme, 50% cancelled curative and/or palliative treatments for LC, and 41.7% modified the liver transplantation programme. Forty-five out of 69 (65.2%) centres in which clinical trials were running modified their treatments in that setting, but 58.1% were able to recruit new patients. The phone call service was modified in 51.4% of centres which had this service before the COVID-19 pandemic (n = 19/37).

Conclusions: The first wave of the COVID-19 pandemic had a tremendous impact on the routine care of patients with liver cancer. Modifications in screening, diagnostic, and treatment algorithms may have significantly impaired the outcome of patients. Ongoing data collection and future analyses will report the benefits and disadvantages of the strategies implemented, aiding future decision-making.

Lay Summary: The coronavirus disease 2019 (COVID-19) pandemic has posed unprecedented challenges to healthcare systems globally. Herein, we assessed the impact of the first wave pandemic on patients with liver cancer and found that routine care for these patients has been majorly disrupted, which could have a significant impact on outcomes.
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http://dx.doi.org/10.1016/j.jhepr.2021.100260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901294PMC
June 2021

Stellate cell computational modeling predicts signal filtering in the molecular layer circuit of cerebellum.

Sci Rep 2021 Feb 16;11(1):3873. Epub 2021 Feb 16.

Department of Brain and Behavioral Sciences, University of Pavia, Via Forlanini 6, 27100, Pavia, Italy.

The functional properties of cerebellar stellate cells and the way they regulate molecular layer activity are still unclear. We have measured stellate cells electroresponsiveness and their activation by parallel fiber bursts. Stellate cells showed intrinsic pacemaking, along with characteristic responses to depolarization and hyperpolarization, and showed a marked short-term facilitation during repetitive parallel fiber transmission. Spikes were emitted after a lag and only at high frequency, making stellate cells to operate as delay-high-pass filters. A detailed computational model summarizing these physiological properties allowed to explore different functional configurations of the parallel fiber-stellate cell-Purkinje cell circuit. Simulations showed that, following parallel fiber stimulation, Purkinje cells almost linearly increased their response with input frequency, but such an increase was inhibited by stellate cells, which leveled the Purkinje cell gain curve to its 4 Hz value. When reciprocal inhibitory connections between stellate cells were activated, the control of stellate cells over Purkinje cell discharge was maintained only at very high frequencies. These simulations thus predict a new role for stellate cells, which could endow the molecular layer with low-pass and band-pass filtering properties regulating Purkinje cell gain and, along with this, also burst delay and the burst-pause responses pattern.
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http://dx.doi.org/10.1038/s41598-021-83209-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886897PMC
February 2021

High levels of 27-hydroxycholesterol results in synaptic plasticity alterations in the hippocampus.

Sci Rep 2021 Feb 12;11(1):3736. Epub 2021 Feb 12.

Department of Neurobiology, Care Sciences and Society, Division of Neurogeriatrics, Center for Alzheimer Research, Karolinska Institutet, Stockholm, Sweden.

Alterations in brain cholesterol homeostasis in midlife are correlated with a higher risk of developing Alzheimer's disease (AD). However, global cholesterol-lowering therapies have yielded mixed results when it comes to slowing down or preventing cognitive decline in AD. We used the transgenic mouse model Cyp27Tg, with systemically high levels of 27-hydroxycholesterol (27-OH) to examine long-term potentiation (LTP) in the hippocampal CA1 region, combined with dendritic spine reconstruction of CA1 pyramidal neurons to detect morphological and functional synaptic alterations induced by 27-OH high levels. Our results show that elevated 27-OH levels lead to enhanced LTP in the Schaffer collateral-CA1 synapses. This increase is correlated with abnormally large dendritic spines in the stratum radiatum. Using immunohistochemistry for synaptopodin (actin-binding protein involved in the recruitment of the spine apparatus), we found a significantly higher density of synaptopodin-positive puncta in CA1 in Cyp27Tg mice. We hypothesize that high 27-OH levels alter synaptic potentiation and could lead to dysfunction of fine-tuned processing of information in hippocampal circuits resulting in cognitive impairment. We suggest that these alterations could be detrimental for synaptic function and cognition later in life, representing a potential mechanism by which hypercholesterolemia could lead to alterations in memory function in neurodegenerative diseases.
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http://dx.doi.org/10.1038/s41598-021-83008-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881004PMC
February 2021

Wnt and Vitamin D at the Crossroads in Solid Cancer.

Cancers (Basel) 2020 Nov 19;12(11). Epub 2020 Nov 19.

Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, 28029 Madrid, Spain.

Abnormal activation of the Wnt/β-catenin pathway is common in many types of solid cancers. Likewise, a large proportion of cancer patients have vitamin D deficiency. In line with these observations, Wnt/β-catenin signaling and 1α,25-dihydroxyvitamin D (1,25(OH)D), the active vitamin D metabolite, usually have opposite effects on cancer cell proliferation and phenotype. In recent years, an increasing number of studies performed in a variety of cancer types have revealed a complex crosstalk between Wnt/β-catenin signaling and 1,25(OH)D. Here we review the mechanisms by which 1,25(OH)D inhibits Wnt/β-catenin signaling and, conversely, how the activated Wnt/β-catenin pathway may abrogate vitamin D action. The available data suggest that interaction between Wnt/β-catenin signaling and the vitamin D system is at the crossroads in solid cancers and may have therapeutic applications.
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http://dx.doi.org/10.3390/cancers12113434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699248PMC
November 2020

Prefiltering based on experimental paradigm for analysis of fMRI complex brain networks.

PLoS One 2020 14;15(10):e0238994. Epub 2020 Oct 14.

Unidad de Diagnóstico por Imagen, Hospital Los Madroños, Madrid, Spain.

Brain networks offers a new insight about connections between function and anatomical regions of human brain. We present results from brain networks built from functional magnetic resonance images during finger tapping paradigm. Pearson voxel-voxel correlation in time and frequency domains were performed for all subjects. Besides this standard framework we have implemented a new approach consisting in filtering the data with respect to the fMRI paradigm (finger tapping) in order to obtain a better understanding of the network involved in the execution of the task. The main topological graph measures have been compared in both cases: voxel-voxel correlation and voxel-paradigm filtering plus voxel-voxel correlation. With the standard voxel-voxel correlation a clearly free-scale network was obtained. On the other hand, when we prefiltered the paradigm we obtained two different kind of networks: 1) free-scale; 2) random-like. To our best knowledge, this behaviour is reported here for first time for brain networks. We suggest that paradigm signal prefiltering can provide more infomation about the brain networks.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0238994PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556450PMC
November 2020

Safety of Chronic Simvastatin Treatment in Patients with Decompensated Cirrhosis: Many Adverse Events but No Liver Injury.

Dig Dis Sci 2020 Oct 9. Epub 2020 Oct 9.

Sección de Hepatología, Hospital de Gastroenterología Dr. Carlos Bonorino Udaondo, Facultad de Medicina, Universidad de Buenos Aires, Av. Caseros 2061 (1264), Ciudad Autónoma de Buenos Aires, Argentina.

Background: The high mortality rate of decompensated cirrhosis underlines the need for new treatments. Experimental models of cirrhosis and its reported relationship with atherosclerotic cardiovascular disease have provided data supporting the rational use of statins in these patients. However, little is known about the safety of statins in this setting.

Aim: We evaluate the safety of chronic simvastatin treatment in patients with decompensated cirrhosis.

Methods: We conducted a prospective, open, uncontrolled, phase 2a trial in 30 patients with Child-Pugh class A (n = 6), B (n = 22), and C (n = 2) decompensated cirrhosis. The patients received standard treatment throughout the trial plus simvastatin 20 mg/day for 2 weeks and thereafter simvastatin 40 mg/day up to 1 year.

Results: Sixteen out of 30 patients (53.3%) showed adverse events, including gastrointestinal toxicity (36.7%), muscle injury (MI) (36.7%), and headache (13.3%). No liver injury was registered. Due to MI alone, simvastatin dosage was reduced in 23.4% of cases and transiently interrupted in 13.3%. Once these adverse events were overcome, simvastatin was resumed until the end of the trial. MI was associated with baseline MELD score > 12 (p = 0.035) and with baseline Child-Pugh class C. No MI was associated with final Child-Pugh score ≤ 6 (p = 0.030) or final Child-Pugh class A (p = 0.020).

Conclusions: Chronic treatment with simvastatin 40 mg/day in patients with decompensated cirrhosis was associated with several adverse events, being MI the only clinically significant one, which appears to be related to the simvastatin dosage and the degree of cirrhosis severity. Noticeably, no liver injury was recorded.
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http://dx.doi.org/10.1007/s10620-020-06630-7DOI Listing
October 2020

Live-cell imaging of rapid calcium dynamics using fluorescent, genetically-encoded GCaMP probes with Aspergillus fumigatus.

Fungal Genet Biol 2021 Jun 24;151:103470. Epub 2020 Sep 24.

Manchester Fungal Infection Group, Division of Infection, Immunity and Respiratory Medicine, University of Manchester, CTF Building, 46 Grafton Street, Manchester M13 9NT, UK.

Calcium signalling plays a fundamental role in fungal intracellular signalling. Previous approaches (fluorescent dyes, bioluminescent aequorin, genetically encoded cameleon probes) with imaging rapid subcellular changes in cytosolic free calcium ([Ca]) in fungal cells have produced inconsistent results. Recent data obtained with new fluorescent, genetically encoded GCaMP probes, that are very bright, have resolved this problem. Here, exposing conidia or conidial germlings to high external Ca, as an example of an external stressor, induced very dramatic, rapid and dynamic [Ca] changes with localized [Ca] transients and waves. Considerable heterogeneity in the timing of Ca responses of different spores/germlings within the cell population was observed.
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http://dx.doi.org/10.1016/j.fgb.2020.103470DOI Listing
June 2021

Calcium homeostasis plays important roles in the internalization and activities of the small synthetic antifungal peptide PAF26.

Mol Microbiol 2020 10 17;114(4):521-535. Epub 2020 Jun 17.

Manchester Fungal Infection Group, Infection, Immunity & Respiratory Medicine, University of Manchester, Manchester, UK.

Fungal diseases are responsible for the deaths of over 1.5 million people worldwide annually. Antifungal peptides represent a useful source of antifungals with novel mechanisms-of-action, and potentially provide new methods of overcoming resistance. Here we investigate the mode-of-action of the small, rationally designed synthetic antifungal peptide PAF26 using the model fungus Neurospora crassa. Here we show that the cell killing activity of PAF26 is dependent on extracellular Ca and the presence of fully functioning fungal Ca homeostatic/signaling machinery. In a screen of mutants with deletions in Ca -signaling machinery, we identified three mutants more tolerant to PAF26. The Ca ATPase NCA-2 was found to be involved in the initial interaction of PAF26 with the cell envelope. The vacuolar Ca channel YVC-1 was shown to be essential for its accumulation and concentration within the vacuolar system. The Ca channel CCH-1 was found to be required to prevent the translocation of PAF26 across the plasma membrane. In the wild type, Ca removal from the medium resulted in the peptide remaining trapped in small vesicles as in the Δyvc-1 mutant. It is, therefore, apparent that cell killing by PAF26 is complex and unusually dependent on extracellular Ca and components of the Ca -regulatory machinery.
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http://dx.doi.org/10.1111/mmi.14532DOI Listing
October 2020

Vitamin D Effects on Cell Differentiation and Stemness in Cancer.

Cancers (Basel) 2020 Aug 25;12(9). Epub 2020 Aug 25.

Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Instituto de Investigación Hospital Universitario La Paz, and CIBERONC, Arturo Duperier 4, 28029 Madrid, Spain.

Vitamin D is the precursor of 1α,25-dihydroxyvitamin D (1,25(OH)D), a pleiotropic hormone that is a major regulator of the human genome. 1,25(OH)D modulates the phenotype and physiology of many cell types by controlling the expression of hundreds of genes in a tissue- and cell-specific fashion. Vitamin D deficiency is common among cancer patients and numerous studies have reported that 1,25(OH)D promotes the differentiation of a wide panel of cultured carcinoma cells, frequently associated with a reduction in cell proliferation and survival. A major mechanism of this action is inhibition of the epithelial-mesenchymal transition, which in turn is largely based on antagonism of the Wnt/β-catenin, TGF-β and EGF signaling pathways. In addition, 1,25(OH)D controls the gene expression profile and phenotype of cancer-associated fibroblasts (CAFs), which are important players in the tumorigenic process. Moreover, recent data suggest a regulatory role of 1,25(OH)D in the biology of normal and cancer stem cells (CSCs). Here, we revise the current knowledge of the molecular and genetic basis of the regulation by 1,25(OH)D of the differentiation and stemness of human carcinoma cells, CAFs and CSCs. These effects support a homeostatic non-cytotoxic anticancer action of 1,25(OH)D based on reprogramming of the phenotype of several cell types.
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http://dx.doi.org/10.3390/cancers12092413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563562PMC
August 2020

Comparative Study of Organoids from Patient-Derived Normal and Tumor Colon and Rectal Tissue.

Cancers (Basel) 2020 Aug 15;12(8). Epub 2020 Aug 15.

Departamento de Biología del Cáncer, Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), 28029 Madrid, Spain.

Colon and rectal tumors, often referred to as colorectal cancer, show different gene expression patterns in studies that analyze whole tissue biopsies containing a mix of tumor and non-tumor cells. To better characterize colon and rectal tumors, we investigated the gene expression profile of organoids generated from endoscopic biopsies of rectal tumors and adjacent normal colon and rectum mucosa from therapy-naive rectal cancer patients. We also studied the effect of vitamin D on these organoid types. Gene profiling was performed by RNA-sequencing. Organoids from a normal colon and rectum had a shared gene expression profile that profoundly differed from that of rectal tumor organoids. We identified a group of genes of the biosynthetic machinery as rectal tumor organoid-specific, including those encoding the RNA polymerase II subunits POLR2H and POLR2J. The active vitamin D metabolite 1α,25-dihydroxyvitamin D3/calcitriol upregulated stemness-related genes (, and ) in normal rectum organoids, while it downregulated differentiation marker genes ( and ). Normal colon and rectum organoids share similar gene expression patterns and respond similarly to calcitriol. Rectal tumor organoids display distinct and heterogeneous gene expression profiles, with differences with respect to those of colon tumor organoids, and respond differently to calcitriol than normal rectum organoids.
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http://dx.doi.org/10.3390/cancers12082302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465167PMC
August 2020

An update on vitamin D signaling and cancer.

Semin Cancer Biol 2020 May 30. Epub 2020 May 30.

Instituto de Investigaciones Biomédicas 'Alberto Sols', Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, CIBERONC and IdiPAZ, E-28029, Madrid, Spain.

A low vitamin D status is associated with an increased risk of various cancers, such as of colon, breast, prostate and hematological cells. The biologically most active vitamin D metabolite 1α,25-dihydroxyvitamin D (1,25(OH)D) is a high affinity ligand of the transcription factor vitamin D receptor (VDR). 1,25(OH)D induces via VDR changes to the epigenome of healthy and neoplastic cells and in this way influences their transcriptome. Ligand-activated VDR binds to more than 10,000 loci within the human genome and affects the transcription of some 1000 target genes in a large proportion of human tissues and cell types. From the evolutionary perspective, the prime role of vitamin D was probably the control of energy metabolism later shifting to modulate innate and adaptive immunity as well as to regulate calcium and bone homeostasis. Since rapidly growing immune and cancer cells both use the same pathways and genes for controlling their proliferation, differentiation and apoptosis, not surprisingly, vitamin D signaling changes these processes also in neoplastic cells. Thus, anti-cancer effects of vitamin D may derive from managing growth and differentiation in immunity. This review provides an update on the molecular basis of vitamin D signaling, i.e., the effects of 1,25(OH)D on the epigenome and transcriptome, and its relationship to cancer prevention and therapy.
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http://dx.doi.org/10.1016/j.semcancer.2020.05.018DOI Listing
May 2020

Targeting Breast Cancer Cells with G4 PAMAM Dendrimers and Valproic Acid Derivative Complexes.

Anticancer Agents Med Chem 2020 ;20(15):1857-1872

Laboratorio de Diseno y Desarrollo de Nuevos Farmacos e Innovacion Biotecnologica de la Escuela Superior de Medicina, Instituto Politecnico Nacional, Mexico. Plan de San Luis Y Diaz Miron S/N, Col. Casco de Santo Tomas, Mexico City, CP 11340, Mexico.

Background: Our research group has developed some Valproic Acid (VPA) derivatives employed as anti-proliferative compounds targeting the HDAC8 enzyme. However, some of these compounds are poorly soluble in water.

Objective: Employed the four generations of Polyamidoamine (G4 PAMAM) dendrimers as drug carriers of these compounds to increase their water solubility for further in vitro evaluation.

Methods: VPA derivatives were subjected to Docking and Molecular Dynamics (MD) simulations to evaluate their affinity on G4 PAMAM. Then, HPLC-UV/VIS, 1H NMR, MALDI-TOF and atomic force microscopy were employed to establish the formation of the drug-G4 PAMAM complexes.

Results: The docking results showed that the amide groups of VPA derivatives make polar interactions with G4 PAMAM, whereas MD simulations corroborated the stability of the complexes. HPLC UV/VIS experiments showed an increase in the drug water solubility which was found to be directly proportional to the amount of G4 PAMAM. 1H NMR showed a disappearance of the proton amine group signals, correlating with docking results. MALDI-TOF and atomic force microscopy suggested the drug-G4 PAMAM dendrimer complexes formation.

Discussion: In vitro studies showed that G4 PAMAM has toxicity in the micromolar concentration in MDAMB- 231, MCF7, and 3T3-L1 cell lines. VPA CF-G4 PAMAM dendrimer complex showed anti-proliferative properties in the micromolar concentration in MCF-7 and 3T3-L1, and in the milimolar concentration in MDAMB- 231, whereas VPA MF-G4 PAMAM dendrimer complex didn't show effects on the three cell lines employed.

Conclusion: These results demonstrate that G4 PAMAM dendrimers are capableof transporting poorly watersoluble aryl-VPA derivate compounds to increase its cytotoxic activity against neoplastic cell lines.
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http://dx.doi.org/10.2174/1871520620666200423073812DOI Listing
June 2021

The number of replicates, and pooling versus individual measurements for analytical imprecision calculations: Does it matter?

Vet Clin Pathol 2020 Mar 11;49(1):112-118. Epub 2020 Mar 11.

Syn Labs, VPG, TDDS, Unit G, The Innovation Centre, Exeter University, Exeter, UK.

Background: Recommendations from the American Society of Veterinary Clinical Pathology (ASVCP) are to calculate the between-run coefficient of variation (CV) based on measuring one replicate per day on quality control materials (QCMs) or pooled patient samples over a minimum of 20 days. However, this recommendation is not always followed by researchers.

Objectives: We aimed to determine if a reduction in the number of replicates using QCM or individual or pooled samples would provide CV results similar to those obtained based on ASVCP recommendations.

Methods: CVs were calculated for three measurands, namely urea, creatinine, and C-reactive protein based on the analytic results of the following groups: (a) QCM measured once daily for 20 days (considered as the reference for comparison), b) QCM measured once daily for 5 days, (c) five different canine serum samples measured once daily for 5 days, and (d) a pooled canine serum measured once daily for 5 days. CVs were calculated for two different measurand concentrations.

Results: Compared with the reference method, significantly different CVs were obtained with all methods except for when the QCM was measured once daily for 5 days. The use of the five different individual samples also provided significantly different CVs compared with the use of a pooled sample.

Conclusions: The results indicate that different protocols for determining between-run imprecision calculations can give different results compared with the reference procedure and that this should be taken into consideration when evaluating the total error associated with a test.
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http://dx.doi.org/10.1111/vcp.12830DOI Listing
March 2020

Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay.

J Vis Exp 2019 12 26(154). Epub 2019 Dec 26.

Medical Oncology Department, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), CIBERONC;

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ECM also participates in cell migration, proliferation, and differentiation. Fibroblasts are the main type of cells modifying ECM fiber arrangement and production. In cancer, CAFs (cancer associated fibroblasts) are in permanent activation status, participating in ECM remodeling, facilitating tumor cell migration, and stimulating tumor-associated angiogenesis, among other pro-tumorigenic roles. The objective of this method is to create a three-dimensional matrix with a fiber composition that is similar to in vivo matrices, using immortalized fibroblasts or human primary CAFs. Fibroblasts are cultured in pre-treated cell culture plates and grown under ascorbic acid stimulation. Then, fibroblasts are removed and matrices are blocked for further cell seeding. In this ECM model, fibroblasts can be activated or modified to generate different kinds of matrix, whose effects can be studied in cell culture. 3D matrices are also shaped by cell signals, like degradation or cross-linking enzymes that might modify fiber distribution. In this context, angiogenesis can be studied, along with other cell types such as epithelial tumor cells.
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http://dx.doi.org/10.3791/60304DOI Listing
December 2019

The Golgi Apparatus of Neocortical Glial Cells During Hibernation in the Syrian Hamster.

Front Neuroanat 2019 19;13:92. Epub 2019 Nov 19.

Laboratorio Cajal de Circuitos Corticales (CTB), Universidad Politécnica de Madrid, Madrid, Spain.

Hibernating mammals undergo torpor periods characterized by a general decrease in body temperature, metabolic rate, and brain activity accompanied by complex adaptive brain changes that appear to protect the brain from extreme conditions of hypoxia and low temperatures. These processes are accompanied by morphological and neurochemical changes in the brain including those in cortical neurons such as the fragmentation and reduction of the Golgi apparatus (GA), which both reverse a few hours after arousal from the torpor state. In the present study, we characterized - by immunofluorescence and confocal microscopy - the GA of cortical astrocytes, oligodendrocytes, and microglial cells in the Syrian hamster, which is a facultative hibernator. We also show that after artificial induction of hibernation, in addition to neurons, the GA of glia in the Syrian hamster undergoes important structural changes, as well as modifications in the intensity of immunostaining and distribution patterns of Golgi structural proteins at different stages of the hibernation cycle.
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http://dx.doi.org/10.3389/fnana.2019.00092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6882278PMC
November 2019

Plocabulin Displays Strong Cytotoxic Activity in a Personalized Colon Cancer Patient-Derived 3D Organoid Assay.

Mar Drugs 2019 Nov 19;17(11). Epub 2019 Nov 19.

Department of Cancer Biology, Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-UAM) 28029 Madrid, Spain and University Hospital La Paz Institute for Health Research (IdiPAZ), 28046 Madrid, Spain.

Plocabulin is a novel microtubule-disrupting antitumor agent of marine origin that is currently undergoing phase II clinical trials. Plocabulin has potent antiproliferative and antiangiogenic actions in carcinoma cell lines and has antitumor activity in xenografted mice. Here, we used three-dimensional (3D) tumor organoids derived from three colorectal cancer (CRC) patients to study the effect of plocabulin in a personalized assay system that ensures dose dependence and high reproducibility. The cytotoxicity of plocabulin was an order of magnitude higher than that of the active irinotecan derivative SN38 (7-ethyl-10-hydroxy-camptothecin) in tumor organoids at different passages. Moreover, plocabulin maintained its strong cytotoxic activity in wash-out experiments, in which a short pulse treatment of tumor organoids was as efficient as continuous treatment. Our data show that plocabulin has a very potent cytotoxic action in CRC patient-derived tumor organoids, supporting ongoing clinical trials with plocabulin and the use of organoid assays to provide personalized validation of antitumor drugs.
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http://dx.doi.org/10.3390/md17110648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891270PMC
November 2019

Urothelial organoids originating from Cd49f mouse stem cells display Notch-dependent differentiation capacity.

Nat Commun 2019 09 27;10(1):4407. Epub 2019 Sep 27.

Epithelial Carcinogenesis Group, Spanish National Cancer Research Centre-CNIO, Madrid, Spain.

Understanding urothelial stem cell biology and differentiation has been limited by the lack of methods for their unlimited propagation. Here, we establish mouse urothelial organoids that can be maintained uninterruptedly for >1 year. Organoid growth is dependent on EGF and Wnt activators. High CD49f/ITGA6 expression features a subpopulation of organoid-forming cells expressing basal markers. Upon differentiation, multilayered organoids undergo reduced proliferation, decreased cell layer number, urothelial program activation, and acquisition of barrier function. Pharmacological modulation of PPARγ and EGFR promotes differentiation. RNA sequencing highlighted genesets enriched in proliferative organoids (i.e. ribosome) and transcriptional networks involved in differentiation, including expression of Wnt ligands and Notch components. Single-cell RNA sequencing (scRNA-Seq) analysis of the organoids revealed five clusters with distinct gene expression profiles. Together, with the use of γ-secretase inhibitors and scRNA-Seq, confirms that Notch signaling is required for differentiation. Urothelial organoids provide a powerful tool to study cell regeneration and differentiation.
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http://dx.doi.org/10.1038/s41467-019-12307-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6764959PMC
September 2019

Slow-Wave Activity in the S1HL Cortex Is Contributed by Different Layer-Specific Field Potential Sources during Development.

J Neurosci 2019 11 23;39(45):8900-8915. Epub 2019 Sep 23.

Departamentos de Neurociencia Traslacional, y

Spontaneous correlated activity in cortical columns is critical for postnatal circuit refinement. We used spatial discrimination techniques to explore the late maturation of synaptic pathways through the laminar distribution of the field potential (FP) generators underlying spontaneous and evoked activities of the S1HL cortex in juvenile (P14-P16) and adult anesthetized rats. Juveniles exhibit an intermittent FP pattern resembling Up/Down states in adults, but with much reduced power and different laminar distribution. Whereas FPs in active periods are dominated by a layer VI generator in juveniles, in adults a developing multipart generator takes over, displaying current sinks in middle layers (III-V). The blockade of excitatory transmission in upper and middle layers of adults recovered the juvenile-like FP profiles. In addition to the layer VI generator, a gamma-specific generator in supragranular layers was the same in both age groups. While searching for dynamical coupling among generators in juveniles we found significant cross-correlation in ∼one-half of the tested pairs, whereas excessive coherence hindered their efficient separation in adults. Also, potentials evoked by tactile and electrical stimuli showed different short-latency dipoles between the two age groups, and the juveniles lacked the characteristic long latency UP state currents in middle layers. In addition, the mean firing rate of neurons was lower in juveniles. Thus, cortical FPs originate from different intra-columnar segments as they become active postnatally. We suggest that although some cortical segments are active early postnatally, a functional sensory-motor control relies on a delayed maturation and network integration of synaptic connections in middle layers. Early postnatal activity in the rodent cortex is mostly endogenous, whereas it becomes driven by peripheral input at later stages. The precise schedule for the maturation of synaptic pathways is largely unknown. We explored this in the somatosensory hindlimb cortex at an age when animals begin to use their limbs by uncovering the laminar distribution of the field potential generators underlying the dominant delta waves in juveniles and adults. Our results suggest that field potentials are mostly generated by a pathway in deep layers, whereas other pathways mature later in middle layers and take over in adults. We suggest that a functional sensory-motor control relies on a delayed maturation and network integration of synaptic connections in middle layers.
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http://dx.doi.org/10.1523/JNEUROSCI.1212-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832678PMC
November 2019

Vitamin D differentially regulates colon stem cells in patient-derived normal and tumor organoids.

FEBS J 2020 01 29;287(1):53-72. Epub 2019 Jul 29.

Departments of Cancer Biology and Biochemistry, Instituto de Investigaciones Biomédicas 'Alberto Sols', Spanish National Research Council (CSIC)-Autonomous University of Madrid (UAM) and IdiPAZ, Madrid, Spain.

Intestine is a major target of vitamin D and several studies indicate an association between vitamin D deficiency and inflammatory bowel diseases (IBD), but also increased colorectal cancer (CRC) risk and mortality. However, the putative effects of 1α,25-dihydroxyvitamin D (calcitriol), the active vitamin D metabolite, on human colonic stem cells are unknown. Here we show by immunohistochemistry and RNAscope in situ hybridization that vitamin D receptor (VDR) is unexpectedly expressed in LGR5 colon stem cells in human tissue and in normal and tumor organoid cultures generated from patient biopsies. Interestingly, normal and tumor organoids respond differentially to calcitriol with profound and contrasting changes in their transcriptomic profiles. In normal organoids, calcitriol upregulates stemness-related genes, such as LGR5, SMOC2, LRIG1, MSI1, PTK7, and MEX3A, and inhibits cell proliferation. In contrast, in tumor organoids calcitriol has little effect on stemness-related genes while it induces a differentiated phenotype, and variably reduces cell proliferation. Concordantly, electron microscopy showed that calcitriol does not affect the blastic undifferentiated cell phenotype in normal organoids but it induces a series of differentiated features in tumor organoids. Our results constitute the first demonstration of a regulatory role of vitamin D on human colon stem cells, indicating a homeostatic effect on colon epithelium with relevant implications in IBD and CRC.
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http://dx.doi.org/10.1111/febs.14998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972655PMC
January 2020

Global Vascular Guidelines on the Management of Chronic Limb-Threatening Ischemia.

Eur J Vasc Endovasc Surg 2019 07 8;58(1S):S1-S109.e33. Epub 2019 Jun 8.

Sheffield Vascular Institute, UK.

Guideline Summary: Chronic limb-threatening ischemia (CLTI) is associated with mortality, amputation, and impaired quality of life. These Global Vascular Guidelines (GVG) are focused on definition, evaluation, and management of CLTI with the goals of improving evidence-based care and highlighting critical research needs. The term CLTI is preferred over critical limb ischemia, as the latter implies threshold values of impaired perfusion rather than a continuum. CLTI is a clinical syndrome defined by the presence of peripheral artery disease (PAD) in combination with rest pain, gangrene, or a lower limb ulceration >2 weeks duration. Venous, traumatic, embolic, and nonatherosclerotic etiologies are excluded. All patients with suspected CLTI should be referred urgently to a vascular specialist. Accurately staging the severity of limb threat is fundamental, and the Society for Vascular Surgery Threatened Limb Classification system, based on grading of Wounds, Ischemia, and foot Infection (WIfI) is endorsed. Objective hemodynamic testing, including toe pressures as the preferred measure, is required to assess CLTI. Evidence-based revascularization (EBR) hinges on three independent axes: Patient risk, Limb severity, and ANatomic complexity (PLAN). Average-risk and high-risk patients are defined by estimated procedural and 2-year all-cause mortality. The GVG proposes a new Global Anatomic Staging System (GLASS), which involves defining a preferred target artery path (TAP) and then estimating limb-based patency (LBP), resulting in three stages of complexity for intervention. The optimal revascularization strategy is also influenced by the availability of autogenous vein for open bypass surgery. Recommendations for EBR are based on best available data, pending level 1 evidence from ongoing trials. Vein bypass may be preferred for average-risk patients with advanced limb threat and high complexity disease, while those with less complex anatomy, intermediate severity limb threat, or high patient risk may be favored for endovascular intervention. All patients with CLTI should be afforded best medical therapy including the use of antithrombotic, lipid-lowering, antihypertensive, and glycemic control agents, as well as counseling on smoking cessation, diet, exercise, and preventive foot care. Following EBR, long-term limb surveillance is advised. The effectiveness of nonrevascularization therapies (eg, spinal stimulation, pneumatic compression, prostanoids, and hyperbaric oxygen) has not been established. Regenerative medicine approaches (eg, cell, gene therapies) for CLTI should be restricted to rigorously conducted randomizsed clinical trials. The GVG promotes standardization of study designs and end points for clinical trials in CLTI. The importance of multidisciplinary teams and centers of excellence for amputation prevention is stressed as a key health system initiative.
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http://dx.doi.org/10.1016/j.ejvs.2019.05.006DOI Listing
July 2019

Vitamin D and Wnt3A have additive and partially overlapping modulatory effects on gene expression and phenotype in human colon fibroblasts.

Sci Rep 2019 05 30;9(1):8085. Epub 2019 May 30.

Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain.

The Wnt/β-catenin signalling pathway is essential for intestinal epithelium homeostasis, but its aberrant activation is a hallmark of colorectal cancer (CRC). Several studies indicate that the bioactive vitamin D metabolite 1α,25-dihydroxyvitamin D (1,25(OH)D) inhibits proliferation and promotes epithelial differentiation of colon carcinoma cells in part through antagonism of the Wnt/β-catenin pathway. It is now accepted that stromal fibroblasts are crucial in healthy and pathologic intestine: pericryptal myofibroblasts are constituents of the stem cell niche and cancer-associated fibroblasts (CAFs) contribute to CRC progression. However, studies on the combined action of 1,25(OH)D and Wnt factors in colon fibroblasts are lacking. Here we show by global transcriptomic studies that 1,25(OH)D and Wnt3A have profound, additive, partially overlapping effects on the gene expression profile of CCD-18Co human colon myofibroblasts. Moreover, 1,25(OH)D and Wnt3A inhibit CCD-18Co cell proliferation and migration, while 1,25(OH)D reduces, but Wnt3A increases, their capacity to contract collagen gels (a marker of fibroblast activation). These data were largely confirmed in patient-derived primary colon normal fibroblasts and CAFs, and in fibroblasts from other origins. Our results indicate that 1,25(OH)D and Wnt3A are strong regulators of colon fibroblast biology and contribute to a better knowledge of intestinal homeostasis and stromal fibroblast action in CRC.
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http://dx.doi.org/10.1038/s41598-019-44574-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542853PMC
May 2019

Extended Triangulenium Ions: Syntheses and Characterization of Benzo-Bridged Dioxa- and Diazatriangulenium Dyes.

J Org Chem 2019 Mar 19;84(5):2556-2567. Epub 2019 Feb 19.

Nano-Science Center and Department of Chemistry , University of Copenhagen , Universitetsparken 5 , Copenhagen 2100 , Denmark.

The very limited class of fluorophores, with a long fluorescence lifetime (>10 ns) and fluorescence beyond 550 nm, has been expanded with two benzo-fused triangulenium derivatives and two cationic [5]-helicene salts. The syntheses of the benzo-bridged dioxa- and diazatriangulenium derivatives (BDOTA and BDATA, respectively) required two different synthetic approaches, which reflect the structural and physiochemical impact on the reactivity of [5]-helicenium precursors. Spectroscopic investigations show that the introduction of the benzo bridge into the triangulenium chromophore significantly redshifts the absorption and emission while maintaining fluorescence lifetimes above 10 ns. The combination of a high quantum yield, long fluorescence lifetime, and emission above 600 nm is possible only if the structural aspects of the triangulenium framework are perfectly harmonized to secure a low rate of nonradiative deactivation. The new benzo bridge may be a general motif to obtain red-shifted derivatives of other dye classes.
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http://dx.doi.org/10.1021/acs.joc.8b02978DOI Listing
March 2019

Measuring Relevant Information in Health Social Network Conversations and Clinical Diagnosis Cases.

Int J Environ Res Public Health 2018 12 9;15(12). Epub 2018 Dec 9.

Biomedical Informatics Group, Departamento de Lenguajes Sistemas Informáticos e Ingeniería de Software & Departamento de Inteligencia Artificial, Escuela Técnica Superior de Ingenieros Informáticos, Universidad Politécnica de Madrid, 28660 Madrid, Spain.

The Internet and social media is an enormous source of information. Health social networks and online collaborative environments enable users to create shared content that afterwards can be discussed. The aim of this paper is to present a novel methodology designed for quantifying relevant information provided by different participants in clinical online discussions. The main goal of the methodology is to facilitate the comparison of participant interactions in clinical conversations. A set of key indicators for different aspects of clinical conversations and specific clinical contributions within a discussion have been defined. Particularly, three new indicators have been proposed to make use of biomedical knowledge extraction based on standard terminologies and ontologies. These indicators allow measuring the relevance of information of each participant of the clinical conversation. Proposed indicators have been applied to one discussion extracted from PatientsLikeMe, as well as to two real clinical cases from the Sanar collaborative discussion system. Results obtained from indicators in the tested cases have been compared with clinical expert opinions to check indicators validity. The methodology has been successfully used for describing participant interactions in real clinical cases belonging to a collaborative clinical case discussion tool and from a conversation from a health social network. This work can be applied to assess collaborative diagnoses, discussions among patients, and the participation of students in clinical case discussions. It permits moderators and educators to obtain a quantitatively measure of the contribution of each participant.
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http://dx.doi.org/10.3390/ijerph15122787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6313597PMC
December 2018

Endothelial cell activation on 3D-matrices derived from PDGF-BB-stimulated fibroblasts is mediated by Snail1.

Oncogenesis 2018 Sep 24;7(9):76. Epub 2018 Sep 24.

Department of Medical Oncology, Hospital Universitario Puerta de Hierro de Majadahonda, Majadahonda, Madrid, Spain.

Carcinomas, such as colon cancer, initiate their invasion by rescuing the innate plasticity of both epithelial cells and stromal cells. Although Snail is a transcriptional factor involved in the Epithelial-Mesenchymal Transition, in recent years, many studies have also identified the major role of Snail in the activation of Cancer-Associated Fibroblast (CAF) cells and the remodeling of the extracellular matrix. In CAFs, Platelet-derived growth factor (PDGF) receptor signaling is a major functional determinant. High expression of both SNAI1 and PDGF receptors is associated with poor prognosis in cancer patients, but the mechanism(s) that underlie these connections are not understood. In this study, we demonstrate that PDGF-activated fibroblasts stimulate extracellular matrix (ECM) fiber remodeling and deposition. Furthermore, we describe how SNAI1, through the FAK pathway, is a necessary factor for ECM fiber organization. The parallel-oriented fibers are used by endothelial cells as "tracks", facilitating their activation and the creation of tubular structures mimicking in vivo capillary formation. Accordingly, Snail1 expression in fibroblasts was required for the co-adjuvant effect of these cells on matrix remodeling and neoangiogenesis when co-xenografted in nude mice. Finally, in tumor samples from colorectal cancer patients a direct association between stromal SNAI1 expression and the endothelial marker CD34 was observed. In summary, our results advance the understanding of PDGF/SNAI1-activated CAFs in matrix remodeling and angiogenesis stimulation.
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http://dx.doi.org/10.1038/s41389-018-0085-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155204PMC
September 2018

Aryl Hydrocarbon Receptor Promotes Liver Polyploidization and Inhibits PI3K, ERK, and Wnt/β-Catenin Signaling.

iScience 2018 Jun 15;4:44-63. Epub 2018 May 15.

Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Badajoz 06071, Spain. Electronic address:

Aryl hydrocarbon receptor (AhR) deficiency alters tissue homeostasis. However, how AhR regulates organ maturation and differentiation remains mostly unknown. Liver differentiation entails a polyploidization process fundamental for cell growth, metabolism, and stress responses. Here, we report that AhR regulates polyploidization during the preweaning-to-adult mouse liver maturation. Preweaning AhR-null (AhR-/-) livers had smaller hepatocytes, hypercellularity, altered cell cycle regulation, and enhanced proliferation. Those phenotypes persisted in adult AhR-/- mice and correlated with compromised polyploidy, predominance of diploid hepatocytes, and enlarged centrosomes. Phosphatidylinositol-3-phosphate kinase (PI3K), extracellular signal-regulated kinase (ERK), and Wnt/β-catenin signaling remained upregulated from preweaning to adult AhR-null liver, likely increasing mammalian target of rapamycin (mTOR) activation. Metabolomics revealed the deregulation of mitochondrial oxidative phosphorylation intermediates succinate and fumarate in AhR-/- liver. Consistently, PI3K, ERK, and Wnt/β-catenin inhibition partially rescued polyploidy in AhR-/- mice. Thus, AhR may integrate survival, proliferation, and metabolism for liver polyploidization. Since tumor cells tend to be polyploid, AhR modulation could have therapeutic value in the liver.
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http://dx.doi.org/10.1016/j.isci.2018.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6147018PMC
June 2018