Analysis Of Several Markers Research Articles

Cardioprotective effect of the anti-ageing protein Klotho on ischemic heart disease

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ISCIII, Ministerio de Universidades, Education and Research Council of Madrid, Biomedicine Network Comunidad de Madrid Background Cardiovascular diseases are the leading cause of death and disability worldwide. Among them, ischemic heart disease, due to myocardial infarction (MI) remains the first cause of cardiovascular death. The anti-ageing protein Klotho is mainly expressed in membrane at renal level. However, Klotho also has a soluble form with different pleiotropic functions that are still undetermined, including a possible cardioprotective role that is currently the target of several studies. Purpose Firstly, we evaluated circulating Klotho levels related to cardiac damage in a cohort of 146 patients after ST-elevation myocardial infarction (STEMI) and in an experimental post-myocardial infarction (PMI) model. Secondly, we investigated soluble Klotho supplementation as a potential novel therapeutic strategy for the prevention of cardiac dysfunction, deleterious remodeling, cardiomyocyte calcium (Ca2+) mishandling and arrhythmias in PMI mice. Methods Study with human samples: we analysed Klotho and NT-proBNP plasma levels in STEMIpatients with preserved renal function. Experimental study: C57BL6 mice subjected to PMI by ligation of the left anterior descending coronary artery were treated with vehicle solution or recombinant Klotho (10 mg/Kg/day) for 15 days after MI. Cardiac function was studied through non-invasive methods including electrocardiogram (ECG) and cardiac magnetic resonance imaging (CMRI). Intracellular Ca2+ handling was analysed using confocal microscopy in isolated ventricular cardiomyocytes. Cardiac remodeling was studied through histological and biomolecular analysis of several markers of cardiac damage and target proteins involved in Ca2+ handling. Results Plasma Klotho levels decrease rapidly after MI in both STEMI patients and PMI mice. Additionally, MI patients in the lowest Klotho tertile showed significantly elevated NT-proBNP levels. Klotho treatment decreased the infarct area in PMI mice accompanied by a decrease in both cardiac hypertrophy and fibrosis. Moreover, reduction in ejection fraction and MI-related ECG alterations were reduced in PMI mice treated with Klotho, including increased QRS, JT, QTc intervals and occurrence of premature ventricular contractions. Klotho also prevented systolic Ca2+ release and cell shortening disturbances in cardiomyocytes from PMI mice without Klotho. Increased diastolic Ca2+ leak was prevented in mice treated with Klotho via CaMKII-dependent pathway by avoiding its phosphorylation and the RyR2 phosphorylation at CaMKII site. Conclusions Klotho supplementation protect against MI by avoiding deleterious cardiac remodeling and ameliorating one of the main complications of MI such as arrhythmias. Thus, recombinant Klotho prevents the intracardiomyocyte Ca2+ mishandling after MI. Our translational findings support the importance of Klotho as a novel biomarker of ventricular damage just after MI and as a novel therapeutic strategy for prevention cardiac events associated to ischemic heart disease.

The association of genetic factors with serum calretinin levels in asbestos-related diseases.

Asbestos exposure is associated with different asbestos-related diseases, including malignant mesothelioma (MM). MM diagnosis is confirmed with immunohistochemical analysis of several markers, including calretinin. Increased circulating calretinin was also observed in MM. The aim of the study was to determine if CALB2 polymorphisms or polymorphisms in genes that can regulate calretinin expression are associated with serum calretinin levels or MM susceptibility. The study included 288 MM patients and 616 occupationally asbestos-exposed subjects without MM (153 with asbestosis, 380 with pleural plaques and 83 without asbestos-related disease). Subjects were genotyped for seven polymorphisms in CALB2, E2F2, MIR335, NRF1 and SEPTIN7 genes using competitive allele-specific polymerase chain reaction (PCR). Serum calretinin was determined with ELISA in 545 subjects. Nonparametric tests, logistic regression and receiver operating characteristic (ROC) curve analysis were used for statistical analysis. Carriers of at least one polymorphic CALB2 rs889704 allele had lower calretinin levels (P = 0.036). Carriers of two polymorphic MIR335 rs3807348 alleles had higher calretinin (P = 0.027), while carriers of at least one polymorphic NRF1 rs13241028 allele had lower calretinin levels (P = 0.034) in subjects without MM. Carriers of two polymorphic E2F2 rs2075995 alleles were less likely to develop MM (odds ratio [OR] = 0.64, 95% confidence interval [CI] = 0.43-0.96, P = 0.032), but the association was no longer significant after adjustment for age (P = 0.093). Optimal serum calretinin cut-off values differentiating MM patients from other subjects differed according to CALB2, NRF1, E2F2, and MIR335 genotypes. The results of presented study suggest that genetic variability could influence serum calretinin levels. These findings could contribute to a better understanding of calretinin regulation and potentially to earlier MM diagnosis.

Mitochondrial DNA diversity and genetic structure of striped dolphin Stenella coeruleoalba in the Northern Ionian Sea

In the framework of global and EU policies focused on stopping the loss of biodiversity process, deepening the genetic variability, especially of populations species identified as threatened, is crucial for defining conservation units and developing appropriate conservation strategies. This is more urgent for cetacean species in the Mediterranean because they assume a key ecological role in the marine food web and are severely affected by numerous and different anthropogenic pressures. This study aims to increase information on the genetic variability of striped dolphin in the Northern Ionian Sea by investigating the population structure, phylogenetic relationships and phylogeographic patterns using two mtDNA markers. From October 2020 to August 2021, a total of 88 skin tissue samples were collected from free-ranging dolphins in the Gulf of Taranto by applying the non-invasive technique of skin swabbing. An acceptable amount of DNA was extracted from 86 samples and used for subsequent genetic analysis conducted on the partial sequences of 421 and 704 bp in length of the cytb gene and D-loop control region, respectively. In addition, the sequences of the two mtDNA markers were joined together to compose a mtDNA concatenated sequence of 1125 bp for each sampled dolphin in order to investigate the genetic variability of the species population in the study area. Genetic analysis highlighted a low nucleotide diversity and high haplotypic diversity of the striped dolphin of the Gulf of Taranto, suggesting a population in rapid expansion after a period of reduction in size and diversity of the initial population. The phylogenetic analyses revealed the presence of at least two different lineages of Stenella coeruleoalba in the Mediterranean Sea, one specific to the Northern Ionian Sea and one shared with the Mediterranean population, confirming results already obtained for the local unit in the Gulf of Taranto. The results point out a potential problem of hybridization between striped and common dolphins which needs to be further investigated. Therefore, increasing the analysis of several markers may increase understanding of the genetic diversity of the population in the Ionian Sea and represent a useful tool to support the implementation of future effective conservation measures.

Differentiated PDGFRα-Positive Cells: A Novel In-Vitro Model for Functional Studies of Neuronal Nitric Oxide Synthase

It is evident that depletion of interstitial cells and dysfunction of nitric oxide (NO) pathways are key players in development of several gastrointestinal (GI) motility disorders such as diabetic gastroparesis (DGP). One of the main limitations of DGP research is the lack of isolation methods that are specific to interstitial cells, and therefore conducting functional studies is not feasible. The present study aims (i) to differentiate telomerase transformed mesenchymal stromal cells (iMSCs) into platelet-derived growth factor receptor-α-positive cells (PDGFRα-positive cells) using connective tissue growth factor (CTGF) and L-ascorbic acids; (ii) to investigate the effects of NO donor and inhibitor on the survival rate of differentiated PDGFRα-positive cells; and (iii) to evaluate the impact of increased glucose concentrations, mimicking diabetic hyperglycemia, on the gene expression of neuronal nitric oxide synthase (nNOS). A fibroblastic differentiation-induction medium supplemented with connective tissue growth factor was used to differentiate iMSCs into PDGFRα-positive cells. The medium was changed every day for 21 days to maintain the biological activity of the growth factors. Gene and protein expression, scanning electron and confocal microscopy, and flow cytometry analysis of several markers were conducted to confirm the differentiation process. Methyl tetrazolium cell viability, nitrite measurement assays, and immunostaining were used to investigate the effects of NO on PDGFRα-positive cells. The present study, for the first time, demonstrated the differentiation of iMSCs into PDGFRα-positive cells. The outcomes of the functional studies showed that SNAP (NO donor) increased the survival rate of differentiated PDGFRα-positive cells whereas LNNA (NO inhibitor) attenuated these effects. Further experimentations revealed that hyperglycemia produced a significant increase in expression of nNOS in PDGFRα-positive cells. Differentiation of iMSCs into PDGFRα-positive cells is a novel model to conduct functional studies and to investigate the involvement of NO pathways. This will help in identifying new therapeutic targets for treatment of DGP.

Abstract 5077: Proteomic profiling reveals chemopreventive targets in esophageal adenocarcinoma

Abstract Esophageal adenocarcinoma (EAC) is characterized by rising incidence rates and high mortality due to late stage diagnosis and a lack of efficacious options for prevention and treatment. While reasons for the rapid increase in EAC are being unraveled, persistent, symptomatic reflux of gastric and duodenal contents, known as gastroesophageal reflux disease is considered the strongest risk factor. Our laboratory utilizes a rat surgical model of reflux-induced EAC to evaluate mechanisms by which cranberry proanthocyanidins (C-PAC) delivered in the drinking water inhibit reflux-induced EAC. Findings show that C-PAC (690ug/rat/day) inhibits EAC formation by 83% at 40 weeks of study and mechanisms of inhibition are under investigation. We utilized nano LC-MS/MS proteomic profiling to identify proteins that were dysregulated due to reflux-induced EAC and reversed with C-PAC treatment. Proteomic profiling revealed that C-PAC treatment restored 63 proteins dysregulated in the context of reflux. EIF3F, PSMD2 and ACTR2 were the top proteins up-regulated by reflux; whereas, top markers restored by C-PAC included GSTT2, OGN and PCMT1. Metacore integrated software showed top pathways up-regulated by reflux and down-regulated C-PAC included Translation_regulation of translation initiation (EIF-linked), Immune response and Regulation of telomere length. Conversely, C-PAC treatment upregulated the glutathione metabolism pathway which is consistent with GSTT2 restoration. Disease enrichment analysis revealed multiple gastrointestinal tract diseases linked to reflux, which C-PAC down regulated supporting that proteomic profiling may inform other potential disease targets for C-PAC. Finally, beyond the 63 reflux-driven proteins C-PAC reversed, we considered proteins, pathways and processes which could not be reversed by C-PAC (n=269). As an example, Process networks upregulated by reflux (not reversed by C-PAC) included Translation initiation (RPL-linked), Elongation, mRNA processing, G2-M and S-phase of the cell cycle. Thus, proteomics profiling may inform the development of complementary preventive combinations for improved cancer inhibitory efficacy, especially for a cancer like EAC, with high mutational burden. Western blot analysis of several markers in the rat esophagus including GSTT2 and COX2 support the data obtained through proteomic profiling. Future directions include interrogating specific pathways that are highly modulated by reflux-induced EAC and restored by C-PAC including DNA damage, repair and extracellular matrix and adhesion. Last, proteomic profiling has also allowed for identification of post-translational modifications which may provide insight into regulation of key dysregulated proteins. Citation Format: Katherine M. Weh, Connor L. Howard, Amy B. Howell, Jennifer L. Clarke, Laura A. Kresty. Proteomic profiling reveals chemopreventive targets in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5077.

Transcriptomic view of detached lettuce leaves during storage: A crosstalk between wounding, dehydration and senescence

Many methods of storage are currently used to delay the postharvest senescence and the ensuing quality losses of detached lettuce leaves. This transcriptome study explores the senescence mechanisms of packaged lettuce leaves over 7 d of cold storage in darkness. At the end of the storage, the detached lettuce leaves showed a 10% water loss and 24% cut-edge discoloration. No chlorophyll fluorescence variation was observed. A total of 1048 and 1846 genes were differentially expressed (DE) after 2 and 7 d of storage, respectively. In terms of gene expression modulation, the data showed a global shutdown of primary-metabolism but no obvious chlorophyll breakdown. Although an early stress-response clearly takes place, the cold storage under darkness appeared to delay senescence by protecting chloroplasts from degradation and enhancing an antioxidative response. Then, a progressive global expression shutdown appeared, concomitantly with the dehydration stress. Among the transcripts that showed an early high expression, the data highlighted many stress-related genes (PIN, bzip TF, Heat-shock, stress A-like), 30 redox-regulation associated transcripts, the majority of the phenylpropanoid pathway markers and ethylene and auxin-related genes. Later (day 7), many dehydration-responsive markers showed an increased expression, as did some abscisic acid related markers. Phytohormones (cytokinins) and transcriptions factors (WRKYs) appeared to play complex roles as senescence progressed. No simple link between cut-edge discoloration and PPO-related genes expression was made. The main known process of senescence induction through the phaephorbide a oxygenase and phospholipase D pathways were not highlighted in this study. To the best of our knowledge, this study is the first to explore the transcriptional response of postharvest senescence in detached lettuce leaves. Deeper analysis of several markers will help to dissect the crosstalk between wounding, dehydration and senescence mechanisms. In addition, post-transcriptional studies are needed to conclude about the described patterns.

Biomarqueurs prédictifs de l’immunothérapie anti-PD1/PD-L1 dans le cancer broncho-pulmonaire non à petites cellules

Immune checkpoint inhibitors (ICI), targeting the PD1/PD-L1 axis has shown their efficacy in lung cancer but only in a restricted population of patients, thus it is mandatory to identify biomarkers predicting the clinical benefit. In this article we will describe and analyzed biomarkers already published, from protein, to RNA and at last DNA markers, discussing each markers feasibility and interest. In the future, combined analysis of several markers will probably be proposed, particularly with the increasing complexity of therapy schema with molecules association.

Imaging mass cytometry - elemental immunohistochemistry for multiparametric imaging and quantitation

Abstract Pathology assessment of tissue sections provides prognostic evaluation and helps select optimum treatment regimens. Digital pathology has made significant strides in the analysis of several markers. There is a growing understanding of cell heterogeneity within tumor tissue, role of microenvironment, and the impact of immune cells on cancer development. Sophisticated tools are needed to provide quantitative information for a large number of biomarkers, low-abundance small molecules and chemotherapeutic drugs, while retaining spatial resolution of cells and tissue architecture. Imaging mass cytometry (IMC) is a novel technology that can simultaneously detect and quantitatively measure more than 50 metal-containing reagents in tissue sections at 1 μm resolution. IMC combines laser ablation with the Helios CyTOF®. We will describe in detail technology, workflow, multiplexing protocols, image analysis and show representative data for human and mouse sections. We will demonstrate the use of metal-containing histological stains for tissue architecture, and endogenous element identification [iodine, platinum]. Combined detection of protein targets and transcripts within cells will be presented. Validation will be demonstrated on sequential tissue sections prepared by conventional immunohistochemistry. Precision medicine is based on access to high-density data (proteomics and genomics) which provides accurate diagnosis and information on the best therapeutic approach. IMC is a highly multiparametric, quantitative method for phenotypic, signaling pathway, and cell state protein identification together with spatial information within tissue sections.

Abstract 865: Evolving biological and clinical concepts of radiation delivery in NSCLC: response to ablative versus fractionated radiotherapy

Abstract Non-small cell lung cancer (NSCLC) patients account for over 80% of all lung cancer cases and have a poor outcome with current radiotherapy regimens. NSCLC has been the subject of many studies characterizing mechanisms of initiation, proliferation, invasion and treatment response. Recent trends in radiotherapy show an increase in the use of ablative radiotherapy (ART) in inoperable small size tumors. Clinical studies have shown response rates up to 90% in NSCLC patients treated with ART compared to 15-20% in patients treated with conventional fractionated radiotherapy (FRT). However, the biological determinants of this response have not been investigated and recent analysis of patterns of failure in patients treated with ART show an increased tendency towards distant metastatic recurrence. We investigated the biological response of NSCLC cell lines with different molecular subtypes including, A549, H1975 and HCC827 to ART and FRT. Radiation doses of 8Gy and 12Gy were delivered in multiple fractions or single fraction. The response to radiation was investigated using several cellular assays including clonogenic survival, cell proliferation, matrigel invasion, wound-healing, morphological characterization and senescence-associated beta-galactosidase. ART significantly reduced cell proliferation and clonogenic survival compared to FRT in A549 cells. In addition, a significant increase in the number of senescent cells as well as large, polynucleated cells was observed in the ART-treated group compared to the FRT-treated group. This differential response to delivery approach (ART vs FRT) was not observed in HCC827 or H1975 cells which harbor EGFR mutations. Both ART and FRT inhibited cell proliferation and clonogenic survival to similar levels in HCC827 and H1975 cells. In contrast to reduced cell proliferation and clonogenicity, ART significantly increased the invasive phenotype of A549 cells, but not HCC827 or H1975 cells compared to FRT. Western blot analysis of several markers of invasion, including cMET, AKT, ERK, SPARC and FAK revealed a significant down regulation of SPARC in A549 cells exposed to ART, but not FRT. Further analysis of cells in Boyden chambers showed that ART-induced senescent cells are capable of migration/invasion. Our results unequivocally demonstrate that response to ART is cell-line dependent. A549 cells, which harbor wild-type EGFR have a differential response to radiotherapy based on delivery approach. Furthermore, A549 cell exposed to ART have significantly increased invasive and migratory capacity compared to FRT. Our findings suggest that the extracellular matrix glycoprotein, SPARC is involved in the modulation of radiation-induced invasion/migration. These findings can have significant implications for NSCLC patients undergoing ART and underscore the importance of understanding the underlying biology for effective disease management. Citation Format: Ayman J. Oweida, Zeinab Sherifi, Yaoxian Xu, Siham Sabri, Bassam Abdulkarim. Evolving biological and clinical concepts of radiation delivery in NSCLC: response to ablative versus fractionated radiotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 865. doi:10.1158/1538-7445.AM2014-865

Study of alternative oxidase as possible molecular marker for phylogenetic analysis of the Botrytis cinerea

Botrytis cinerea (teleomorph Botryotinia fuckeliana (de Bary) Whetzel) is able to attack several economically important plants causing gray rot. Botrytis cinerea species complex includes two cryptic species (B. cinerea and B. pseudocinerea) that tolerate fungicides differently. On the basis of classical taxonomic markers, the two related species are very difficult to be distinguished; therefore, their separation is usually performed using molecular methods based on the time-consuming molecular analysis of several markers. Our goal was to find markers, which are suitable for the differentiation. Testing the nucleotide sequences of the alternative oxidase encoding gene, B. cinerea and B. pseudocinerea strains were clearly differentiated. Moreover, the analysis of the protein sequences of the enzyme with the maximum likelihood method reflected well the taxonomic relationships of the different fungi.

Pharmacogenetic Analysis in Neoadjuvant Chemoradiation for Rectal Cancer: High Incidence of Somatic Mutations and their Relation with Response

The identification of predictive markers of response to chemoradiotherapy treatment remains a promising approach for patient management in order to obtain the best response with minor side effects. Initially, we investigated whether the analysis of several markers previously studied and others not yet evaluated could predict response to 5-fluorouracil- and capecitabine-based neoadjuvant treatment in locally advanced rectal cancer. We studied germline and tumoral samples of 65 stage II/III rectal patients. A panel of pharmacogenetic markers was genotyped in paired peripheral blood samples and rectal cancer tumors. Our results seem to confirm the previously described association of thymidylate synthase and the prediction of chemoradiotherapy response in rectal cancer. However, it failed to confirm the clinical utility proposed for XRCC1, ERCC1, ERCC2, MTHFR and EGFR polymorphisms in blood/germline samples. Subsequently, with the aim of improving prediction of individual response and assessing the role of studied polymorphisms in response to treatment, we determined if changes in tumor response to these markers could predict clinical outcome. We found a high degree of changes between germline and tumor samples, mainly somatic mutations without microsatellite instability, and a minor frequency of loss-of-heterozygosity events. In tumoral samples, XRCC1 appeared to be significantly associated (p = 0.006) with downstaging of the tumor (odds ratio: 7.93; 95% CI: 1.03-60.83), but the increasing of TYMS low-expression alleles contradict the previous results observed in germline samples. The detection of somatic mutations in rectal cancer tumors led us to re-evaluate the utility of the tests performed in blood samples for these polymorphisms in rectal cancer. Furthermore, studies aimed at assessing the influence of pharmacogenetic markers in treatment response performed in blood samples should take into account the particular pattern of hypermutability present in each tumor type. We hypothesize that different patterns of hypermutability present in each tumor type would be related to the different results in association studies related to response to the treatment.

Ribonuclear inclusions and MBNL1 nuclear sequestration do not affect myoblast differentiation but alter gene splicing in myotonic dystrophy type 2

Myotonic dystrophy type 2 (DM2) is an autosomal dominant multisystemic disorder caused by a CCTG expansion in intron 1 of the zinc finger protein 9 gene on chromosome 3. Mutant transcripts are retained in muscle nuclei producing ribonuclear inclusions, which can bind specific RNA-binding proteins leading to a reduction in their activity. The nuclear sequestration of muscleblind-like proteins appears to be involved in splicing defects of genes directly related to the myotonic dystrophy phenotypes. Experimental evidence suggests that ribonuclear inclusions and muscleblind-like protein 1 (MBNL1) sequestration are strongly involved in DM2 pathogenesis. By using fluorescence in situ hybridization in combination with MBNL1-immunofluorescence, we have observed the presence of ribonuclear inclusions and MBNL1 nuclear sequestration at different time points of in vitro myoblast differentiation in each DM2 patient examined. Immunofluorescence and Western blot analysis of several markers of skeletal muscle differentiation reveal that the degree of differentiation of DM2 myoblasts is comparable to that observed in controls. Nevertheless the splicing pattern of the insulin receptor and MBNL1 transcripts, directly related to the DM2 phenotype, appears to be altered in in vitro differentiated DM2 myotubes. Our data seem indicate that the presence of ribonuclear inclusions and MBNL1 nuclear foci are involved in alteration of alternative splicing but do not impair DM2 myogenic differentiation.

Translational repression by MSY4 inhibits spermatid differentiation in mice.

In developing male germ cells, newly synthesized protamine mRNAs are stored for up to 7 days before translational activation. Translational repression of protamine 1 (Prm1) mRNA requires sequences present in its 3' untranslated region (UTR) and substantial evidence suggests a role for the murine Y-box protein MSY4 in this process. To determine if MSY4 can mediate translational repression in vivo, we generated transgenic mice in which the temporal window of MSY4 expression was extended during spermatogenesis. Expression of MSY4 disrupted the normal completion of spermatogenesis and caused dominant sterility. Immunocytochemical analysis of several markers, including the protamines, indicated that MSY4 prevented normal activation of translation. mRNAs whose translation was inhibited contained at least one MSY4 RNA recognition site, suggesting sequence-dependent translational repression. Altered translational activation resulted in defective processing of protamine 2 and severe defects in sperm morphogenesis. These results suggest that MSY4 plays an active role in translational repression of several mRNAs in differentiating spermatids.

Development of multiplex sets of simple sequence repeat DNA markers covering the soybean genome

Multiplexing involves the analysis of several markers in a single gel lane that is based on the allele size range of marker loci. Multiplex SSR marker analysis is conducted with primers that are labeled with one of three dyes. The development of an SSR multiplex system requires estimates of the allele size range of markers to strategize primer labeling and for grouping markers into multiplex sets. A method is presented that describes the development of multiplex sets of SSR markers in soybean (Glycine max (L.) Merr.) by the selective placement of primer sites and by the analysis of diverse germplasm. Primer sites were placed at specific distances from the SSR to adjust the allele size range of marker loci. The analysis of pooled DNA samples comprising diverse soybean genotypes provided robust estimates of the allele size range of marker loci that enabled the development of multiplex sets. Eleven multiplex sets comprising 74 SSR markers distributed across the 20 linkage groups of soybean were developed. Multiplex sets constructed from the analysis of diverse soybean germplasm should have a wide range of genotyping applications. The procedures used in this study were systematic and rapid and should be applicable for multiplex development in any species with SSR marker technology.