There is a paucity of data characterizing right ventricular performance in heart failure with preserved ejection fraction (HFpEF) using the gold standard of cardiovascular magnetic resonance imaging (CMR). We aimed to assess the proportion of right ventricular systolic dysfunction (RVD) in HFpEF and the relation to clinical outcomes. As part of a single-centre, prospective, observational study, 183 subjects  underwent extensive characterization with CMR. transthoracic echocardiography, blood sampling and six-minute walk testing. Patients were followed for the composite endpoint of death or HF hospitalization. RVD (defined as right ventricular ejection fraction < 47%) controls was present in 19% of HFpEF. Patients with RVD presented more frequently with lower systolic blood pressure, atrial fibrillation, radiographic evidence of pulmonary congestion and raised cardiothoracic ratio and larger right ventricular volumes. During median follow-up of 1429 days, 47% (n = 64) of HFpEF subjects experienced the composite endpoint of death (n = 22) or HF hospitalization (n = 42). RVD was associated with an increased risk of composite events (Log-Rank p = 0.001). In multivariable Cox regression analysis, RVD was an independent predictor of adverse outcomes  along with indexed extracellular volume  and E/E’ . RVD as assessed by CMR is prevalent in nearly one-fifth of HFpEF patients and is independently associated with death and/or hospitalization with HF.The trial was registered retrospectively on ClinicalTrials.gov (Identifier: NCT03050593). The date of registration was February 06, 2017.The online version of this article (10.1007/s10554-020-01953-y) contains supplementary material, which is available to authorized users. The importance of right ventricular (RV) function and its impact upon functional status  and outcCardiovascular magnetic resonance imaging (CMR) is the gold standard for RV volumetric and functional assessment, providing excellent accuracy and reproducibility , 7. Howe1] < 30% predicted or forced vital capacity [FVC] < 50% predicted); estimated glomerular filtration rate (eGFR) < 30 ml/min/m2 and standard contraindications to CMR.HFpEF patients were recruited as part of the Developing Imaging And plasMa biOmarkers iN Describing HFpEF (DIAMOND-HFpEF) study: an observational, cohort study conducted at a single tertiary cardiac centre . The NatA control group of 48 asymptomatic, age- and sex-matched subjects without known cardiac disease were also recruited. Since hypertension is highly prevalent in the general population without heart failure and is also strongly associated with incident HFpEF development, we included a subset of hypertensive controls (n = 22) in order to account for this potential confounder. All study participants underwent comprehensive clinical evaluation, blood sampling, TTE, six-minute walk testing (6MWT) and CMR during a solitary study visit.Blood was sampled for measurement of B-type natriuretic peptide , haematocrit, haemoglobin and renal function .As reported previously , 12 imagTo provide an objective metric of exercise capacity, the 6MWT distance was measured in all subjects according to standardized protocols .The radiology reports of the most recent chest X-ray prior to the study visits were sourced from the hospital computerized reporting system . The preThe CMR protocol used has been reported previously , 14, 15.cvi42 software  by a single experienced observer (PK), blinded to all clinical data. All volumetric and mass data were indexed to body surface area (BSA). Ventricular volumes . In patients experiencing multiple events, the time to first event was used as the censored outcome. Outcome data were obtained from Hospital databases. All patients had a minimum of 12 months follow-up, post study entry.t test, Mann–Whitney U test and the Chi-square test as appropriate. BNP, creatinine, 6MWT distance and RVEF were log10 transformed before analysis.Statistical analyses were performed using SPSS V22. A p value of < 0.05 was considered significant. Normality for continuous data was assessed using histograms, Q-Q plots and the Shapiro–Wilk test. Continuous data are presented as mean (± standard deviation) or median (25–75% interquartile range or range). Categorical data are presented as absolute numbers or percentages. Between group differences were compared using the Spearman’s rank correlations were performed to check for important associations of other continuous variables with RVEF. Intra-observer and inter-observer variability assessments for RV parameters were undertaken (by PK and JRA), a minimum of 4 weeks apart on 10 randomly selected patients.Event rates were calculated from Kaplan–Meier survival analysis. Survival curve differences were assessed using the Log-Rank test. Univariable Cox regression modeling was initially performed to identify variables associated with outcomes. Only parameters associated with endpoints at p < 0.1 were entered into subsequent multivariable analysis to identify independent predictors using both backwards and forwards stepwise elimination methods. In cases of collinearity, only variables with the highest coefficients or which have shown historically stronger prognostic importance based upon published literature were entered into multivariable analysis. To further minimize over-fitting, Cox regression models were limited to one parameter per approximately 10 composite events. Four separate, clinically relevant models were created including a final model comprising the strongest predictors. Continuous variables were further Z-standardized to enable comparison of hazard ratios (HR) based upon one standard deviation increase in the predictor variable. The accuracy of the final independent Cox model to predict events was then tested by receiver operator characteristics (ROC) analysis.Two hundred and thirty two subjects were enrolled , of whom 49 were excluded  and sex. Approximately two-thirds of HFpEF patients had experienced prior hospitalization for HF or had radiographic evidence of pulmonary congestion. HFpEF was frequently associated with co-morbidities including obesity, diabetes, hypertension, atrial fibrillation (AF), renal dysfunction and anaemia. Significant minorities of HFpEF also had known ischaemic heart disease (22%) and lung disease (16%). Furthermore, HFpEF patients had worse exercise capacity (shorter 6MWT distance) and nearly a third were classed as NYHA III/IV.Metrics of diastolic dysfunction , and LV mass) were higher in HFpEF. Compared to control subjects (58 ± 5%), LVEF was marginally lower in HFpEF , albeit preserved overall. More concentric remodeling (higher LV mass/volume) and diffuse fibrosis (iECV) was also evident in HFpEF. LA remodeling  and dysfunction (lower LAEF) was highly prevalent in HFpEF, irrespective of cardiac rhythm. RVEF in the control group had a narrow range  in contrast to HFpEF , but the difference was not statistically significant (p = 0.090). No significant differences in RV volumes between HFpEF and controls were noted.RVD  of patients with HFpEF. Compared to patients without RVD, those with RVD presented more frequently with lower systolic blood pressure, AF, radiographic evidence of pulmonary congestion and elevated cardiothoracic ratio. RVD was associated with larger right ventricular and LA (LAVImin) volumes, and lower LAEF (irrespective of AF or sinus rhythm). Furthermore, prior hospitalization with decompensated HF was also more prevalent in this sub-group. There were no statistically significant differences between groups in terms of medical history, biochemical profiles and prescribed cardiac pharmacotherapies. While measures of functional status were worse in the RVD group i.e. greater proportion of NYHA III/IV and shorter 6 MWT distance walked, these differences did not reach statistical significance.Statistically significant correlations of RVEF with other variables are shown in Table During median follow-up of 1429 days (1153–1654), 47% of HFpEF subjects (n = 64) experienced the composite endpoint of death (n = 22) or hospitalization with HF (n = 42). There were no events in the control group.Kaplan–Meier survival curves stratified according to the presence or absence of RVD in HFpEF are shown in Fig. This is the first prospective study to analyze RV systolic performance and remodeling with CMR in both age- and sex-matched HFpEF and control groups. The principal findings in our study are that in HFpEF: (1) RVD is present in a significant minority; (2) RVEF is associated with RV/LV/LA volumes and LA function; and (3) RVD is independently associated with the risk of death or re-hospitalization with HF.To date, the reportedly wide range of prevalence of RVD in HFpEF of 4 to 44% has been derived almost exclusively from echocardiographic data . FactorsCMR is the established gold standard for RV assessment , 7. To dIn HFrEF, the presence of RVD portends poorer functional status, exercise capacity , 20 and Our work however adds to findings from the only 2 CMR-based HFpEF outcome studies to date , 9 and cIn line with previous studies, the RVD sub-group in our HFpEF cohort was also noted to have lower systolic blood pressure, more frequent AF , 27, higOther authors have previously suggested a clear relationship between RVD and the severity of left heart disease as reflected by NYHA class, natriuretic peptides or LV systolic function . HoweverAlthough the observational nature of our study precludes determination of causation, AF was significantly associated with RVD, suggesting a contributory role. Our findings of a higher AF prevalence are consistent with similar reports from previous HFpEF studies , 22, 24.Our results, through the gold standard medium of CMR reinforce previous data that RVD is present in a significant minority of HFpEF. Furthermore, the presence of RVD alone and not necessarily more severe RVD is associated with heightened risk in HFpEF. RVD may drive recurrent HF hospitalizations and mortality. Identifying RVD is potentially important for multiple reasons. HF hospitalizations are associated with significant morbidity and are a drain on healthcare resources . ImportaOur study is one of the largest to date evaluating RV performance utilizing CMR and also benefits from having the longest follow-up to gauge the impact of RVD on clinical outcomes. Furthermore, we also have a comparator control group which is a particular strength. While RVEF measurement is reportedly more reproducible using axial slice orientations , we deliThis is a single centre, observational study and therefore should be replicated in additional cohorts. The association between RVD and outcome does not imply causality. We also do not have outcome data for deaths categorized as cardiovascular versus non-cardiovascular. Our definition of HFpEF was not in accordance with latest European Society of Cardiology guidelines . In partRVD as assessed by CMR is present in a significant proportion of HFpEF and is independently associated with death and or HF hospitalizations.Supplementary file1 (PDF 383 kb)Below is the link to the electronic supplementary material.


Gordonia. Jodelie19 is a lytic phage found in Gordonia rubripertincta NRRL B-16540. The temperate phage BlingBling and lytic phage Burnsey were both isolated using the host bacterium Gordonia terrae 3612.Jodelie19, BlingBling, and Burnsey are bacteriophages identified using host bacteria of the genus  Gordonia. Jodelie19 is a lytic phage found in Gordonia rubripertincta NRRL B-16540. The temperate phage BlingBling and lytic phage Burnsey were both isolated using the host bacterium Gordonia terrae 3612.Jodelie19, BlingBling, and Burnsey are bacteriophages identified using host bacteria of the genus  Actinobacteria, like Gordonia spp., due to their ability to serve as human pathogens. Siphoviridae Jodelie19 was collected from damp soil located by a pond in Las Vegas . Jodelie19 was discovered from direct isolation and displayed cloudy plaques 1 mm in diameter   (http://www.phrap.org/consed/consed.html) to verify accuracy and complete genome circularization and to determine genomic termini as previously described (All three bacteriophages were collected as part of the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program . The SEAdiameter . Siphovi950639W) . Siphovi026757W) . The bachttp://cobamide2.bio.pitt.edu/computer.htm), Starterator v1.2 (https://github.com/SEA-PHAGES/starterator), Phamerator (phamerator.org) (phagesdb.org/blast) (https://blog.kbrinsgd.org/), GeneMark v2.5p  , PhagesDg/blast) , PECAAN rk v2.5p , Glimmerrk v2.5p , ARAGORNrk v2.5p , tRNAscark v2.5p , TMHMM (rk v2.5p , and SOSrk v2.5p .Gordonia bacteriophages are clustered together if they share at least 35% of their genes (Putative functions were predicted for 23 of 91 of Jodelie19’s genes. Bioinformatic analysis of BlingBling’s 76 genes allowed for a putative function assignment for 35 genes and 30 genes out of 70 genes for Burnsey. ir genes . JodelieMT498052 and SRR12707452, respectively. BlingBling GenBank and SRA accession numbers are MT553336 and SRR12707454, respectively. Burnsey GenBank and SRA accession numbers are MT889378 and SRR12707453, respectively.Jodelie19 GenBank and SRA accession numbers are


The identification of soluble fibroblast growth factor (FGF) receptors in blood and the extracellular matrix has led to the prediction that these proteins modulate the diverse biological activities of the FGF family of ligands in vivo. A recent structural characterization of the soluble FGF receptors revealed that they are primarily generated by proteolytic cleavage of the FGFR-1 ectodomain. Efforts to examine their biological properties are now focused on understanding the functional consequences of FGFR-1 ectodomain shedding and how the shedding event is regulated. We have purified an FGFR-1 ectodomain that is constitutively cleaved from the full-length FGFR-1(IIIc) receptor and released into conditioned media. This shed receptor binds FGF-2; inhibits FGF-2-induced cellular proliferation; and competes with high affinity, cell surface FGF receptors for ligand binding. FGFR-1 ectodomain shedding downregulates the number of high affinity receptors from the cell surface. The shedding mechanism is regulated by ligand binding and by activators of PKC, and the two signaling pathways appear to be independent of each other. Deletions and substitutions at the proposed cleavage site of FGFR-1 do not prevent ectodomain shedding. Broad spectrum inhibitors of matrix metalloproteases decrease FGFR-1 ectodomain shedding, suggesting that the enzyme responsible for constitutive, ligand-activated, and protein kinase C-activated shedding is a matrix metalloprotease. In summary, shedding of the FGFR-1 ectodomain is a highly regulated event, sharing many features with a common system that governs the release of diverse membrane proteins from the cell surface. Most importantly, the FGFR ectodomains are biologically active after shedding and are capable of functioning as inhibitors of FGF-2. Drosophila and mammals leads to a wide variety of developmental and functional defects, emphasizing the importance of shedding throughout the phylogenetic tree thymidine , followed by incubation for 5 h. The cultures were then processed for scintillation counting, as previously described [Swiss 3T3 cells were grown in 96-well dishes (4000 cells/well) for 2 days in DMEM containing 10% calf serum, at which time they were confluent. After washing with serum-free DMEM, they were incubated for a further 2 days in DMEM with 0.5% calf serum. The rate of DNA synthesis was measured 24 h after the addition of FGF-2 and soluble FGF ectodomains by the addition of 0.2 mCi/well of [methyl-escribed .The 3.5 kb cDNA fragment encoding human FGFR-1 in the mammalian expression vector pcDNA3 (FGFR-1/pcDNA3) was used to generate the mutants employed in this study. A 138 bp HincII-KpnI fragment containing a portion of the third Ig-domain and the transmembrane region was subcloned into pBluescript for PCR mutagenesis. The first mutant—Bingo (B1)—was made by deleting a stretch of seven amino acids called PAVMTSP  containing the putative Val-Met cleavage site ,47, locaThe primers used to generate the mutants (upstream and downstream sequences) were as follows: 5′ CTGGAAGAGAGGCTGTACCTGGAGATC 3′ and 5′ CAGCCTCTCTTCCAGGGCTTC 3′ for Bingo (B1); 5′ CGGACCCAGGGCTTCCAGAACGGT 3′ and 5′ GTTCTGGAAGCCCTGGGTCCGCTGTACCTGGAGATCATC 3′ for P2; and 5′ AACGGTCAACCATGCAGAGTG 3′ and 5′ GCATGGTTGACCGTTCTGTACCTGGAGATCATC 3′ for L4. The mutations were confirmed by sequencing before subcloning back into the HindIII-KpnI site of pcDNA3/FGFR-1 (h1/p3) for further studies.


Major depressive disorder (MDD) is the most severe depression type and one of the leading causes of morbidity worldwide. Animal models are widely used to understand MDD etiology, pathogenesis, and treatment, but the efficacy of this research for patients has barely been systematically evaluated. Such evaluation is important given the resource consumption and ethical concerns incurred by animal use. We used the citation tracking facilities within Web of Science and Scopus to locate citations of original research papers on rats related to MDD published prior to 2013—to allow adequate time for citations—identified in PubMed and Scopus by relevant search terms. Resulting citations were thematically coded in eight categories, and descriptive statistics were calculated. 178 publications describing relevant rat studies were identified. They were cited 8,712 times. More than half  of their citations were by other animal studies. 794 (less than 10%) were by human medical papers. Citation analysis indicates that rat model research has contributed very little to the contemporary clinical understanding of MDD. This suggests a misuse of limited funding hence supporting a change in allocation of research and development funds targeting this disorder to maximise benefits for patients. Depression is the leading cause of disability worldwide . NowadayCurrently, and according to DSM-5 , there aMDD etiology is not completely understood yet. Most authors agree that there is a combination of biological and environmental factors that determine the triggering of the disorder . BiologiEvidence suggests that there are genetic factors involved, but even though more than 100 candidate genes have been investigated, a clear connection between specific genes and MDD has not yet been established . FurtherRecent studies also suggest that there is a link between inflammation and MDD, suggesting that MDD has an inflammatory subtype , but theAmong the most documented environmental factors linked to MDD are childhood traumas, which also cause biological changes in the brain of MDD patients , stressfIt is not always possible to determine a proximal cause. MDD may have seasonal or peri-partum onset, as well as being induced by other disorders  or substance ingestion, but it can also emerge without an obvious reason .Major depressive disorder pathogenesis is as diverse as its etiology. Even though there are several different treatment courses available  .Randomized controlled trials (RCTs) are considered to be the gold standard for empirical research , namely,In this regard, advanced magnetic resonance imaging techniques used in patients and healthy controls can be a powerful tool regarding the physiological and metabolic characterization of brain tissue, in the same way that single photon emission computed tomography and positron emission tomography imaging modalities provide valuable data on brain function and activity .in vitro emerge : “Depressive Disorder, Major” AND “rat” OR “rodent.” MeSH terms are a comprehensive list of key terms made available by PubMed designed to identify all relevant studies in an area . So, seaSince our goal was to evaluate the contribution of animal models—particularly rat models—to current clinical knowledge of MDD, we manually excluded from our analysis all the papers that reported animal and human data, as well as papers reporting other species’ data . Hence, we included only original papers focused on MDD, exclusively using data obtained from rats. We excluded papers describing other disorders, data from other species along with data from rats, and papers that did not present first hand data . Out of 237 papers originally located through the search engines’ filters, we manually excluded 59 because they met exclusion criteria.The retrieved papers were subjected to a subsequent citation analysis using the cited reference search facility within Scopus and Web of Science.Web of Science is a major scientific citation indexing service that encompasses over 50,000 scholarly books, 12,000 journals, and 160,000 conference proceedings. Scopus is the largest citation database; it covers nearly 36,377 titles from approximately 11,678 publishers.For each rat study, we recorded the total number of times it was cited, and allocated each citation to one or more of seven categories, defined prospectively:–Animals. This category included all animal studies from observational ethological studies to invasive procedures as defined by –Humans. This category included papers that used human participants. They included clinical or treatment trials , papers aiming to explore psychological, social, biochemical, physiological, genetic, or neurological variables related to MDD or other human disorders; as well as papers aiming to understand the relationship between MDD and other disorders (co-morbidities) in human patients. We recorded within this category which human papers focused on MDD and which focused on other disorders.–Reviews. This category included narrative reviews, systematic reviews, meta-analysis, as well as extensive opinion papers that did not report original empirical data. We included reviews focused on animals and on humans alike.–Editorials. This category included editorials, comments, and clinical guides.–In vitro. This category included exclusively cell-line data. Whenever the source of the tissue or cell was a human participant  or a laboratory animal killed for such purpose the paper was allocated into “human paper” or “animal research paper,” respectively.–In silico. This category included data obtained via computer simulations of human data.–Social. This category included human surveys or other social perception papers.Whenever it was not possible to define the category of the citing paper , the paper was denoted “not available” and removed from the sample. If more than one category could be assigned to a citing paper , then that paper was allocated to every appropriate category.t-test. Results were considered statistically significant when P < 0.05. The analyses were performed in R 3.6.1  studies were never cited in subsequent publications describing human studies on MDD, and 53 (30%) were never cited in any publications related to human research, either focused on MDD, or on other disorders such as post-traumatic stress disorder or bipolar disorder.in vitro papers (211), editorials (58), in silico papers (57), and human social papers (one). 230 citations were unavailable to us due to access or language barriers. These were removed from further analysis.As shown in p < 0.001). Beyond the statistical significance, this is certainly a considerable practical difference that reflects almost 100∗(53.3–9.1)/53.3 less citations by human papers than by animal papers.The proportion of citations by human medical papers was 9.1% while the proportion of citations by animal papers was 53.3%. This corresponds to a mean difference between the proportions of citations by human and by animal papers of −46%  of the original papers retrieved were never cited in subsequent papers related to MDD in humans, and in fact about a third (30%) were never cited in any subsequent human studies.Our citation analysis revealed that only around a tenth (9.1%) of the total number of citations were by human medical papers. This result contradicted the hypothesis that the proportion of citations by human medical papers would be substantially higher when compared to other research categories. This hypothesis was based on the assumption that animal models are essential to clinical research, as promulgated by several authors e.g., . Hence, The results of our study are in agreement with previous studies that empirically evaluated the contribution of animal models to human healthcare either through citation analysis e.g.,  either tThis suggests that biomedical research resorting to animal models is not normally considered significant, or particularly visible to, the human medical research community.Supporters of animal models of human disorders claim that this happens: (a) due to differences in the way basic animal work and human clinical trials are conducted, and propose a change to a translational biomarker-based approach within early steps of pre-clinical research ; or thatOpponents of the use of animal models point out that animal models lack external validity, i.e., findings derived in one setting, population, or species cannot be reliably applied to other settings, populations, and species, which is unavoidable since animal models: (a) oversimplify complex human disorders and the conditions in which they occur ; (b) areThis paradigm change toward human-based research is gaining more and more supporters both for ethical and scientific reasons e.g., . But thein vitro and in silico) which received more citations by human medical papers in a small study previously conducted  of the citations our target papers received were by subsequent animal papers, which strengthens the likelihood of the path dependency phenomenon described above. It can be argued that there is a need to have a substantial amount of animal research before achieving a critical mass that can lead to useful breakthroughs in human health, which might explain the high level of citations of animal papers by subsequent animal papers. Nonetheless, it does not explain the low level of citations by human papers, especially when this does not appear to be a trend in human-based approaches  or may have been excluded due to human error, as only one person selected which retrieved papers met exclusion criteria.Similarly, we did not take note of certain citations such as self-citations, in-house citations , and content-irrelevant citations . Such citation types were not considered significant in several studies and were similarly dismissed in recent studies .Also, we did not analyze the quality of citing papers as in previous studies , 2019. TFinally, we acknowledge that being cited by human medical papers is not the only indicator of the clinical usefulness of research. Uncited studies may also contribute, for example, through direct transfer of knowledge between scientists and clinicians. Most clinicians are not involved in research, i.e., they do not publish; hence, it is possible that practitioners involved in the treatment of MMD are informed by or utilize findings from rat studies through direct transfer of knowledge. A citation analysis does not measure this effect. However, it is also possible that clinicians are less aware of research results, than researchers. Thus, a survey on practitioners—which we did not perform—would have been an interesting complement to our results.Review papers on depression that might inspire authors likely cite animal papers, nonetheless if the animal paper is really relevant for the authors’ hypothesis or method it is likely the primary paper will be cited and not solely the review paper that referred to it.Citation rates may also be affected by factors such as article length, number of authors, their country, and university of affiliation . DespiteAccordingly, we believe that our citation analysis provides a valid and critical assessment of the contribution of rat models to current clinical knowledge of MDD. Such evaluation of the efficacy of research approaches is important to ensure the most responsible allocation of research funds and scientific resources, to maximize the likelihood of clinical benefit to patients, and to minimize the consumption of animal lives in research where such benefits are unlikely.The raw data supporting the conclusions of this article will be made available by the authors on request.CC developed the study concept with the supervision of AK, TM, and LV. CC and FP performed data collection. CC and TM made data analysis. All authors contributed to the study design and the draft of the manuscript and approved the final version of the article for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To select the appropriate drug carrier concentration, a series of solid dispersions were prepared in the drug carrier weight ratios of 1:1, 1:2 and 1:4 by fusion method. The solid dispersions composed of drug carrier at 1:4 weight ratio showed highest dissolution with the time required for the release of 50% of the drug <15 min compared to the supplied FFH (>120 min). The intestinal absorption study presented a significant improvement in the absorption of drug from the solid dispersions composed of poloxamer 188 than PEG 20,000. In summary, the solid dispersions of FFH prepared using PEG 20,000 and poloxamer 188 demonstrated improved dissolution and absorption than supplied FFH and could be used to improve the oral bioavailability of fexofenadine.The objective of this study was to enhance dissolution and permeation of a low soluble, absorbable fexofenadine hydrochloride (FFH) by preparing solid dispersions using polyethylene glycol 20,000  and poloxamer 188 as carriers. The phase solubility measurement for the supplied FFH revealed a linear increase in the solubility of fexofenadine with increasing carrier concentration in water (1.45 mg/mL to 11.78 mg/mL with 0%  Allergic rhinitis (AR), an immunoglobulin E-mediated inflammation of the nasal mucosa, is estimated to affect approximately 10% to 25% of the worldwide population  at 14.1°, 16.0°, 17.9°, 18.3°, 19.5° and 19.9°, matching with the diffraction pattern of crystalline FFH reported by Kumar et al. (2009) [(1:4) and PM-FP188(1:4)) and solid dispersions (SD-FP20K(1:4) and SD-FP188(1:4)) produced a diffraction pattern that exactly matched with the superimposed diffractograms of the supplied FFH and respective carrier  indicating the crystalline nature of drug in its solid dispersion/physical mixture form. The absence of new peaks in the prepared solid dispersions and physical mixtures ruled out the formation of new crystalline phase [. (2009) . PEG 20,. (2009)  and Ige,. (2009) , respectne phase .(1:4) and PM-FP188(1:4)) and the solid dispersions (SD-FP20K(1:4) and SD-FP188(1:4)). The FTIR spectrum of FFH showed characteristic peaks (cm−1) at 3296 , 2935 (ν O–H carboxylic acid), 1705 (ν C=O carboxylic acid), 1278 (ν C–N amine), 744 and 702 (δ C–H aromatic); PEG 20,000 at 3300 to 3600 (ν O–H), 2800 to 2900 (ν C–H of OC2H5) and 1000 to 1200 (ν C–O); poloxamer 188 at 3502 (ν O–H), 2885 (ν C–H), and 1112 (ν C–O). All the characteristic peaks of FFH and respective carrier  were conserved at the same position in their physical mixture (PM-FP20K(1:4) and PM-FP188(1:4)) and the solid dispersions (SD-FP20K(1:4) and SD-FP188(1:4)) indicating the absence of interactions between FFH and carrier in the physical mixtures and solid dispersions.eff(rat)) from the dispersions of supplied FFH, solid dispersions SD-FP20K(1:4) and SD-FP188(1:4) was found to be 7.04 ± 0.56 (×10−6) cm/s, 14.40 ± 0.27 (×10−6) cm/s and 21.30 ± 0.45 (×10−6) cm/s, respectively.The oral bioavailability of FFH in rats was reported to be about 4.2%  due to ieff(human)) and absorption rate constant (Ka) values also indicate significantly (p < 0.05) higher rate of absorption of the drug from prepared solid dispersions compared to the supplied FFH. The enhancement ratio (ER) for SD-FP20K(1:4) and SD-FP188(1:4) was found to be 2.04 and 3.03, respectively which is greater than 1 indicating enhanced permeation of drug from the solid dispersions. Microscopic observation of the intestinal segments without and with treatment of supplied fexofenadine, SD-FP20K(1:4) and SD-FP188(1:4) (The estimated human effective permeability coefficient (P188(1:4)  showed a(1:4) and SD-FP188(1:4) were able to significantly enhance (p < 0.05) the intestinal permeability compared to that of supplied FFH dispersion. However, the extent of enhancement was significantly higher with non-ionic surfactant, poloxamer 188 than PEG 20,000 which could be attributed to the inhibition of P-gp efflux pump along with faster dissolution rate. Several studies have reported that poloxamer 188 inhibits P-gp mediated efflux activity and enhance the permeability of P-gp substrate drugs across rat intestinal segments [Both SD-FP20Ksegments ,50. Ther(1:4) and SD-FP188(1:4), showed 2–3 fold improvement in fexofenadine absorption compared to the control drug only dispersion. However, the extent of enhancement of intestinal absorption was significantly higher with poloxamer 188 than PEG 20,000 which could be attributed to the inhibition of P-gp efflux pump along with faster dissolution rate. The results of this study demonstrated that poloxamer 188 could serve as a better carrier to improve the oral bioavailability of FFH by enhancing dissolution as well as intestinal permeability.In this study, solid dispersions of fexofenadine hydrochloride were successfully prepared using PEG 20,000 and poloxamer 188 following fusion method. The phase solubility and in vitro dissolution studies revealed an improved solubility and dissolution rate of FFH with increased concentration of carriers compared to the supplied drug. The solid-state characterization using DSC and XRPD analysis suggested the crystalline nature of fexofenadine in the prepared solid dispersions. The FTIR observation supported the absence of the physicochemical interactions between drug carrier combinations studied. The in situ single pass intestinal perfusion study was carried out to evaluate the effect of the carriers in the intestinal absorption of fexofenadine from the prepared solid dispersions. Both the solid dispersions, SD-FP20K


In contrast to the wealth of proven therapies for heart failure with reduced ejection fraction (HFrEF), therapeutic efforts in the past have failed to improve outcomes in heart failure with preserved ejection fraction (HFpEF). Moreover, to this day, diagnosis of HFpEF remains controversial. However, there is growing appreciation that HFpEF represents a heterogeneous syndrome with various phenotypes and comorbidities which are hardly to differentiate solely by LVEF and might benefit from individually tailored approaches. These hypotheses are supported by the recently presented PARAGON-HF trial. Although treatment with LCZ696 did not result in a significantly lower rate of total hospitalizations for heart failure and death from cardiovascular causes among HFpEF patients, subanalyses suggest beneficial effects in female patients and those with an LVEF between 45 and 57%. In the future, prospective randomized trials should focus on dedicated, well-defined subgroups based on various information such as clinical characteristics, biomarker levels, and imaging modalities. These could clarify the role of LCZ696 in selected individuals. Furthermore, sodium-glucose cotransporter-2 inhibitors have just proven efficient in HFrEF patients and are currently also studied in large prospective clinical trials enrolling HFpEF patients. In addition, several novel disease-modifying drugs that pursue different strategies such as targeting cardiac inflammation and fibrosis have delivered preliminary optimistic results and are subject of further research. Moreover, innovative device therapies may enhance management of HFpEF, but need prospective adequately powered clinical trials to confirm safety and efficacy regarding clinical outcomes. This review highlights the past, present, and future therapeutic approaches in HFpEF. Heart failure (HF) poses a growing burden for health systems worldwide as incidence and prevalence is rising annually. Typically, the term HF was applied to patients with reduced ejection fraction only, until the first reports on patients suffering from symptoms of HF despite having normal left-ventricular ejection fraction (LVEF) and small hearts emerged –3. InitiE/e′ ratio > 9, and echocardiographic pulmonary artery systolic pressure > 35 mmHg has been shown to substantially identify patients at high risk of HFpEF that should undergo further evaluation [HFpEF mostly affects older patients, predominantly females. Depending on various factors , the proportion of HFpEF among HF patients ranges from 22 to 73% . Also, taluation . Accordialuation , a step-aluation . If untraluation , 13. Thealuation .2−) instead of NO. These processes result in a vasoconstricting, pro-inflammatory, and pro-thrombotic state of endothelial dysfunction [Currently, the precise pathophysiological processes in HFpEF are incompletely resolved, since animal models are sparse. This is due to a high prevalence of comorbidities in HFpEF patients, which is difficult to be translated into animal models, which are typically younger and less comorbid . Howeverfunction . Furtherfunction , 21. Forfunction . Besidesfunction . Endothefunction , these cfunction , 25. In function , 27. Morfunction . Finallyfunction .Fig. 1Cu2 and 21% had a BMI between 23.5 and 26.4% kg/m2 [2 and in those with BMI ≥ 35 kg/m2. Both physical activity (PA) and caloric restriction are important non-pharmacological approaches to reduce obesity and deconditioning and have shown to be associated with prognostic effects. In a post hoc analysis of the TOPCAT trial, risk of HF hospitalization and mortality was lower in physically high-active HFpEF patients than in intermediate-active and poorly active patients [VO2 in a cohort of 100 obese HFpEF patients, most of which were female (81%). In addition, the effects were even greater when patients also had to join supervised exercise sessions three times a week, suggesting the combination of PA and diet to provide additive effects [Clinical findings suggest that prognosis in patients with HFpEF is highly influenced by comorbidities –32. This4% kg/m2 . Moreovepatients . The ongpatients . Further effects . Another effects . In a sm effects . Interes effects . Enhanci effects , 42. Two effects  and need effects . Myocard effects . Therefo effects . Accordi effects . While c effects , 49, it  effects . In a sm effects . HoweverIn past trials, there have been significant differences regarding the definition of HFpEF. In contrast to the ESC definition (LVEF ≥ 50%), major clinical trials such as the TOPCAT trial  or the rStimulation of AT1 receptors induces myocardial hypertrophy and fibrosis which can then lead to HF . ACE inhE/e′-ratio and decreased left-ventricular (LV) hypertrophy and NT-proBNP levels [p value for the treatment-by-region-interaction was not significant (p = 0.12) [Mineralocorticoid receptor antagonists (MRAs) prevent the maladaptive effects of aldosterone. Aldosterone mediates myocardial fibrosis , contribP levels . SurprisP levels . HoweverP levels  and theiP levels . These aP levels . FurtherP levels . As a reP levels . Critica = 0.12) . Moreove = 0.12) , 69, butIn the future, new studies such as the German prospective SPIRIT-HF trial (2017-000697-11) and the large registry-randomized clinical trial SPIRRIT-HF (NCT02901184) will reevaluate therapy with spironolactone in HFpEF patients. In SPIRIT-HF, particular emphasis will lie on patient characterization and selection. Novel MRAs, such as nonsteroidal aldosterone antagonists, will also be evaluated , 71.1-selective beta-blocker, in patients with impaired and preserved LV function  [VO2) in 116 HFpEF patients [High heart rate (HR) predicts poor outcome in patients with HFpEF and sinus rhythm, but does not apply for those in atrial fibrillation, as shown in a post hoc analysis of the I-PRESERVE trial . These fF > 35%) . In the patients . A largepatients . In the patients . Howeverpatients  and the patients  did not The angiotensin receptor neprilysin inhibitor LCZ696, combining the two acting agents valsartan and sacubitril, has revolutionized treatment of HFrEF. By inhibition of neprilysin, sacubitril increases ANP-, BNP- and CNP-plasma levels . These pIn conclusion, results from the PARAGON-HF trial support the heterogeneity of the HFpEF syndrome as well as the importance of an individually tailored approach in HFpEF therapy. In this context, identifying specific causes of HFpEF by an aetiological work-up is of great importance. Moreover, LCZ696 might be associated with beneficial effects in female patients and those with a LVEF between 45–57% which would include both HFmEF and HFpEF patients. This aspect may underline the limitations of subdividing HF phenotypes solely by LVEF. As the primary endpoint of PARAGON-HF was neutral, new prospective randomized studies in dedicated subgroups might scrutinize efficacy of LCZ696 in selected individuals.E/e′ ratio, exercise tolerance, and NT-proBNP levels in HFpEF patients [In a mouse model of HFpEF, established by diabetic mice (db/db), β-adrenergic receptor-independent reduction of HR with ivabradine, an inhibitor of the funny current, improved vascular stiffness, as well as systolic and diastolic function . Howeverpatients . Apparenpatients .In the DIG trial, cardiac glycosides were able to decrease the risk for overall hospitalization and hospitalization due to worsening HF in patients with HFrEF and HFpEF (LVEF > 45%) . On the All main approaches regarding device and pharmacological therapy in HFpEF patients are highlighted in Fig. 3 adrenergic receptor (β3-AR)-selective agonists, or endothelial nitric oxide synthase (eNOS) enhancer have been studied -axis represents a new promising approach in treatment of HFpEF. Experimental data suggest that disturbance of this signal cascade poses a specific pathomechanism in HFpEF, which promotes myocardial fibrosis, eventually leading to diastolic dysfunction , 88. TheNO-donating drugs Direct NO donators, for instance organic nitrates (isosorbide-nitrate), are not recommended in HFpEF patients. In the multicenter trial Neat-HFpEF on 110 patients with HFpEF, isosorbide mononitrate treatment even resulted in decreased activity levels [E/e′), and NT-proBNP levels [y levels . One majy levels . Moreovey levels . These ey levels . HoweverP levels . As for β3AR-selective agonists Conventional beta-blockers mainly target β1- and β2-adrenoreceptors (β1-AR/β2-AR), which can mediate maladaptive effects of prolonged catecholamine exposure including cardiac remodeling [3-AR, has been identified in human hearts [1-AR and β2-AR, these receptors prevent the myocardial hypertrophic response to neurohormonal stimulation [3-AR with the selective agonist mirabegron as a therapeutic option in HFpEF is currently studied in two clinical trials .modeling . Moreoven hearts . In contmulation . As a reEndothelial nitric oxide synthase (eNOS) activators Enhancing eNOS activity by the transcription amplifier AVE3085 results in increased production of NO and was shown to be associated with a significant improvement in diastolic function in a rat model [at model . HoweverPotential limitations of enhancing NO bioavailability According to a recent mouse model, nitrosative stress needs to be acknowledged as one of the main drivers in HFpEF rather than the limited bioavailability of NO [ty of NO . In thisPhosphodiesterase-5 inhibitors Therapy with the PDE5 inhibitor sildenafil did not improve pVO2 in HFpEF patients without evidence of pulmonary hypertension (PH) [ion (PH)  and failion (PH) . Howeverion (PH) . TranslaSoluble guanylyl cyclase stimulators and activators Vericiguat and riociguat, primarily used to treat PH, have been analyzed in HF patients in phase 2 clinical studies. As the SOCRATES-PRESERVED trial has shown, vericiguat improved QOL, but failed to reduce NT-proBNP levels or left-atrial volumes [ volumes . CurrentSodium-glucose cotransporter-2 inhibitors After empagliflozin led to a striking reduction of CV events in patients with type 2 diabetes at high CV risk in the EMPA-REG OUTCOME study [ME study , treatmeME study . Among tME study , 105. HoME study . CurrentME study .Incretins Modulation of the incretin system includes mimicking glucagon-like peptide 1 (GLP-1) effects and inhibition of the GLP-1-degrading enzyme dipeptidyl peptidase-4 (DPP-IV) [(DPP-IV) . GLP-1, (DPP-IV) . The cor(DPP-IV) . In larg(DPP-IV) , 111. CuPirfenidone represents an anti-fibrotic drug which targets the TGF-β signaling pathway and is mainly used in idiopathic pulmonary fibrosis [fibrosis . By actifibrosis . The PIRCross-link breakers Cross-link breakers target advanced glycation endproducts (AGEs), which are formed by proteins and carbohydrates that underwent “cross-linking” with the extracellular matrix [r matrix . Productr matrix . In a smr matrix . A similr matrix . In mousr matrix . ClinicaMicro-RNAs Micro-RNAs (miRNAs) are small non-coding RNA molecules which can interfere with gene expression on a post-transcriptional level by binding to messenger-RNA [nger-RNA . There inger-RNA , 120. Fonger-RNA . TherefoCytokine inhibitors Derived from the pathophysiological model of systemic inflammation being one of the main mediators in the development of HFpEF, cytokine inhibitors have been tested as therapeutic options. In the D-HART2 trial [VO2 and ventilatory efficiency. However, in a sub-analysis of the large RCT CANTOS [T2 trial , interleT CANTOS  includinT CANTOS .Cell therapy Cell therapy targets myocardial inflammation and myocardial fibrosis in HFpEF. In rat models, application of cardiosphere-derived cells (CDCs) decreased LV fibrosis and inflammatory infiltrates achieving normalization of LV relaxation and diastolic pressures and, therefore, led to an improvement in survival [+ cells, collected by apheresis after G-CSF stimulation, resulted in an enhancement in diastolic function (E/e′), and decreased NT-proBNP levels [+ cell therapy in patients with HFpEF is currently under further evaluation in the CELL-pEF-trial (NCT02923609). However, cell therapy has been evaluated as a promising therapy for CV diseases in numerous past trials without delivering consistent and convincing results. Questions about optimal cell type, dose, and delivery route are still inadequately answered [survival . The safP levels . CD34+ canswered .Szeto-Schiller peptides “Szeto-Schiller peptides (SS peptides)” belong to a new class of antioxidant peptides that bind to the cardiolipin, an important phospholipid in the inner mitochondrial membrane. SS peptides protect cardiolipin from oxidation and, thereby, prevent the damage of oxidative stress to mitochondria, maintaining ATP production and reducing further oxidative stress [e stress . The mose stress . In the e stress . In patie stress . CurrentAdenosine A1 receptor agonists The partial adenosine A1 receptor agonist Neladenoson bialanate is thought to yield several beneficial effects in the heart and also in the skeletal muscle. These compromise improvement in mitochondrial function and energy substrate utilization, enhanced SERCA2a activity, reversion of ventricular remodeling, and providing anti-ischemic properties [operties . In the operties .Levosimendan According to ESC guidelines, levosimendan, a calcium sensitizer and PDE3 inhibitor with vasodilative properties [operties , can be operties . The posoperties . Moreoveoperties . Thus, tIn patients with group 2 PH and HFpEF, administration of inhaled iloprost led to an acute reduction of PAP and pulmonary vascular resistance (PVR) during right heart catheterization . The twoFluid overload in patients with HFpEF can rapidly reduce QOL by causing dyspnea and peripheral edema or even lead to hospitalization due to cardiac decompensation. Since hospitalization for HF is associated with a higher mortality risk , monitorThe idea of creating artificial left–right shunts to reduce left-atrial pressure originates from a publication from the early twentieth century. The so-called Lutembacher syndrome was used to describe the finding that patients with an untreated mitral stenosis and resulting increase in left-atrial pressure benefit from a concomitant atrial septal defect . In a piIn HFpEF patients, LV mechanical dyssynchrony has been suggested to contribute to an impairment of longitudinal systolic and diastolic LV function and to be associated with higher LV-filling pressures and worse clinical status in terms of NYHA functional class . On the CCM aims to trigger molecular remodeling by delivering electrical signals into the septum during the refractory period and has been associated with numerous beneficial effects in chronic HF such as increased titin and troponin phosphorylation, and reduced expression of proteins that mark cardiac fibrosis . In two Renal denervation (RDN), a catheter-based, radiofrequency ablation of the renal sympathetic nerves, has been shown to effectively lower both systolic and diastolic blood pressure , 150. FuFluid overload can cause preload increase and as a result cardiac decompensation. Patients may suffer from peripheral edema and signs of congestion such as dyspnea. Therefore, diuretics, which are established drugs to treat fluid overload and signs of congestion, are a cornerstone in the symptomatic therapy of HFpEF . TreatmeThe 2016 ESC/HFA guidelines  acknowle


To investigate the effects of adalimumab pretreatment on the lipopolysaccharide-mediated myocardial injury. Twenty-eight Wistar rats were randomized into four groups (n=7). Control (C) group animals were injected once a day with intraperitoneal (i.p) 0.9 % saline for two days. In the Adalimumab (Ada) group, adalimumab was injected at a dose of 10 mg/kg/ day (i.p) for two days. Lipopolysaccharide (Lps) group rats were injected with a dose of 5 mg/kg (i.p) lipopolysaccharide. Lipopolysaccharide + Adalimumab (Lps+Ada) group rats received adalimumab before the administration of lipopolysaccharide. The animals were sacrificed 24 h after the last injection and blood samples were obtained for determination of biochemical cardiac injury markers and circulating levels of TNF-α and interleukin-6 (IL-6). Hearts were harvested for histological examination. Endotoxin exposure resulted in significant increases in serum cardiac injury markers, serum cytokines and histological myocardial injury scores in the Lps group. The levels of circulating cytokines, cardiac injury markers and histological injury scores for myocardial necrosis, perivascular cell infiltration, and inflammation were significantly reduced in Lps+Ada as compared to Lps group (p<0.05). Adalimumab pretreatment reduces endotoxin-induced myocardial damage in rats. This beneficial effect is thought to be related to the reduction of cytokine release. Systemic inflammation may be triggered by lipopolysaccharide (Lps) components of gram negative bacterial wall which are commonly known as endotoxins2 . Human intestinal flora produces a considerable amount of endotoxins but the endothelial cell lining in intestines restrict the transfer of these endotoxins into circulation and prevent the systemic inflammation in normal conditions. In the course of major surgical procedures including cardiac surgery with cardiopulmonary bypass and aortic aneurysm repair, the disruption of endothelial cell barrier may permit the passage of endotoxins through the mucosal epithelium to the adjacent tissues or distal organs and provoke a systemic inflammatory response leading to increased postoperative complications3 .Systemic inflammatory response syndrome is a phenomenon in which various stimuli- such as severe trauma, sepsis, acute mesenteric ischemia and major surgery- induce a widespread activation of inflammatory cascades leading to multi-organ failure7 . Cytokines have been suggested to be responsible for endotoxin-mediated myocardial injury9 . Increased myocardial and serum levels of TNF-α and IL-6 during Lps-mediated systemic inflammatory responses have shown to be associated with both cellular and functional cardiac deterioration11 . Furthermore, inhibition of TNF-α has been observed to attenuate endotoxin induced myocardial injury13 .The heart is among the most affected organs by the systemic inflammatory response14 . It is used in various chronic immune-mediated diseases such as rheumatoid arthritis, inflammatory bowel disease, psoriatic arthritis, vasculitis or ankylosing spondylitis to modify the inflammatory responses15 . However, the effect of adalimumab in cytokine-induced inflammatory myocardial damage has not been previously studied. The aim of our experimental study is to investigate the effects of systemic adalimumab treatment on circulating cytokines and myocardial injury in Lps-mediated systemic inflammation.Adalimumab is a recombinant human monoclonal anti-TNF-α antibody that blocks the interaction of TNF-α with the cell surface receptorsThe animal care was conducted in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals. The animals used in the experiment were obtained from the Aydın Adnan Menderes University Experimental Animals Research and Production Laboratory. Adult female Wistar rats (170-270g) were fed a standard rat chow diet and water ad libitum and kept in cages in a temperature (22°C ± 2°C) and humidity (45-50%) controlled room with a 12-h dark-light cycle and acclimatized for a week before the study. The experimental design and protocol were approved by the Animal Care Committee of Adnan Menderes University . The study was performed in the Adnan Menderes University Faculty of Medicine, Experimental Animals Laboratory, Aydın, Turkey. A total of 28 animals were randomly allocated into one of the four following groups each consisting of seven animals: Control (C), Adalimumab (Ada), Lipopolysaccharide (Lps), Lipopolysaccharide + Adalimumab (Lps+Ada) groups.Control (C) Group (n=7): Animals were injected once a day with intraperitoneal (i.p) 0.9% saline for two days. After the injection, they were returned to cages and allowed standard rat chow and water ad libitum for 24 h.Adalimumab (Ada) Group (n=7): Adalimumab  was injected at a dose of 10 mg / kg / day (i.p) for two days. The animals were returned to cages and allowed standard rat chow and water ad libitum for 24 h after last injection.Lipopolysaccharide (Lps) Group (n=7): Animals were injected with lipopolysaccharide (LPS) , with a dose of 5 mg/kg (i.p) then returned to their cages and were allowed standard rat chow and water ad libitum for 24 h17 .Lipopolysaccharide + Adalimumab (Lps+Ada) Group (n=7): Animals were given Adalimumab treatment as described above before the injection of lipopolysaccharide. Then, they were returned to cages and allowed standard rat chow and water ad libitum for 24 h after the last injection.0 until analysis of serum cytokines.In each group, 24 hours after the last injection animals were anesthetized with a combination of ketamine hydrochloride and xylazine injection (i.p). Then, a median sternotomy was performed and 1 cc of blood was sampled through right atrium puncture via 30 G x 8 mm cannula. After blood sampling, the rats were sacrificed by cervical dislocation and immediately after that, the heart was dissected free from the surrounding tissues, harvested for histopathological examination and fixed in 10% phosphate-buffered formalin. Blood samples were centrifuged at 1000g for 10 min to obtain the serum and were stored at – 80 C18 .Serum Troponin I and creatine kinase muscle-brain (CK-MB) analysis was made by Chemiluminescent micro particle immunoassay (CMIA) method with Autoanalyser  on the same day of samplingThe serum concentrations of IL-6 and TNF-α were measured by performing enzyme-linked immunosorbent assay (ELISA) analyses. Inflammatory mediators from each sample were quantified using rat specific ELISA kits . Results were calculated using ELISA micro plate reader  using standard curves and expressed as picograms per milliliter (pg/mL) of serum.Tissue samples were embedded in paraffin blocks and 4-µm thick sections were obtained across the ventricles. The sections were stained with hematoxylin-eosin and examined under light microscope  by a blinded pathologist. Myocardial tissue samples were evaluated in terms of congestion, inflammation, necrosis, and perivascular cell infiltration. Histopathological changes were scored on a 4-point semi quantitative scale as follows: none (0), mild (1), moderate (2), severe (3).Research data were evaluated using the SPSS 24.0 statistical program . The Kolmogorov-Smirnov test was used to evaluate whether the distribution of continuous variables were normal. As continuous variables were normally distributed, descriptive analyses were presented as means ± standard deviations. One-way ANOVA was used to compare parameters among study groups. Levene’s test was used to assess the homogeneity of variances. Post-hoc Tukey test was performed to determine the significance of pairwise differences using the Bonferroni correction to adjust for multiple comparisons. A 5% type-1 error level was used to infer statistical significance.Levels of biochemical cardiac injury markers among study groups are demonstrated in Serum cytokine levels in study groups are demonstrated in The results of histopathological injury scores are shown in In the present study, we used a model of lipopolysaccharide-induced experimental myocardial injury. Our findings indicate that adalimumab treatment significantly reduces circulating cytokines (TNF-α and IL-6), which are released as a response to lipopolysaccharide administration in rats. In animals that are subjected to lipopolysaccharide, adalimumab treatment resulted in a significant reduction in serum levels of cardiac injury markers and amelioration of histopathologic signs of myocardial injury. These findings suggest that TNF- α blocker adalimumab attenuates endotoxin-induced myocardial injury by suppression of cytokine release into the circulation. Furthermore, we also observed that the administration of adalimumab at the selected dose in rats did not cause any cardiac injury whilst slightly reducing the levels of circulating cytokines.19 . It acts as a mediator in local homeostasis by regulating cellular growth, cytoprotection, and innate immunity when produced as a part of a physiologic adaptive response. On the other hand; TNF-α plays a crucial role in various cardiovascular pathologies such as atherosclerosis, myocarditis and heart failure as a key molecule of the inflammation20 . It is postulated that an uncontrolled increase of TNF-α production or sustained accumulation of TNF-α leads to detrimental effects on the myocardium. These effects include the induction of apoptosis, enhancement of cytokine production, accumulation of inflammatory cells and decrement of myocardial contractility as a consequence of the deterioration in myocardial calcium metabolism20 . Therefore; blockage of TNF-α has been proposed to be a preventive or alleviating therapeutic target particularly in cardiac pathologies in which inflammation plays a role21 .Tumor necrosis factor-alpha is a multifunctional cytokine that has pleiotropic effects in the cardiovascular system. It is primarily produced by lymphocytes and macrophages; however, various other cell types such as cardiomyocytes, resident cardiac macrophages and vascular smooth muscle cells are also capable of TNF-α production22 . The major cytokines that induce cardiac damage during endotoxemia are shown to include TNF-α and IL-611 . Xianchu et al. demonstrated that in mice, myocardial TNF-α and IL-6 levels are increased in lipopolysaccharide-mediated myocardial damage and suppression of these cytokines using rutin extract reduces the inflammatory response and damage12 . In another experimental study, Chung et al. reported that estrogen causes cardiovascular protective effects via inhibition of TNF-α and IL-6 release in a lipopolysaccharide-induced sepsis model in rats23 . Similarly, our findings in the present study indicate that lipopolysaccharide administration results in myocardial damage, which is associated with a significant increment in circulating TNF-α and IL-6 levels in rats. We observed that adalimumab treatment before the administration of lipopolysaccharide causes attenuation of myocardial damage as determined by a significant reduction in cardiac injury markers and the severity of the histopathologic injury.In experimental and clinical conditions, endotoxin-mediated myocardial injury has been demonstrated to be related to increased cytokines24 . It has a beneficial disease-modifying effect in various chronic inflammatory pathologies by blocking the activity of TNF-α; however, its effect on serum TNF-α levels is inconsistent. Adalimumab treatment was not observed to cause a significant change in serum TNF-α profile in some clinical studies26 . On the other hand, it was reported to result in a significant decrement in circulating IL-6 levels in several inflammatory diseases26 . The reason for these findings may be the differences in individual genetic features and immunological processes affecting the response to the drug28 .Adalimumab binds with the soluble and trans-membrane TNF- α molecules with high affinity without binding to the other cytokines29 . It has been shown that adalimumab treatment reduces serum TNF–α levels in diabetic rats and leads to a decrement in both TNF–α and IL-6 serum levels in obese non-diabetic rats30 . In addition to this, Cure et al .31 reported that adalimumab decreases renal tissue cytokine levels and renal damage in a rat model of aortic clamping induced ischemia-reperfusion injury. Adalimumab has also been demonstrated to attenuate the ischemia-reperfusion mediated lung injury through inhibition of the increase in tissue TNF-α levels in rats32 . Furthermore, in an experimental setting of non-alcoholic steatohepatitis in rats, adalimumab has been declared to significantly decrease the serum TNF–α and IL-6 levels33 . In our experimental model, we found that the increment in levels of TNF–α and IL-6 in circulation as a response to lipopolysaccharide administration is significantly suppressed in adalimumab treated rats. We suggest that the myocardial protective effect of adalimumab, which we observed in this study, is the result of the reduction in circulating cytokines; however, this effect might also be related to the blockage of TNF–α in cardiac tissue.Although adalimumab has been reported not to bind with rat TNF-α in vitro, various in-vivo experimental studies have established that it can significantly inhibit the overproduction of TNF-α in rats34 . According to the product information provided by the manufacturer, the highest dose of adalimumab that has been evaluated during clinical trials is multiple intravenous doses of 10 mg/kg29 . Based on this knowledge, we preferred to use a dose of 10 mg /kg/day (i.p) and we have repeated the dose for two consecutive days, because the drug absorption and distribution following a single dose are relatively slow in the clinical setting24 . In order to determine the effect of the selected dose of adalimumab on serum cytokine profile and myocardium, we have administered the drug without endotoxin challenge in the Ada group. We did not observe any biochemical or histological evidence of myocardial damage associated with adalimumab treatment in our study. Although there was a slight decrease in the levels of serum cytokines in the adalimumab treated rats, the differences were not statistically significant as compared with the control group animals.Adalimumab has been usually used at a single intraperitoneal (i.p) dose of 50 mg/kg in experimental studies in rats, but in clinical practice, it is often delivered as a 40 mg single bolus dose via subcutaneous injection in adultsThere are some limitations of our study; firstly, although we aimed to mimic the production of TNF-α resulting from enhanced bowel permeability and bacterial translocation, which occurs during major surgical procedures, our experimental model of lipopolysaccharide injection does not fully meet the clinical situation. Secondly, we focused mainly on the circulating TNF-α and IL-6 levels but we have not studied the levels of or gene expressions of these cytokines in the myocardium and the levels of other inflammatory mediators that are likely to play a role in myocardial injury. Thirdly, we have not determined the changes in functional cardiac status by means of specific methods such as echocardiography. Therefore, the results of the present study should be interpreted with caution.The results of this study showed that adalimumab pretreatment reduces endotoxin-induced myocardial damage in rats. The beneficial effect of adalimumab observed in this study is thought to be related to the reduction of cytokine release. However, further investigation is needed to determine the effect of adalimumab in acute inflammatory conditions that damage the heart and enlighten the possible cardio protective role of adalimumab.


In this work, we have studied, formulated, prepared, and characterized the rheological and electrical behavior of a composite material based on an epoxy resin Diglycidyl Ether of Bisphenol A (DGEBA) reinforced with hexaglycidyl cyclotriphosphazene (HGCP). The epoxy system was cured with 4,4’-methylene dianiline (MDA). DGEBA-HGCP-MDA epoxy composite materials with reinforced HGCP which varied from 5% to 10% by weight were prepared by mixing in the molten state. The morphology was evaluated by SEM. The rheological behavior was studied using small deformation rheology. The electrical characterization was carried out with a frequency variation range from 1 Hz to 100 KHz at room temperature. These measurements revealed that the rheological and electrical behaviors strongly depend on the quantity of HGCP in the DGEBA matrix. The linear viscoelastic properties study reveals that the modulus of elasticity G’ is dependent on the amount of HGCP present in the epoxy resin DGEBA. The capacitance-frequency measurements suggest a distribution of localized states in the band gap of the blends.  Epoxy resins are thermosetting polymers that have been extensively employed in various areas such as aerospace, coatings, adhesives, electronic devices, laminates and encapsulations due to the characteristics of outstanding mechanical and electrical properties, relatively low curing shrinkage, superior adhesion to substrates as well as good thermal, chemical and corrosion resistance ,5,6,7,8.Hexachlorocyclotriphosphazene is a procured oligomer usually used for the synthesis of phosphazene-based polymers ,10. The In this work the three epoxy components (DGEBA-HGCP MDA) was reinforced by various amount of hexaglycidyl of cyclotriphosphazene (HGCP). The amounts of HGCP ranged from 5% to 10%. The epoxy system was cured with the MDA. Produced thermosetting network was characterized by various techniques that include morphology by SEM, rheology by HAAKE MARS Modular Advanced Rheometer System and electrical study by Keithley 3330LCZ impedance meter.The chemical structures of the materials studied in this work are represented in One night at 70 °C3 h at 100 °C2 h at 120 °C1 h at 140 °C30 min at 150 °CMixing the polyepoxide and the hardener (curing agent) produced a bridged three-dimensional network as shown in Numerous studies have already demonstrated that the physical properties largely depend on the degree of dispersion of the nanoparticles in the polymer matrices. Mainly, the influence of the degree of dispersion on the rheological and mechanical properties is well known. The absence of chemical bonds and compatibility between the polymer and the particle, which has a link with the dispersion, can influence the mechanical properties of the composites because a strong interfacial bond can effectively transfer the load from the matrix to the reinforcement. Often used are the chemical surface modification of the inorganic nanoparticle, the modification of the deposition reaction, the chemical modification of mechanical force, the modification by high energy and the modification by polymer surface grafting and intercalation. These changes resolve the heterogeneous aggregation issues. The interactions between the reactive groups of the polymer and the nanoparticles depend on the chemical structure of the polymer and the surface charge of the nanoparticles. Good dispersion was expected due to the organic group on the HGCP and since DGEBA is epoxy resin. Due to the compatibility between two species, the flame retardant additive dispersed in the DGEBA resin and no agglomerate was observed  18]..18].2]3-DGEBA), the aggregations were observed in the latter composite. This suggests that the modified chlorocyclophosphazene may be well dispersed in DGEBA, which resulted from improved compatibility with DGEBA resin and the crosslinked structure of cyclophosphazene. In this work, while HGCP had good dispersion in DGEBA epoxy resin, the study will focus on the effect of HGCP in the physical behavior of materials based on DGEBA epoxy resin.Compared to the composite  of the JEOL-JSM-5500 type , whose maximum resolution is 3.5 nm with a magnification ranging from 18 to 300,000 times and automatic focus adjustment , the acceleration voltage varies from 0.5 to 30 KV. A computer control system allowed images to be digitized using image processing software .For the measurement of rheological properties, an imposed constraint Rheometer of the HAAKE MARS Modular Advanced Rheometer System type  was used. This instrument is equipped with different measuring bodies. Our study is carried out with a plane-plane geometry with a diameter of 25 mm and a thickness of 1.8 mm. The dynamic oscillatory measurements were made using a frequency sweep ranging from 0.1 to 100 rad/s with a constant applied strain of 0.1% which is in the linear viscoelastic region. All measurements were taken at a constant temperature of 100 °C.The electrical measurements were carried out at room temperature on samples. The impedance measurements (capacitance-frequency) were carried out using a Keithley 3330LCZ impedance meter . All instruments are controlled by a computer via a GPIB card.The SEM micrograph of DGEBA sample mixed with HGCP is shown in Work on the rheological behavior of composites, i.e., macroscopic dynamic viscoelastic behavior, has often made it possible to better understand the structures and relationships responsible for the reinforcement of polymers by HGCP particles. Rheology therefore places itself here on the border between structural determination and physical properties of composites; moreover, this technique is often the key in understanding and improving the implementation. The elastic modulus G’ (solid symbols) and the loss modulus G “(open symbols) of the DGEBA / HGCP / MDA composites were measured in the linear range at 100 °C and in a frequency range from 0.1–100 rad/s, the results are given in the These measurements were obtained by applying a constant deformation of 0.1%, which is in the linear viscoelastic region. The results show that all the 0%, 5% and 10% mixtures of HGCP dispersion behaved like a solid, with G ‘and G “almost independent of frequency and G’> G”. The value of G’ and G “decreased with increasing amount of HGCP in the epoxy resin DGEBA. At high frequency, we can observe the crossover frequency when G’ = G”. The latter increased with the increase in the quantity of HGCP, which reflects the elastic behavior of the samples. These effects were also observed on other polymers . These cIn all cases, an increase in the linear field was observed when the amount of HGCP increased. For these systems where the HGCP was well dispersed in the DGEBA, the curves did not show any particular characteristic compared to the linear field for a pure DGEBA.The stress-strain curves for the DGEBA samples with different quantities of HGCP  are illustrated in the The mechanical properties of the DGEBA material modified by the addition of HGCP exhibited better ductility compared to pure DGEBA-MDA and also he observed that the breaking strain of the DGEBA-HGCCP-MDA material was significantly higher than that of the DGEBA resin pure. The presence of HGCP in the DGEBA resin results in the appearance of a plastic deformation zone from 74.33% deformation. However, the tensile strength decreased after the introduction of 10% HGCP into the DGEBA resin and the modulus of elasticity by a factor of 2 which can be attributed to the increase in knots in the matrix. In other cases, the result indicates that the introduction of HGCP into the DGEBA resin makes the samples more flexible. This increased flexibility of the samples can be attributed to the free volume presented by the HGCP / MDA / DGEBA network, promote molecular movement and improve the tensile strength .Various composite materials based on the epoxy resin DGEBA, MDA as hardener and the epoxy resin HGCP as an additive were prepared and formulated. The presence of a small amount (5% and 10% by weight) of HGCP showed high improvement in the mechanical and electrical properties of the DGEBA based epoxy materials. The chemical modification of cyclophosphazene facilitated better dispersion by optimizing the compatibility between the organic matrix and the cyclophosphazene. The presence of HGCP in the DGEBA resin showed a significant improvement in flexural modulus and hardness, which could be attributed to the flexibility of HGCP within a matrix stretched via the free volume present in the DGEBA network and also appearance of a plastic deformation zone from 74.33% deformation. However, the tensile strength decreased after the introduction of 10% HGCP into the DGEBA resin and the modulus of elasticity by a factor of 2. In addition, for 5% and 10% of the quantity of HGCP in the DGEBA, the capacity of the samples increases. This could be due to the increase in nods in the samples.


RNASEH2A is the catalytic subunit of the ribonuclease (RNase) H2 ternary complex that plays an important role in maintaining DNA stability in cells. Recent studies have shown that the RNASEH2A subunit alone is highly expressed in certain cancer cell types. Via a series of bioinformatics approaches, we found that RNASEH2A is highly expressed in human proliferative tissues and many cancers. Our analyses reveal a possible involvement of RNASEH2A in cell cycle regulation in addition to its well established role in DNA replication and DNA repair. Our findings underscore that RNASEH2A could serve as a biomarker for cancer diagnosis and a therapeutic target.RNASEH2A being the catalytic subunit. RNASEH2A expression levels have been shown to be upregulated in transformed and cancer cells. In this study, we used a bioinformatics approach to identify RNASEH2A co-expressed genes in different human tissues to underscore biological processes associated with RNASEH2A expression. Our analysis shows functional networks for RNASEH2A involvement such as DNA replication and DNA damage response and a novel putative functional network of cell cycle regulation. Further bioinformatics investigation showed increased gene expression in different types of actively cycling cells and tissues, particularly in several cancers, supporting a biological role for RNASEH2A but not for the other two subunits of RNase H2 in cell proliferation. Mass spectrometry analysis of RNASEH2A-bound proteins identified players functioning in cell cycle regulation. Additional bioinformatic analysis showed that RNASEH2A correlates with cancer progression and cell cycle related genes in Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) Pan Cancer datasets and supported our mass spectrometry findings.Ribonuclease (RNase) H2 is a key enzyme for the removal of RNA found in DNA-RNA hybrids, playing a fundamental role in biological processes such as DNA replication, telomere maintenance, and DNA damage repair. RNase H2 is a trimer composed of three subunits,  The development of high-throughput tools to monitor gene expression levels in a specific cell type and tissue has allowed the characterization of gene expression patterns throughout the human body. Such an approach was utilized in several studies to identify molecular signatures of tissues and cells ,2,3. GenRNASEH2A, together with RNASEH2B and RNASEH2C, composes the holoenzyme ribonuclease (RNase) H2 [RNASEH2A, which serves as the catalytic subunit of the RNase H2 complex [RNASEH2A comes from examining its expression levels in cancer compared to normal tissues. A previous study showed that, in human mesenchymal stem cells transformed by the over-expression of several oncogenes, RNASEH2A was among the genes with the highest and the earliest up fold change in their expression [RNASEH2A in cancer compared to normal tissues and cells were also reported in cervical cancer [RNASEH2A orthologous gene in yeast Saccharomyces cerevisiae (RNH201), peaking at S and G2 phases of the cell cycle, supporting the hypothesis that RNASEH2A plays a role in cancer progression. This is in contrast with the yeast RNASEH1 ortholog, RNH1, activity and level of which are not altered throughout the cell cycle [RNASEH2A in cancer, we analyzed the prevalence of patients with copy number alterations in the RNASEH2A gene in The Cancer Genome Atlas (TCGA) Pan Cancer studies in different cancer tissues, hypothesizing that identifying patterns of its expression can shed new insights on its function.Nase) H2 ,5. RNASE complex . The fun complex , DNA rep complex , and rep complex . RNA-DNA complex ,11,12,13 complex . An exam complex ,16. Gene complex . However complex , highligpression . In addil cancer , prostatl cancer , colorecl cancer , and tril cancer . In suppl cancer  highlighll cycle . With thhttps://gtexportal.org/home/datasets; accessed date 21 September 2019) which were obtained through RNA-Seq for 53 different tissues. The dataset contained total 56,202 transcripts, out of which 42,548 transcripts had greater than zero transcripts per million (TPM) values in at least one tissue of the 53 different tissues. We calculated Pearson correlation coefficients for every transcript with RNASEH transcripts data using R [We obtained expression data from GTEx portal v7   and 20 co-expressed proteins for each from the STRING database, we ended up with a list of 200 proteins. We removed any duplicate proteins listed and counted how many proteins were in the top five percentile of the Pearson’s correlation coefficient distribution from the co-expression correlation analysis that was to be validated.To validate the correlation co-expression coefficients of different genes with work v11 . We lookRNASEH genes in different type of cancers compared to normal tissues and in cancers at different stage of progression.Data were obtained from Cancer RNA-Seq Nexus (CRN) database providing phenotype-specific coding-transcript/lncRNA expression profiles, and mRNA-lncRNA co-expression networks in cancer cells  were useRNASEH2A, we used The Cancer Genome Atlas (TCGA) Pan Cancer studies [To analyze copy number alterations (CNAs) in  studies  involvin studies ,30. Afte2 incubator at 37 °C and were passaged once a week to maintain cell growth. Whole cell lysate from HEK293 was extracted with NP-40 lysis buffer  together with a cocktail of protease inhibitor  followed by centrifugation at 14,000 rpm for 15 min at +4 °C to pellet the debris.Human embryonic kidney, HEK293 were grown in Dulbecco’s Modified Eagle Medium (DMEM) medium  containing 10% fetal bovine serum (FBS)  and 1% penicillin-streptomycin  in 5% CORNASEH2A in HEK293 cell line, we used the pEGFP-RNASEH2A plasmid . As control, we cloned the eGFP plasmid by restricting the pEGFP-RNASEH2A using HincII  that cut upstream and downstream of the RNASEH2A gene but left the EGFP intact. Then, the plasmid was ligated by T4 ligase  and transformed into Agilent XL1-Blue Electroporation-Competent Cells  by electroporation using Gene Pulser Xcell electroporation system . Isolated colonies were grown in Luria-Bertani (LB) medium overnight; plasmids were extracted using the miniprep kit  and sent to DNA sequencing to verify the plasmid sequence.To over-express 6 per 10 cm plate. The following day, 5 µg of the plasmids were incubated with 25 µL of the Lipofectamine transfection reagent  for 10–15 min at room temperature in Opti-MEM media , and the plasmid-reagent mix was distributed to the cells. The next day, transfected cells were fed with fresh media and harvested 48 h after transfection.For transient transfection, HEK293 cells were plated at 1–3 × 10eGFP or the pEGFP-RNASEH2A plasmid was incubated with 30 µL of GFP-Trap Magnetic Agarose beads  at 4 °C with end to end rotation for 1 h. Then, the magnetic beads–GFP protein complex were separated from the rest of the protein extract using a magnetic stand. The beads were washed twice with wash buffer I  and an additional 2 washes with wash buffer II . Then, the beads were resuspended in SDS loading buffer and boiled at 95 °C for 10 min. Finally, the beads were centrifuged at 13,000 rpm for 5 min at room temperature, and the eluted proteins were collected and used for either Western blot or mass spectrometry analysis.Whole cell lysate (1 mg) from HEK293 cells transfected with either the anti-RNASEH2A  and anti-GFP (B2) . After washing 3 × 10 min with TBS-T, the membrane was incubated with a mouse secondary antibody conjugated with horseradish peroxidase  at room temperature for 1 h. Following washing 3 × 10 min with TBS-T, protein signals were visualized using the electrochemiluminescence (ECL) method according to the manufacture’s recommendations  and exposed on autoradiograph films .Total protein concentrations from HEK293 lysate were quantified , and 15 µg of the protein extracts was separated on a 10% SDS-polyacrylamide electrophoresis gel, transferred to 0.45 µm nitrocellulose membrane . The blotted membrane was then blocked in 5% non-fat dry milk tris-buffered saline and Tween 20 (TBS-T) (10 mM Tris-Cl (pH 7.5), 100 mM NaCl, 0.1% Tween-20) at room temperature for 1 h and incubated overnight with specific primary antibodies: Uniprot.com, 37,941 entries, downloaded 22 May 2019), the human reference database , the GFP-RNASEH2A protein, and a contaminant database  via Proteome Discoverer 2.1. A 1% false discovery rate (FDR) (“High Confidence”) was used for both the peptides and the proteins. At least 3 peptide sequences for the RNASEH2A protein were considered for the analysis.The gel lanes were excised from the gel, and the proteins were reduced, alkylated, and digested with trypsin as previously described . The pepHomo sapiens, added a count of 1 and log2 transformed the spectral counts. We then used these transformed data to get differentially present protein candidates in immunoprecipitation product of HEK293 with GFP-RNASEH2A vs. HEK293 with GFP using linear fitting and empirical Bayes method from limma, a bioconductor package [p value < 0.05.Spectral count data were analyzed for mass spectrometry runs on two sets of immuno-precipitation experiments described previously in the methods. Counts in each run were normalized based on mean of total spectral counts in all runs in a respective set of the experiment. We then filtered hits specific to  package ,34,35. TRNASEH2A and 39 other genes, including common cancer proliferative markers with few genes involved in each cell cycle phase as well as genes upregulated and downregulated in cancer as controls. We also included the genes for the proteins we identified as putative binding partners from mass spectrometry analysis. We used Broad Institute Cancer Cell Line Encyclopedia (CCLE) dataset containing RNA-Seq expression levels in Reads Per Kilobase of transcript, per Million mapped reads (RPKM) in 1019 cancer cell lines from 26 different tissues of origin [To validate the results of our study, we further made use of 2 large cancer related datasets to investigate the correlations between f origin . The secf origin . We addef origin . We alsoRNASEH2A function based on co-expressed genes, we used an approach (To study approach  that utiapproach .p = 2.77 × 10−5) and DNA replication (p = 2.66 × 10−4), compatible with the known functions of RNASEH2A. In addition, we found enrichment of genes involved in mitotic cell-cycle process (p = 2.80 × 10−14), regulation of microtubule cytoskeleton organization (p = 1.39 × 10−7), and chromosome segregation (p = 9.00 × 10−4) (p = 9.59 × 10−6) but also microtubule binding (p = 4.25 × 10−5) and kinase binding (p = 3.28 × 10−4) (−8), spindle pole (p = 1.13 × 10−6), as well as nucleoplasm (p = 3.52 × 10−6), midbody (p = 5.01 × 10−6), microtubule organizing center (p = 5.13 × 10−6), and condensed chromosomes outer kinetochore (p = 8.72 × 10−6)  with highest correlation coefficient, using the GOrilla analysis tool , we obse × 10−4) . For mol × 10−4) . For cel × 10−6) . Based oRNASEH2A at the protein expression level using the STRING protein–protein association network, we looked at 10 transcripts having high correlation with RNASEH2A (including RNASEH2A) that also have at least 20 known and characterized co-expressed proteins in the STRING network . After filtering top 5% correlated genes, we could identify processes such as regulation of telomerase RNA and telomeres maintenance , as expected for this RNase H2 subunit. For RNASEH2C, we could not find any functional network either in the 2% or the 5% analyses, as there were no GO terms with an enrichment p-value below the specified value p < 0.001 (data not shown).Analysis of the top 2% co-expressed correlated genes with ntenance , which wRNASEH genes using the GTEx v.7 database, which allowed us to compare the expression levels of RNASEH1, RNASEH2A, RNASEH2B, and RNASEH2C genes in 53 different human tissues, including two cell lines  [MYBL2 and SCARA5 genes, which are reported to be upregulated and downregulated in cancer, respectively [MYBL2 was mostly upregulated in cancers such as triple negative breast cancer, bladder urothelial carcinoma stages 2 and 4, and lung adenocarcinoma stages 2 and 4, while SCARA5 was downregulated in cancers such as bladder urothelial carcinoma stages 2 and 4; thyroid carcinoma stage 1, and rectum adenocarcinoma stage 2A   in 29 diectively . As expestage 2A . RNASEH2 tissues .RNASEH genes throughout the progression of cancer, we examined the expression levels of the genes at different stages of lung squamous adenocarcinoma, breast invasive carcinoma, and bladder urothelial carcinoma. In all these cancers, the expression of RNASEH2A changed drastically from a normal stage to early cancer stages and remained high with the progression of the disease  interacting with RNASEH2A  and DnaJ homologue subfamily A member 1 (DNAJA1) and apoptosis-inducing factor mitochondrion-associated 1 (AIFM1) proteins with a gene correlation coefficient with RNASEH2A of 0.84, 0.585, and 0.378 respectively. These three potential RNASH2A binding partners have shown to play a role in cell cycle regulation and mitosis [To identify proteins that interact with RNASEH2A a. We als protein b. After RNASEH2A . RNASEH2 mitosis ,43,44,45RNASEH2A in cancer, we examined the correlations between RNASEH2A and 39 additional genes in CCLE and TGCA Pan Cancer datasets , Gap 2 (G2), G2/Mitosis (M), M, and M/G1 phases and a comparatively lower or no correlation with genes unique to G1 and S phases  inhibitors compared to knocking out RNASEH2B [RNASEH2A levels constitute an additional layer of regulation of RNASEH2A activity, which is not dictated by RNASEH2B or RNASEH2C levels, and this is why the co-expression correlation analysis did not show a functional network of mitotic cell regulation for RNASEH2B and RNASEH2C genes. Following this notion, it would be intriguing to examine the levels of the three RNase H2 genes throughout the cell cycle in human cells and study how perturbing their levels affects RNase H2 function and/or cell cycle progression. Similar studies have been performed on yeast showing a cell cycle dependent expression of RNH201 (orthologous gene of RNASEH2A), peaking at S and G2 phases [Among the three RNASEH2C . This noRNASEH2B . We prop2 phases ,49, and RNASEH2A level in actively cycling tissues as well as different cancer tissues compared to normal ones.Rejins et al. , using aRNASEH2A is involved: DNA replication, DNA damage repair, and regulation of chromosome segregation in mitosis. Hyper-proliferation of cells results in excessive addition of RNA Okazaki fragments into the genome during DNA replication, increases the number of events when replicative DNA polymerases insert ribonucleotides instead of deoxyribonucleotides into the genomic DNA [RNASEH2A and link RNASEH2A activity to cell cycle regulation. In support of this notion, we identified several cellular pathways among the highest RNASEH2A co-expressed genes such as the minichromosome maintenance (MCM) complex, a replicative eukaryotic helicase that, in yeast, has been demonstrated to be activated upon accumulation of RNA-DNA hybrids at the G2-M checkpoint. This finding suggests that targeting the RNASEH2A level and/or activity could prevent the DNA damage occurring during replication, which leads to mitotic catastrophe and cell death [NDC80 complex, the CENP family, and the KIF family, have all been shown to function in cell cycle regulation through regulation of the microtubule filaments with the kinetochore [Using our co-expression correlation approach, we identified three main functional networks in which omic DNA ,52, and ll death ,54. The etochore ,56,57.RNASEH genes  using the GTEx v.7 database and revealed a marked contrast in RNASEH2A expression levels between low and high proliferative tissues. RNASEH2A showed low expression levels in low proliferative tissues such as brain, kidney (cortex), and heart and high expression levels in highly proliferative tissues such as skin, esophagus, small intestine, testis, cervix, as well as in transformed fibroblasts and transformed lymphocytes. Moreover, in line with the initial study by Flanagan et al. [RNASEH2A in transformed mesenchymal stem cells, our analysis of 29 different randomly selected tumors in different tissues compared to their healthy controls revealed that RNASEH2A is the only RNASEH gene displaying increased expression levels. Further analyses of RNASEH expression levels throughout different stages of lung squamous adenocarcinoma, breast invasive carcinoma, and bladder urothelial carcinoma again highlighted specific upregulation of RNASEH2A from normal tissues to early stages and more advanced stages of these cancers. It is interesting to note that Dai et al. reported on a possible role of RNASEH2A in glioma cell proliferation. Their findings suggested a role of RNASEH2A upregulation in cell growth and apoptosis, contributing to glioma-genesis and cancer progression [RNASEH2A overexpression was associated with cancer cell resistance to chemotherapy in vitro and with aggressiveness and poor outcomes in breast cancers of estrogen receptor (ER)-positive subtypes [RNASEH2A gene that we performed in our study further supports the role of RNASEH2A in cancer development. We demonstrated that RNASEH2A gene amplifications have higher incidence in multiple cancer types when compared to deep deletions and normal diploid samples suggesting RNASEH2A as a target for cancer diagnosis and therapy. Interestingly, cancers from tissues with high cell turnover, such as the reproductive system, showed a maximum percentage of patients with RNASEH2A amplification, suggesting a possible role of adult stem cells in the overexpression of RNASEH2A. In fact, deregulation of the adult stem cell niche is considered a key event in the etiology of cancer [Our analyses of expression levels focused on the n et al. , showinggression . More resubtypes . The anaf cancer .RNASEH2A. The top two proteins observed were RNASEH2B and RNASEH2C, confirming the authenticity of the results. Additional proteins that we identified in the context of RNASEH2A biology and mitosis regulation were CCT8, DNAJA1, and AIFM1. CCT8 is part of the CCT/TRiC complex that was reported to regulate telomerase function by mediating its trafficking from the cytoplasm to the telomeres [RNASEH2A functional network in mitosis regulation. DNAJA1 belongs to the family of DnaJ heat shock protein family (Hsp40); it has been shown to be activated by E2F transcription factor 1 (E2F1) and to promote cell cycle by stabilizing cell division protein 45 (CDC45) [RNASEH2A by E2F1 has been also reported in human papillomavirus cervical cancers [Our mass spectrometry analysis shows five protein candidates that specifically interact with elomeres . In addielomeres , support (CDC45) . Of inte cancers . AIFM1 i cancers .RNASEH2A expression with genes that play a role in cancer development and cell cycle progression validate our study, showing that RNASEH2A plays a role in cancer transformation and progression. In addition, our final analysis supports the mass spectrometry findings and indicates CCT8, DNAJA1, and AIFM1 as binding partners of RNASEH2A.The high correlation of RNASEH2A subunit and the candidate proteins found in our experiments. In our in vitro experiments, we used the HEK293 cell line. Depending on a number of factors, this cell line in culture can either show the phenotype of a normal or of a cancer cell line [RNASEH2A requires a more appropriate model system.The analysis and the experimental approaches used in this works have some limitations. In this respect, we made use of the Cancer RNA-Seq Nexus database for which patient information is not available. Factors such as age, gender, and medical treatment versus surgery could have influenced the results of our in-silico analysis. In addition, although mass spectrometry is a highly specific and sensitive technique, further investigation is needed to confirm the specific interactions between the ell line . AlthougRNASEH2A in cell cycle regulation that appears independent from its function as part of the RNase H2 whole enzyme. The presented findings stimulate new research directions such as investigating the differential expression of RNASEH2A during cell cycle phases to better understanding and characterizing the function of RNASEH2A in cell proliferation in healthy, adults stem cells and cancer cells and to support further exploring RNASEH2A as a target for cancer diagnosis and therapy.Overall, our study suggests an emerging role of