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Psoriatic arthritis (PsA) is a chronic, progressive, inflammatory arthropathy associated with psoriasis as well as a complex pathogenesis. Genetic and environmental factors trigger the development of the immune-mediated auto-inflammatory response in different sites: skin, bone marrow, entheses and synovial tissues. Studies of the last two decades have changed the view of PsA from a mild, non-progressive arthritis to an inflammatory systemic disease with serious health consequences, not only associated with joint dysfunction, but also with an increased risk of cardiovascular disease and socioeconomic consequences with significantly reduced quality of life. The joint damage starts early in the course of the disease, thus early recognition and treatment with modern biological treatments, which may modify the natural history and slow down progression of this debilitating disease, is essential for the patient long-term outcome.

Psoriasis is a systemic disease that is strictly connected with metabolic disorders (insulin resistance, atherogenic dyslipidemia, arterial hypertension, and cardiovascular diseases). It occurs more often in patients with a more severe course of the disease. Obesity is specially an independent risk factor and it is associated with a worse treatment outcome because of the high inflammatory activity of visceral fatty tissue and the production of inflammatory mediators involved in the development of both psoriasis and metabolic disorders. However, in psoriasis the activation of the Th17/IL-17 and the abnormalities in the Th17/Treg balance axis are observed, but this pathomechanism does not fully explain the frequent occurrence of metabolic disorders. Therefore, there is a need to look for better biomarkers in the diagnosis, prognosis and monitoring of concomitant disorders and therapeutic effects in psoriasis. In addition, the education on the use of a proper diet as a prophylaxis for the development of the above disorders is an important element of holistic care for a patient with psoriasis. Diet may affect gene expression due to epigenetic modification which encompasses interactions of environment, nutrition and diseases. Patients with psoriasis should be advised to adopt proper diet and dietician support.

Endoplasmic reticulum (ER) stress conditions promote a cellular adaptive mechanism called the unfolded protein response (UPR) that utilizes three stress sensors, inositol-requiring protein 1, protein kinase RNA-like ER kinase, and activating transcription factor 6. These sensors activate a number of pathways to reduce the stress and facilitate cell survival. While much is known about the mechanisms involved that modulate apoptosis during chronic stress, less is known about the transition between the prosurvival and proapoptotic factors that determine cell fate. Here, we employed a genetic screen that utilized three different pharmacological stressors to induce ER stress in a human-immortalized airway epithelial cell line, immortalized human bronchial epithelial cells. We followed the stress responses over an 18-h time course and utilized real-time monitoring of cell survival, next-generation sequencing, and quantitative real-time PCR to identify and validate genes that were upregulated with all three commonly employed ER stressors, inhibitor of calpain 1, tunicamycin, and thapsigargin. growth arrest and DNA damage-inducible alpha (GADD45A), a proapoptotic factor, and regulator of calcineurin 1 (RCAN1) mRNAs were identified and verified by showing that small interfering RNA (siRNA) knockdown of GADD45A decreased CCAAT-enhancer-binding protein homologous protein (a.k.a DDIT3), BCL2-binding component 3 (a.k.a. BBC3), and phorbol-12-myristate-13-acetate-induced protein 1 expression, 3 proapoptotic factors, and increased cell viability during ER stress conditions, whereas siRNA knockdown of RCAN1 dramatically decreased cell viability. These results suggest that the relative levels of these two genes regulate cell fate decisions during ER stress independent of the type of ER stressor.

Psoriasis is a systemic disease that is strictly connected with metabolic disorders (insulin resistance, atherogenic dyslipidemia, arterial hypertension, and cardiovascular diseases). It occurs more often in patients with a more severe course of the disease. Obesity is specially an independent risk factor and it is associated with a worse treatment outcome because of the high inflammatory activity of visceral fatty tissue and the production of inflammatory mediators involved in the development of both psoriasis and metabolic disorders. However, in psoriasis the activation of the Th17/IL-17 and the abnormalities in the Th17/Treg balance axis are observed, but this pathomechanism does not fully explain the frequent occurrence of metabolic disorders. Therefore, there is a need to look for better biomarkers in the diagnosis, prognosis and monitoring of concomitant disorders and therapeutic effects in psoriasis. In addition, the education on the use of a proper diet as a prophylaxis for the development of the above disorders is an important element of holistic care for a patient with psoriasis. Diet may affect gene expression due to epigenetic modification which encompasses interactions of environment, nutrition and diseases. Patients with psoriasis should be advised to adopt proper diet and dietician support.

Psoriasis is a multifactorial disease in which genetic, environmental and epigenetic factors regulating gene expression play a key role. In the “genomic era”, genome-wide association studies together with target genotyping platforms performed in different ethnic populations have found more than 50 genetic susceptible markers associated with the risk of psoriasis which have been identified so far. Up till now, the strongest association with the risk of the disease has been proved for HLA-C*06 gene. The majority of other psoriasis risk SNPs are situated near the genes encoding molecules involved in adaptive and innate immunity, and skin barrier function. Many contemporary studies indicate that the epigenetic changes: histone modification, promoter methylations, long non-coding and micro-RNA hyperexpression are considered as factors contributing to psoriasis pathogenesis as they regulate abnormal keratinocyte differentiation and proliferation, aberrant keratinocytes – inflammatory cells communication, neoangiogenesis and chronic inflammation. The circulating miRNAs detected in the blood may become specific markers in the diagnosis, prognosis and response to the treatment of the disease. The inhibition of expression in selected miRNAs may be a new promising therapy option for patients with psoriasis.

The role of microRNAs in controlling angiogenesis is recognized as a promising therapeutic target in both cancer and cardiovascular disorders. However, understanding a miRNA’s pleiotropic effects on angiogenesis is a limiting factor for these types of therapeutic approaches. Using genome-wide next-generation sequencing, we examined the role of an antiangiogenic miRNA, miR-200b, in primary human endothelial cells. The results indicate that miR-200b has complex effects on hypoxia-induced angiogenesis in human endothelia and importantly, that many of the reported miR-200b effects using miRNA overexpression may not be representative of the physiological role of this miRNA. We also identified the antiangiogenic KLF2 gene as a novel target of miR-200b. Our studies indicate that the physiological changes in miR-200b levels during acute hypoxia may actually have a proangiogenic effect through Klf2 downregulation and subsequent stabilization of HIF-1 signaling. Moreover, we provide a viable approach for differentiating direct from indirect miRNA effects in order to untangle the complexity of individual miRNA networks.

Small noncoding microRNAs (miRNAs) post-transcriptionally regulate a large portion of the human transcriptome. miRNAs have been shown to play an important role in the unfolded protein response (UPR), a cellular adaptive mechanism that is important in alleviating endoplasmic reticulum (ER) stress and promoting cell recovery. Another class of small noncoding RNAs, the Piwi-interacting RNAs (piRNAs) together with PIWI proteins, was originally shown to play a role as repressors of germline transposable elements. More recent studies, however, indicate that P-element induced WImpy proteins (PIWI proteins) and piRNAs also regulate mRNA levels in somatic tissues. Using genome-wide small RNA next generation sequencing, cell viability assays, and caspase activity assays in human airway epithelial cells, we demonstrate that ER stress specifically up-regulates total piRNA expression profiles, and these changes correlate with UPR-induced apoptosis as shown by up-regulation of two pro-apoptotic factor mRNAs, CHOP and NOXA. Furthermore, siRNA knockdown of PIWIL2 and PIWIL4, two proteins involved in piRNA function, attenuates UPR-related cell death, inhibits piRNA expression, and inhibits the up-regulation of CHOP and NOXA mRNA expression. Hence, we provide evidence that PIWIL2 and PIWIL4 proteins, and potentially the up-regulated piRNAs, constitute a novel epigenetic mechanism that control cellular fate during the UPR.

Somatic mosaicism for DNA copy-number alterations (SMC-CNAs) is defined as gain or loss of chromosomal segments in somatic cells within a single organism. As cells harboring SMC-CNAs can undergo clonal expansion, it has been proposed that SMC-CNAs may contribute to the predisposition of these cells to genetic disease including cancer. Herein, the gross genomic alterations (>500 kbp) were characterized in uninvolved mammary glandular tissue from 59 breast cancer patients and matched samples of primary tumors and lymph node metastases. Array-based comparative genomic hybridization showed 10% (6/59) of patients harbored one to 359 large SMC-CNAs (mean: 1,328 kbp; median: 961 kbp) in a substantial portion of glandular tissue cells, distal from the primary tumor site. SMC-CNAs were partially recurrent in tumors, albeit with considerable contribution of stochastic SMC-CNAs indicating genomic destabilization. Targeted resequencing of 301 known predisposition and somatic driver loci revealed mutations and rare variants in genes related to maintenance of genomic integrity: BRCA1 (p.Gln1756Profs*74, p.Arg504Cys), BRCA2 (p.Asn3124Ile), NCOR1 (p.Pro1570Glnfs*45), PALB2 (p.Ser500Pro), and TP53 (p.Arg306*). Co-occurrence of gross SMC-CNAs along with point mutations or rare variants in genes responsible for safeguarding genomic integrity highlights the temporal and spatial neoplastic potential of uninvolved glandular tissue in breast cancer patients.

The nitric oxide (NO) secreted by vascular endothelium is required for the maintenance of cardiovascular homeostasis. Diminished release of NO generated by endothelial NO synthase contributes to endothelial dysfunction. Hypoxia and ischemia reduce endothelial eNOS expression via posttranscriptional mechanisms that result in NOS3 transcript destabilization. Here, we examine whether microRNAs contribute to this mechanism. We followed the kinetics of hypoxia-induced changes in NOS3 mRNA and eNOS protein levels in primary human umbilical vein endothelial cells (HUVECs). Utilizing in silico predictive protocols to identify potential miRNAs that regulate eNOS expression, we identified miR-200b as a candidate. We established the functional miR-200b target sequence within the NOS3 3′UTR, and demonstrated that manipulation of the miRNA levels during hypoxia using miR-200b mimics and antagomirs regulates eNOS levels, and established that miR-200b physiologically limits eNOS expression during hypoxia. Furthermore, we demonstrated that the specific ablation of the hypoxic induction of miR-200b in HUVECs restored eNOS-driven hypoxic NO release to the normoxic levels. To determine whether miR-200b might be the only miRNA that had this effect, we utilized Next Generation Sequencing (NGS) to follow hypoxia-induced changes in the miRNA levels in HUVECS and found 83 novel hypoxamiRs, with two candidate miRNAs besides miR-200b that could potentially influence eNOS levels. Taken together, the data establish miR-200b-eNOS regulation as a first hypoxamiR-based mechanism that limits NO bioavailability during hypoxia in endothelial cells, and show that hypoxamiRs could become useful therapeutic targets for cardiovascular diseases and other hypoxic-related diseases including various types of cancer. Electronic supplementary material The online version of this article (10.1007/s10456-018-9620-y) contains supplementary material, which is available to authorized users.

Understanding the cellular pathways that regulate endothelial nitric oxide (eNOS, NOS3) expression and consequently nitric oxide (NO) bioavailability during hypoxia is a necessary aspect in the development of novel treatments for cardiovascular disorders. eNOS expression and eNOS-dependent NO cellular signaling during hypoxia promote an equilibrium of transcriptional and posttranscriptional molecular mechanisms that belong to both proapoptotic and survival pathways. Furthermore, NO bioavailability results not only from eNOS levels, but also relies on the presence of eNOS substrate and cofactors, the phosphorylation status of eNOS, and the presence of reactive oxygen species (ROS) that can inactivate eNOS. Since both NOS3 levels and these signaling pathways can also be a subject of posttranscriptional modulation by microRNAs (miRNAs), this class of short noncoding RNAs contribute another level of regulation for NO bioavailability. As miRNA antagomirs or specific target protectors could be used in therapeutic approaches to regulate NO levels, either by changing NOS3 mRNA stability or through factors governing eNOS activity, it is critical to understand their role in governing eNOS activity during hypoxa. In contrast to a large number of miRNAs reported to the change eNOS expression during hypoxia, only a few miRNAs modulate eNOS activity. Furthermore, impaired miRNA biogenesis leads to NOS3 mRNA stabilization under hypoxia. Here we discuss the recent studies that define miRNAs’ role in maintaining endothelial NO bioavailability emphasizing those miRNAs that directly modulate NOS3 expression or eNOS activity.

Hypoxia-inducible factors (HIF) are heterodimeric transcription factors that allow cells to adapt and survive during hypoxia. Regulation of HIF1A and HIF2A mRNA is well characterized, whereas HIF3A mRNA regulation and function are less clear. Using RNA-Seq analysis of primary human umbilical vein endothelial cells, we found two isoforms of HIF3A were expressed, HIF3A2 and HIF3A3. Comparing HIF3A expression profiles to HIF1A mRNA during 48 hours of hypoxia revealed that HIF1A message peaked at 4 hours, whereas HIF3A expression increased while HIF1A was decreasing. Given that HIF1A mRNA is regulated by miR-429, we tested miR-429 effects on both HIF3A isoforms and found that they too were regulated by miR-429. Analysis of a HIF-3 target, DNA-damage-inducible transcript 4, a key survival gene, indicated that DDIT4 mRNA is induced by HIF-3 and negatively regulated by miR-429 through miR-429’s actions on HIF3A message. This provides a compelling model for how hypoxia-induced miR-429 regulates the switch between HIF-1 adaptive responses to HIF-3 survival responses by rapidly decreasing HIF1A levels while simultaneously slowing the progression of HIF3A expression until the miR-429 levels drop below normoxic levels. Since HIF-1 drives HIF3A and miR-429 expression, this establishes a regulatory network in which miR-429 plays a pivotal role.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify individuals at risk of prolonged paralysis following the administration of neuromuscular blocking agents, like succinylcholine, pesticides and nerve agents. In this study, the activity of BChE and its sensitivity to inhibition by dibucaine and fluoride was evaluated in 1200 Polish healthy individuals. In addition, molecular analysis of all exons, exon-intron boundaries and the 3'UTR sequence of the BCHE gene was performed in a group of 72 subjects with abnormal BChE activity (2000 U/L and5745 U/L) or with DN (Dibucaine Number) or FN (Fluoride-Number) values outside the reference range (DN 78 and FN lower than wild type). In a studied group, BChE activity range was similar to those observed in other populations. BChE activity screening allowed to detect UA and UF phenotypes in 26 (2.2%) and 15 (1.2%) individuals, respectively. Observed UA or UF phenotypes were confirmed by direct sequencing and heterozygous c.293A G or c.1253G T substitutions were identified in all cases. Nine out of 18 (50%) individuals with BChE activity below 2000 U/L had a mutation in 5'UTR (32G/A), intron 2 (c.1518-121T/C) or exon 4 (c.1699G/A; the K variant mutation). Majority of the individuals with BChE activity ≥6000 U/L were wild type. To summarize, the range of BChE activity in a Polish population is similar to those observed in other countries. We conclude that the BChE phenotyping assay is a reliable method for identification of individuals with the UA and UF genotypes.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman’s method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.

Synonymous or silent mutations are often overlooked in genetic analyses for disease-causing mutations unless they are directly associated with potential splicing defects. More recent studies, however, indicate that some synonymous single polynucleotide polymorphisms (sSNPs) are associated with changes in protein expression, and in some cases, protein folding and function. The impact of codon usage and mRNA structural changes on protein translation rates and how they can affect protein structure and function is just beginning to be appreciated. Examples are given here that demonstrate how synonymous mutations alter the translational kinetics and protein folding and/or function. The mechanism for how this occurs is based on a model in which codon usage modulates the translational rate by introducing pauses caused by nonoptimal or rare codons or by introducing changes in the mRNA structure, and this in turn influences co-translational folding. Two examples of this include the multidrug resistance protein (p-glycoprotein) and the cystic fibrosis transmembrane conductance regulator gene (CFTR). CFTR is also used here as a model to illustrate how synonymous mutations can be examined using in silico predictive methods to identify which sSNPs have the potential to change protein structure. The methodology described here can be used to help identify “non-silent” synonymous mutations in other genes. Electronic supplementary material The online version of this article (doi:10.1186/s11658-016-0025-x) contains supplementary material, which is available to authorized users.