Extracellular Vesicles in Biological Fluids. A Biomarker of Exposure to Cigarette Smoke and Treatment with Chemopreventive drugs

Extracellular vesicles (EVs) are released from cells and enter into body fluids thereby providing a toxicological mechanism of cell-cell communication. The present study aimed at assessing (a) the presence of EVs in mouse body fluids under physiological conditions, (b) the effect of exposure of mice to cigarette smoke for 8 weeks, and (c) modulation of smoke-related alterations by the nonsteroidal anti-inflammatory drug celecoxib, a selective cyclooxygenase-2 inhibitor. To this purpose, ICR (CD-1) mice were either unexposed or exposed to cigarette smoke, either treated or untreated with oral celecoxib. EVs, isolated from bronchoalveolar lavage fluid (BALF), blood serum, and urines, were analyzed by nanoparticle tracking analysis and flow cytometry. EVs baseline concentrations in BALF were remarkably high. Larger EVs were detected in urines. Smoking increased EVs concentrations but only in BALF. Celecoxib remarkably increased EVs concentrations in the blood serum of both male and female smoking mice. The concentration of EVs positive for EpCAM, a mediator of cell-cell adhesion in epithelia playing a role in tumorigenesis, was much higher in urines than in BALF, and celecoxib significantly decreased their concentration. Thus, the effects of smoke on EVs concentrations were well detectable in the extracellular environment of the respiratory tract, where they could behave as delivery carriers to target cells. Celecoxib exerted both protective mechanisms in the urinary tract and adverse systemic effects of likely hepatotoxic origin in smoke-exposed mice. Detection of EVs in body fluids may provide an early diagnostic tool and an end-point exploitable for preventive medicine strategies.

Relativistic Particle Beams as a Resource to Solve Outstanding Problems in Space Physics

The Sun\u27s connection with the Earth\u27s magnetic field and atmosphere is carried out through the exchange of electromagnetic and mass flux and is regulated by a complex interconnection of processes. During space weather events, solar flares, or fast streams of solar atmosphere strongly disturb the Earth\u27s environment. Often the electric currents that connect the different parts of the Sun-Earth system become unstable and explosively release the stored electromagnetic energy in one of the more dramatic expressions of space weather—the geomagnetic storm and substorm. Some aspects of the magnetosphere-ionosphere connection that generates auroral arcs during space weather events are well-known. However, several fundamental problems remain unsolved because of the lack of unambiguous identification of the magnetic field connection between the magnetosphere and the ionosphere. The correct mapping between different regions of the magnetosphere and their foot-points in the ionosphere, coupled with appropriate distributed measurements of plasma and fields in focused regions of the magnetosphere, is necessary to establish unambiguously that a given magnetospheric process is the generator of an observed arc. We present a new paradigm that should enable the resolution of the mapping ambiguities. The paradigm calls for the application of energetic electron beams as magnetic field tracers. The three most important problems for which the correct magnetic field mapping would provide closure to are the substorm growth phase arcs, the expansion phase onset arcs and the system of arcs that emerge from the magnetosphere-ionosphere connection during the development of the early substorm expansion phase phenomenon known as substorm current wedge (SCW). In this communication we describe how beam tracers, in combination with distributed measurements in the magnetosphere, can be used to disentangle the mechanisms that generate these critical substorm phenomena. Since the application of beams as tracers require demonstration that the beams can be injected into the loss cone, that the spacecraft potentials induced by the beam emission are manageable, and that sufficient electron flux reaches the atmosphere to be detectable by optical or radio means after the beam has propagated thousands of kilometers under competing effects of beam spread and constriction as well as effects of beam-induced instabilities, in this communication we review how these challenges are currently being addressed and discuss the next steps toward the realization of active experiments in space using relativistic electron beams

Flow cytometry based techniques to study testicular acidophilic granulocytes from the protandrous fish gilthead seabream (Sparus aurata L.)

The gilthead seabream is a protandrous seasonal breeding teleost that is an excellent model for studying the testicular regression process which occurs in both seasonal testicular involution and sex reversion. Little is known about the cell types and the molecular mechanisms involved in such processes, mainly because of the lack of appropriate methods for testis dissociation, and testicular cell isolation, culture and functional characterization. We have previously reported that gilthead seabream acidophilic granulocytes infiltrate the testis at post-spawning stage, settle close to the spermatogonia and accumulate intracellular interleukin-1β. In this paper, we report several flow cytometry based assays which allow to establish the role played by gilthead seabream testicular acidophilic granulocytes and permits their quantification

Quantifying the Quiet Epidemic: Diagnosing Dementia in Twentieth Century Britain

During the late 20(th) century numerical rating scales became central to the diagnosis of dementia and helped transform attitudes about its causes and prevalence. Concentrating largely on the development and use of the Blessed Dementia Scale, I argue that rating scales served professional ends during the 1960s and 1970s. They helped old age psychiatrists establish jurisdiction over conditions such as dementia and present their field as a vital component of the welfare state, where they argued that ‘reliable modes of diagnosis’ were vital to the allocation of resources. I show how these arguments appealed to politicians, funding bodies and patient groups, who agreed that dementia was a distinct disease and claimed research on its causes and prevention should be designated ‘top priority’. But I also show that worries about the replacement of clinical acumen with technical and depersonalized methods, which could conceivably be applied by anyone, led psychiatrists to stress that rating scales had their limits and could be used only by trained experts

Mutation spectrum of MLL2 in a cohort of kabuki syndrome patients

ABSTRACT: BACKGROUND: Kabuki syndrome (Niikawa-Kuroki syndrome) is a rare, multiple congenital anomalies/mental retardation syndrome characterized by a peculiar face, short stature, skeletal, visceral and dermatoglyphic abnormalities, cardiac anomalies, and immunological defects. Recently mutations in the histone methyl transferase MLL2 gene have been identified as its underlying cause. METHODS: Genomic DNAs were extracted from 62 index patients clinically diagnosed as affected by Kabuki syndrome. Sanger sequencing was performed to analyze the whole coding region of the MLL2 gene including intron-exon junctions. The putative causal and possible functional effect of each nucleotide variant identified was estimated by in silico prediction tools. RESULTS: We identified 45 patients with MLL2 nucleotide variants. 38 out of the 42 variants were never described before. Consistently with previous reports, the majority are nonsense or frameshift mutations predicted to generate a truncated polypeptide. We also identified 3 indel, 7 missense and 3 splice site. CONCLUSIONS: This study emphasizes the relevance of mutational screening of the MLL2 gene among patients diagnosed with Kabuki syndrome. The identification of a large spectrum of MLL2 mutations possibly offers the opportunity to improve the actual knowledge on the clinical basis of this multiple congenital anomalies/mental retardation syndrome, design functional studies to understand the molecular mechanisms underlying this disease, establish genotype-phenotype correlations and improve clinical management

Evaluation of Allele-Specific Somatic Changes of Genome-Wide Association Study Susceptibility Alleles in Human Colorectal Cancers

Tumors frequently exhibit loss of tumor suppressor genes or allelic gains of activated oncogenes. A significant proportion of cancer susceptibility loci in the mouse show somatic losses or gains consistent with the presence of a tumor susceptibility or resistance allele. Thus, allele-specific somatic gains or losses at loci may demarcate the presence of resistance or susceptibility alleles. The goal of this study was to determine if previously mapped susceptibility loci for colorectal cancer show evidence of allele-specific somatic events in colon tumors.We performed quantitative genotyping of 16 single nucleotide polymorphisms (SNPs) showing statistically significant association with colorectal cancer in published genome-wide association studies (GWAS). We genotyped 194 paired normal and colorectal tumor DNA samples and 296 paired validation samples to investigate these SNPs for allele-specific somatic gains and losses. We combined analysis of our data with published data for seven of these SNPs.No statistically significant evidence for allele-specific somatic selection was observed for the tested polymorphisms in the discovery set. The rs6983267 variant, which has shown preferential loss of the non-risk T allele and relative gain of the risk G allele in previous studies, favored relative gain of the G allele in the combined discovery and validation samples (corrected p-value = 0.03). When we combined our data with published allele-specific imbalance data for this SNP, the G allele of rs6983267 showed statistically significant evidence of relative retention (p-value = 2.06×10(-4)).Our results suggest that the majority of variants identified as colon cancer susceptibility alleles through GWAS do not exhibit somatic allele-specific imbalance in colon tumors. Our data confirm previously published results showing allele-specific imbalance for rs6983267. These results indicate that allele-specific imbalance of cancer susceptibility alleles may not be a common phenomenon in colon cancer

Integrated Ugi-Based Assembly of Functionally, Skeletally, and Stereochemically Diverse 1,4-Benzodiazepin-2-ones

A practical, integrated and versatile U-4CR-based assembly of 1,4-benzodiazepin-2-ones exhibiting functionally, skeletally, and stereochemically diverse substitution patterns is described. By virtue of its convergence, atom economy, and bond-forming efficiency, the methodology documented herein exemplifies the reconciliation of structural complexity and experimental simplicity in the context of medicinal chemistry projects.This work was financially supported by the Galician Government (Spain), Projects: 09CSA016234PR and GPC-2014-PG037. J.A. thanks FUNDAYACUCHO (Venezuela) for a predoctoral grant and Deputación da Coruña (Spain) for a postdoctoral research grant. A.N.-V. thanks the Spanish government for a Ramón y Cajal research contract

Overexpression of S100A4 in human cancer cell lines resistant to methotrexate

Methotrexate is a chemotherapeutic drug that is used in therapy of a wide variety of cancers. The efficiency of treatment with this drug is compromised by the appearance of resistance. Combination treatments of MTX with other drugs that could modulate the expression of genes involved in MTX resistance would be an adequate strategy to prevent the development of this resistance. Methods: The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. A global comparison of all the studied cell lines was performed in order to find out differentially expressed genes in the majority of the MTX-resistant cells. S100A4 mRNA and protein levels were determined by RT-Real-Time PCR and Western blot, respectively. Functional validations of S100A4 were performed either by transfection of an expression vector for S100A4 or a siRNA against S100A4. Transfection of an expression vector encoding for β-catenin was used to inquire for the possible transcriptional regulation of S100A4 through the Wnt pathway. Results: S100A4 is overexpressed in five out of the seven MTX-resistant cell lines studied. Ectopic overexpression of this gene in HT29 sensitive cells augmented both the intracellular and extracellular S100A4 protein levels and caused desensitization toward MTX. siRNA against S100A4 decreased the levels of this protein and caused a chemosensitization in combined treatments with MTX. β-catenin overexpression experiments support a possible involvement of the Wnt signaling pathway in S100A4 transcriptional regulation in HT29 cells. Conclusions: S100A4 is overexpressed in many MTX-resistant cells. S100A4 overexpression decreases the sensitivity of HT29 colon cancer human cells to MTX, whereas its knockdown causes chemosensitization toward MTX. Both approaches highlight a role for S100A4 in MTX resistanc

Evidence for protandry in Polydactylus quadrifilis in the Kwanza Estuary, Angola, and its implications for local fisheries

A total of 141 Polydactylus quadrifilis were sampled from the Kwanza Estuary in Angola ranging in size from 436 to 1360 mm fork-length (FL). Of these, 124 were male, six intersex and 11 female. Female fish were significantly longer (mm, FL) and heavier (kg) than males and had significantly higher gonadosomatic indices (GSI’s) than those of males and intersex fish. Transitional (intersex) gonads were delimited, with testicular and ovarian regions separated by connective tissue. The first signs of ovarian tissue appeared on the outer ventral surface of the gonad. A second layer of ovarian tissue was first noticeable at either end of the initial ovarian region and developed back towards the centre of the ventral wall to form a luminal space. Early-stage oocytes were commonly found in the outer area of male regions and residual late-stage spermatids and spermatozoa were found in the luminal space of ovarian regions, suggesting a process of sex change from the outside inwards. A loss of male function was noted with increased ovarian prevalence. Based on this evidence it is suggested that P. quadrifilis in the Kwanza Estuary are protandrous. Owing to the reliance of P. quadrifilis on large highly fecund females for egg production, it is likely that they will be sensitive to fishing practices that target larger individuals within the population

Adherence to antibiotic treatment guidelines and outcomes in the hospitalized elderly with different types of pneumonia

Background: Few studies evaluated the clinical outcomes of Community Acquired Pneumonia (CAP), Hospital-Acquired Pneumonia (HAP) and Health Care-Associated Pneumonia (HCAP) in relation to the adherence of antibiotic treatment to the guidelines of the Infectious Diseases Society of America (IDSA) and the American Thoracic Society (ATS) in hospitalized elderly people (65 years or older). Methods: Data were obtained from REPOSI, a prospective registry held in 87 Italian internal medicine and geriatric wards. Patients with a diagnosis of pneumonia (ICD-9 480-487) or prescribed with an antibiotic for pneumonia as indication were selected. The empirical antibiotic regimen was defined to be adherent to guidelines if concordant with the treatment regimens recommended by IDSA/ATS for CAP, HAP, and HCAP. Outcomes were assessed by logistic regression models. Results: A diagnosis of pneumonia was made in 317 patients. Only 38.8% of them received an empirical antibiotic regimen that was adherent to guidelines. However, no significant association was found between adherence to guidelines and outcomes. Having HAP, older age, and higher CIRS severity index were the main factors associated with in-hospital mortality. Conclusions: The adherence to antibiotic treatment guidelines was poor, particularly for HAP and HCAP, suggesting the need for more adherence to the optimal management of antibiotics in the elderly with pneumonia