Functional analysis of germline ETV6 W380R mutation causing inherited thrombocytopenia and secondary acute lymphoblastic leukemia or essential thrombocythemia.

Germline mutations in ETV6 gene cause inherited thrombocytopenia with leukemia predisposition. Here, we report on functional validation of ETV6 W380R mutation segregating with thrombocytopenia in a family where two family members also suffered from acute lymphoblastic leukemia (ALL) or essential thrombocythemia (ET). In-silico analysis predicted impaired DNA binding due to W380R mutation. Functional analysis showed that this mutation prevents the ETV6 protein from localizing into the cell nucleus and impairs the transcriptional repression activity of ETV6. Based on the germline ETV6 mutation, ET probably started with somatic JAK2 V617F mutation, whereas ALL could be caused by diverse mechanisms: high-hyperdiploidity; somatic deletion of exon 1 IKZF1 gene; or somatic mutations of other genes found by exome sequencing of the ALL sample taken at the diagnosis.

RGDS-Modified Superporous Poly(2-Hydroxyethyl Methacrylate)-Based Scaffolds as 3D In Vitro Leukemia Model.

Superporous poly(2-hydroxyethyl methacrylate-co-2-aminoethyl methacrylate) (P(HEMA-AEMA)) hydrogel scaffolds are designed for in vitro 3D culturing of leukemic B cells. Hydrogel porosity, which influences cell functions and growth, is introduced by adding ammonium oxalate needle-like crystals in the polymerization mixture. To improve cell vitality, cell-adhesive Arg-Gly-Asp-Ser (RGDS) peptide is immobilized on the N-(γ-maleimidobutyryloxy)succinimide-activated P(HEMA-AEMA) hydrogels via reaction of SH with maleimide groups. This modification is especially suitable for the survival of primary chronic lymphocytic leukemia cells (B-CLLs) in 3D cell culture. No other tested stimuli (interleukin-4, CD40 ligand, or shaking) can further improve B-CLL survival or metabolic activity. Both unmodified and RGDS-modified P(HEMA-AEMA) scaffolds serve as a long-term (70 days) 3D culture platforms for HS-5 and M2-10B4 bone marrow stromal cell lines and MEC-1 and HG-3 B-CLL cell lines, although the adherent cells retain their physiological morphologies, preferably on RGDS-modified hydrogels. Moreover, the porosity of hydrogels allows direct cell lysis, followed by efficient DNA isolation from the 3D-cultured cells. P(HEMA-AEMA)-RGDS thus serves as a suitable 3D in vitro leukemia model that enables molecular and metabolic assays and allows imaging of cell morphology, interactions, and migration by confocal microscopy. Such applications can prospectively assist in testing of drugs to treat this frequently recurring or refractory cancer.

Activation-induced deaminase and its splice variants associate with trisomy 12 in chronic lymphocytic leukemia.

Activation-induced cytidine deaminase (AID) is a mutator enzyme essential for somatic hypermutation (SHM) and class switch recombination (CSR) during effective adaptive immune responses. Its aberrant expression and activity have been detected in lymphomas, leukemias, and solid tumors. In chronic lymphocytic leukemia (CLL) increased expression of alternatively spliced AID variants has been documented. We used real-time RT-PCR to quantify the expression of AID and its alternatively spliced transcripts (AIDΔE4a, AIDΔE4, AIDivs3, and AIDΔE3E4) in 149 CLL patients and correlated this expression to prognostic markers including recurrent chromosomal aberrations, the presence of complex karyotype, mutation status of the immunoglobulin heavy chain variable gene, and recurrent mutations. We report a previously unappreciated association between higher AID transcript levels and trisomy of chromosome 12. Functional analysis of AID splice variants revealed loss of their activity with respect to SHM, CSR, and induction of double-strand DNA breaks. In silico modeling provided insight into the molecular interactions and structural dynamics of wild-type AID and a shortened AID variant closely resembling AIDΔE4, confirming its loss-of-function phenotype.

MicroRNA and mesial temporal lobe epilepsy with hippocampal sclerosis: Whole miRNome profiling of human hippocampus.

OBJECTIVE: Mesial temporal lobe epilepsy (mTLE) is a severe neurological disorder characterized by recurrent seizures. mTLE is frequently accompanied by neurodegeneration in the hippocampus resulting in hippocampal sclerosis (HS), the most common morphological correlate of drug resistance in mTLE patients. Incomplete knowledge of pathological changes in mTLE+HS complicates its therapy. The pathological mechanism underlying mTLE+HS may involve abnormal gene expression regulation, including posttranscriptional networks involving microRNAs (miRNAs). miRNA expression deregulation has been reported in various disorders, including epilepsy. However, the miRNA profile of mTLE+HS is not completely known and needs to be addressed. METHODS: Here, we have focused on hippocampal miRNA profiling in 33 mTLE+HS patients and nine postmortem controls to reveal abnormally expressed miRNAs. In this study, we significantly reduced technology-related bias (the most common source of false positivity in miRNA profiling data) by combining two different miRNA profiling methods, namely next generation sequencing and miRNA-specific quantitative real-time polymerase chain reaction. RESULTS: These methods combined have identified and validated 20 miRNAs with altered expression in the human epileptic hippocampus; 19 miRNAs were up-regulated and one down-regulated in mTLE+HS patients. Nine of these miRNAs have not been previously associated with epilepsy, and 19 aberrantly expressed miRNAs potentially regulate the targets and pathways linked with epilepsy (such as potassium channels, γ-aminobutyric acid, neurotrophin signaling, and axon guidance). SIGNIFICANCE: This study extends current knowledge of miRNA-mediated gene expression regulation in mTLE+HS by identifying miRNAs with altered expression in mTLE+HS, including nine novel abnormally expressed miRNAs and their putative targets. These observations further encourage the potential of microRNA-based biomarkers or therapies.

Non-coding RNAs in diseases with a focus on osteoarthritis.

Osteoarthritis (OA) is a frequent musculoskeletal disorder affecting millions of people worldwide. Despite advances in understanding the pathogenesis of OA, prognostic biomarkers or effective targeted treatment are not currently available. Research on epigenetic factors has yielded some new insights as new technologies for their detection continue to emerge. In this context, non-coding RNAs, including microRNAs, long non-coding RNAs, circular RNAs, piwi-interacting RNAs, and small nucleolar RNAs, regulate intracellular signaling pathways and biological processes that have a crucial role in the development of several diseases. In this review, we present current knowledge on the role of epigenetic factors with a focus on non-coding RNAs in the development, prediction and treatment of OA. This article is categorized under: RNA in Disease and Development > RNA in Disease.

Menopausal Transition: Prospective Study of Estrogen Status, Circulating MicroRNAs, and Biomarkers of Bone Metabolism.

Objective: Osteoporosis is associated with an impaired balance between bone resorption and formation, which in turn leads to bone loss and fractures. Many recent studies have underlined the regulatory role of microRNAs (miRNAs) in bone remodeling processes and their potential as biomarkers of osteoporosis. The purpose of this study was to prospectively examine the association of circulating miRNAs and bone biomarkers with estrogen status in women before and after oophorectomy, as well as in oophorectomized women on estrogen therapy. Methods: In this prospective study, we included 11 women before oophorectomy and hysterectomy and at 201 ± 24 days after the surgery. Another 11 women were evaluated 508 ± 127 days after oophorectomy and hysterectomy and after an additional 203 ± 71 days of estradiol treatment. Serum miRNAs were profiled by sequencing. Estrogen status and biomarkers of bone metabolism were quantified. Bone mineral density was assessed in the lumbar spine. Results: Our analysis revealed 17 miRNAs associated with estrogen levels. Of those miRNAs that were upregulated with estrogen deficiency and downregulated after estrogen therapy, miR-422a correlated with serum beta-carboxy-terminal type I collagen crosslinks (ß-CTX) and procollagen 1 N-terminal propeptide (P1NP); and miR-1278 correlated with serum ß-CTX, P1NP, osteocalcin, sclerostin, and Dickkopf-1(Dkk1). In contrast, we found an inverse association of miR-24-1-5p with estrogen status and a negative correlation with serum ß-CTX, P1NP, osteoprotegerin, and sclerostin levels. Conclusion: The reported miRNAs associated with estrogen status and bone metabolism could be potential biomarkers of bone pathophysiology and would facilitate studies on the prevention of postmenopausal osteoporosis. Our findings require validation in an extended cohort.

Dynamic miRNA changes during the process of epileptogenesis in an infantile and adult-onset model.

Temporal lobe epilepsy (TLE) is the most common epilepsy type. TLE onset in infancy aggravates features like severity, drug responsiveness, or development of comorbidities. These aggravations may arise from altered micro RNA (miRNA) expression specific to the early onset of the disease. Although the miRNA involvement in TLE is widely studied, the relationship between the onset-age and miRNA expression has not been addressed. Here, we investigated the miRNA profile of infantile and adult-onset TLE in rats combining sequencing and PCR. Since miRNA expression changes with the disease progression, we scrutinized miRNA dynamics across three stages: acute, latent, and chronic. We report that infantile-onset TLE leads to changes in the expression of fewer miRNAs across these stages. Interestingly, the miRNA profile in the acute stage of infantile-onset TLE overlaps in dysregulation of miR-132-5p, -205, and -211-3p with the chronic stage of the disease starting in adulthood. The analysis of putative targets linked the majority of dysregulated miRNAs with pathways involved in epilepsy. Our profiling uncovered miRNA expression characteristic for infantile and adulthood-onset epileptogenesis, suggesting the distinct biology underlying TLE in the onset age-dependent matter. Our results indicate the necessity of addressing the onset age as an important parameter in future epilepsy research.

S100A11 (calgizzarin) is released via NETosis in rheumatoid arthritis (RA) and stimulates IL-6 and TNF secretion by neutrophils.

S100A11 (calgizzarin), a member of S100 family, is associated with several autoimmune diseases, including rheumatoid arthritis (RA). Neutrophil extracellular traps (NETs) are implicated in the pathogenesis of RA and in the externalization of some S100 family members. Therefore, we aimed to determine the association between S100A11 and NETs in RA. For this purpose, the levels of S100A11 and NETosis markers were detected in the RA synovial fluid by immunoassays. The expression of S100A11 by neutrophils in the RA synovial tissue was assessed. Neutrophils isolated from peripheral blood were exposed to S100A11 or stimulated to release NETs. The levels of NETosis- and inflammation-associated proteins were analysed by immunoassays. NETs were visualized by immunofluorescence. We showed that S100A11 was expressed by the neutrophils in the RA synovial tissue. Moreover, S100A11 in the RA synovial fluid correlated with several NETosis markers. In vitro, S100A11 was abundantly released by neutrophils undergoing NETosis compared to untreated cells (p < 0.001). Extracellular S100A11 increased the secretion of IL-6 (p < 0.05) and TNF (p < 0.05) by neutrophils but did not induce NETosis. This study demonstrates, for the first time, that the release of S100A11 is dependent on NETosis and that extracellular S100A11 augments the inflammatory response by inducing pro-inflammatory cytokines in neutrophils.

Complexes of glutathione with heavy metal ions as a new biochemical marker of aquatic environment pollution.

Reduced glutathione (GSH) plays a number of key roles in many biochemical pathways. This peptide is highly reactive and forms conjugates with other molecules via its sulfhydryl moiety. The interactions of the common heavy metal pollutant Cd(II) with GSH were determined by using the Brdicka reaction to evaluate whether this technique would be suitable as a biomarker. After GSH interaction with Cd(II) ions, two characteristic changes in the measured voltammogram were observed: Cat2 signal height decreased, and a new signal called P1 was found. The observed signal probably relates to the formation of a GSH-heavy metal ion complex adsorbed on the surface of the working electrode. When the interaction of GSH with cisplatin was studied, the same characteristic changes in the voltammogram were observed, which confirmed our hypothesis. Moreover, changes in the height of P1 and Cat2 signals with increasing time of GSH interaction with Cd(II) ions and/or cisplatin were also investigated. Cat2 peak height decreased proportionally with increasing time of interaction. This decrease can be explained by shielding of free sulfhydryl moiety by heavy metal ions, so it cannot catalyze the evolution of hydrogen from the supporting electrolyte. In addition, we found that, with increasing time of the interaction, the P1 signal was enhanced and shifted to more positive potentials for both Cd(II) ions and cisplatin.

Chemokine and Cytokine Profiles in Patients with Hand Osteoarthritis.

BACKGROUND: The development of hand osteoarthritis (HOA) and its progression into the erosive subset are unclear, but inflammation is suspected to be the main source. To verify the involvement of inflammation in HOA pathogenesis, we evaluate serum inflammatory mediators and their association with HOA-related clinical features in patients. METHODS: 153 participants (50 non-erosive HOA patients, 54 erosive HOA patients, and 49 healthy control subjects) were included in this study. All patients underwent clinical examination, which included assessment of tender and swollen small hand joints, ultrasound (US) examination, and self-reported measures (e.g., AUSCAN or algofunctional indexes). Serum inflammatory mediators were quantified using human cytokine 27-plex immunoassay. We employed linear modelling, correlation analysis, and resampling statistics to evaluate the association of these mediators to HOA. RESULTS: We identified increased levels of nine inflammatory mediators (e.g., eotaxin, monocyte chemoattractant protein 1, interleukin-8, and tumour necrosis factor) in HOA patients compared to healthy controls. Increased mediators correlated with ultrasound findings as well as with clinically tender and swollen joint counts in patients with erosive HOA. However, none of the mediators distinguished between erosive and non-erosive HOA subtypes. CONCLUSION: Our findings support the hypothesis on the involvement of inflammation in HOA.

Epilepsy miRNA Profile Depends on the Age of Onset in Humans and Rats.

Temporal lobe epilepsy (TLE) is a severe neurological disorder accompanied by recurrent spontaneous seizures. Although the knowledge of TLE onset is still incomplete, TLE pathogenesis most likely involves the aberrant expression of microRNAs (miRNAs). miRNAs play an essential role in organism homeostasis and are widely studied in TLE as potential therapeutics and biomarkers. However, many discrepancies in discovered miRNAs occur among TLE studies due to model-specific miRNA expression, different onset ages of epilepsy among patients, or technology-related bias. We employed a massive parallel sequencing approach to analyze brain tissues from 16 adult mesial TLE (mTLE)/hippocampal sclerosis (HS) patients, 8 controls and 20 rats with TLE-like syndrome, and 20 controls using the same workflow and categorized these subjects based on the age of epilepsy onset. All categories were compared to discover overlapping miRNAs with an aberrant expression, which could be involved in TLE. Our cross-comparative analyses showed distinct miRNA profiles across the age of epilepsy onset and found that the miRNA profile in rats with adult-onset TLE shows the closest resemblance to the profile in mTLE/HS patients. Additionally, this analysis revealed overlapping miRNAs between patients and the rat model, which should participate in epileptogenesis and ictogenesis. Among the overlapping miRNAs stand out miR-142-5p and miR-142-3p, which regulate immunomodulatory agents with pro-convulsive effects and suppress neuronal growth. Our cross-comparison study enhanced the insight into the effect of the age of epilepsy onset on miRNA expression and deepened the knowledge of epileptogenesis. We employed the same methodological workflow in both patients and the rat model, thus improving the reliability and accuracy of our results.

Study of Interactions between Metallothionein and Cisplatin by using Differential Pulse Voltammetry Brdickás reaction and Quartz Crystal Microbalance.

Treatment strategies for tumour diseases are progressively focusing on personalization of medicine. However, this focus requires methods revealing the early general biological mechanisms, including the formation anti-cancer drugs' resistance. The low molecular mass protein metallothionein is thought to be the crucial for the formation of resistance in tumour treatment based on the platinum-cytostatics. The interactions between metallothionein (MT) and cisplatin were determined by the adsorptive transfer stripping technique coupled with the differential pulse votlammetry Brdickás reaction. The signals related to the MT-cisplatin complex appeared at -0.9 V. The formation of this complex depended on the time of interaction between cisplatin and MT. The complex formation was consequently confirmed by quartz crystal microbalance analyses. The formation of this complex was detectable even after a 20 s long interaction. Moreover, we detected presence of MT-cisplatin complex in the blood of male rats treated with this drug.

Affecting of aquatic vascular plant Lemna minor by cisplatin revealed by voltammetry.

Within the context of application of platinum derivates based effective cytostatics, we can suppose that these risk metals can get into aquatic ecosystems where they can show biologic availability for food chain. In the present work we report on investigation of affecting of duckweed (Lemna minor) by various doses of cisplatin (0, 5, 10, 20, 40, 80 and 160 microM) for 4 days. The toxic influence of cisplatin was evaluated on the basis of growth inhibition expressed as number of leaves, growth rate, and total amount of biomass. The result value of 96hEC50, calculated from growth inhibition with comparison of growth rates, was 6.93 microM. Moreover we aimed on determination of cisplatin content using differential pulse voltammetry. The highest content of cisplatin (320 ng g(-1) of fresh weight) was determined in plants treated by 80 microM at the second day of treatment. Plants protect themselves against heavy metals by means of synthesis of cysteine-rich peptides such as glutathione and phytochelatins. Thus thiol determination in the treated plants by means of Brdicka reaction followed. The marked increase in thiol concentration detected is associated with defence reaction of the plant against stress caused by cisplatin.

An Electrochemical Detection of Metallothioneins at the Zeptomole Level in Nanolitre Volumes.

An Electrochemical Detection of Metallothioneins at the Zeptomole Level in Nanolitre VolumesWe report on improvement of the adsorptive transfer stripping technique (AdTS) coupled with the differential pulse voltammetry Brdicka reaction to determine a thiol-protein. The current technique has been unable to generate reproducible results when analyzing very low sample volumes (nanolitres). This obstacle can be overcome technically by modifying the current transfer technique including cooling step of the adsorbed analyte. We tested the technique on determination of a promising tumour disease marker protein called metallothionein (MT). The detection limit (3 S/N) of MT was evaluated as 500 zeptomoles per 500 nL (1 pM) and the quantification limit (10 S/N) as 1,500 zeptomoles per 500 nL (3 pM). Further, the improved AdTS technique was utilized to analyze blood serum samples from patients with breast cancer. Based on the results obtained it can be concluded that the improved technique can be used to detect a thiolprotein in very low sample volumes and can also prevent interferences during the washing and transferring step.

Electrochemical Determination of Low Molecular Mass Thiols Content in Potatoes (Solanum tuberosum) Cultivated in the Presence of Various Sulphur Forms and Infected by Late Blight (Phytophora infestans).

In the present paper potato plants were cultivated in the presence of ammonium sulphate or elemental sulphur supplementation into the soil to reveal the effects of different sulphur forms on content of nitrogen, phosphorus, potassium, calcium, magnesium and sulphur, and yield of tubers. During the investigation of the influence of different sulphur forms on yield of potato tubers we did not observe significant changes. Average weight of tubers of control plants per one experimental pot was 355 g. Application of sulphur in both forms resulted in moderate potato tubers weight reduction per one experimental pot compared to control group; average value ranged from 320 to 350 g per one experimental pot. Further we treated the plants with two different supplementation of sulphur with cadmium(II) ions (4 mg of cadmium(II) acetate per kilogram of the soil). The significantly lowest cadmium content (p < 0.05) was determined in tissues of plants treated with the highest dosage of elemental sulphur (0.64 mg Cd/kg) compared to control plants (0.82 mg Cd/kg). We also aimed our attention on the cadmium content in proteins, lipids or soluble carbohydrates and ash. Application of sulphate as well as elemental sulphur resulted in significant cadmium content reduction in lipid fraction compared to control plants. In addition to this we quantified content of low molecular mass thiols in potatoes tissues. To determine the thiols content we employed differential pulse voltammetry Brdicka reaction. After twelve days of the treatment enhancing of thiols level was observed in all experimental groups regardless to applied sulphur form and its concentration. Finally we evaluated the effect of sulphur supplementation on Phytophora infestans infection of potato plants.

Electrochemical determination of Ag-ions in environment waters and their action on plant embryos.

We utilized liquid chromatography coupled with electrochemical detector (HPLC-ED) for analyzing of silver ions. The optimization of basic chromatographic parameters has been done. The detection limit (3 S/N) obtained were 20 nmol/dm(3). Influence of different interferences (anions and cations) on current response of silver ions has been described. Moreover, we used HPLC-ED to analyze waters of different purity including photographic emulsion, which naturally contained silver ions. We found out that content of silver ions in the emulsion was 1.57 x 0.03 mmol/dm(3). Moreover, we investigated influence of silver ions on early somatic embryos of Blue Spruce. We were interested in the issue how much silver ions can embryos uptake during four days long treatment. For this purpose, we used optimized HPLC-ED technique. The content increased with increasing treatment time and applied concentration. We also studied how silver ions can influence thiols content in the treated embryos. For these purposes we used adsorptive transfer stripping voltammetry in connection with differential pulse voltammetry--Brdicka reaction. It clearly follows from the obtained results that content of thiols increased with increasing treatment time and applied concentration.

Characterization of the HMA7 gene and transcriptomic analysis of candidate genes for copper tolerance in two Silene vulgaris ecotypes.

Silene vulgaris possesses ecotype-specific tolerance to high levels of copper in the soil. Although this was reported a few decades ago, little is known about this trait on a molecular level. The aim of this study was to analyze the transcription response to elevated copper concentrations in two S. vulgaris ecotypes originating from copper-contrasting soil types - copper-tolerant Lubietova and copper-sensitive Stranska skala. To reveal if plants are transcriptionally affected, we first analyzed the HMA7 gene, a known key player in copper metabolism. Based on BAC library screening, we identified a BAC clone containing a SvHMA7 sequence with all the structural properties specific for plant copper-transporting ATPases. The functionality of the gene was tested using heterologous complementation in yeast mutants. Analyses of SvHMA7 transcription patterns showed that both ecotypes studied up-regulated SvHMA7 transcription after the copper treatment. Our data are supported by analysis of appropriate reference genes based on RNA-Seq databases. To identify genes specifically involved in copper response in the studied ecotypes, we analyzed transcription profiles of genes coding Cu-transporting proteins and genes involved in the prevention of copper-induced oxidative stress in both ecotypes. Our data show that three genes (APx, POD and COPT5) differ in their transcription pattern between the ecotypes with constitutively increased transcription in Lubietova. Taken together, we have identified transcription differences between metallifferous and non-metalliferous ecotypes of S. vulgaris, and we have suggested candidate genes participating in metal tolerance in this species.

Expression response of duplicated metallothionein 3 gene to copper stress in Silene vulgaris ecotypes.

Metallothioneins (MTs) were identified as important players in metal metabolism. MT3 gene presents a key metallothionein controlling copper homeostasis in plants. We have selected one cupricolous and one non-cupricolous ecotype to isolate and analyse the MT3 gene in Silene vulgaris. For expression data comparison, we have also included other metal-tolerant ecotypes. Based on a S. vulgaris BAC library screening, we have identified and sequenced a genomic clone containing MT3 gene (SvMT3). We found that SvMT3 gene has been locally duplicated in a tandem arrangement. Expression analysis and complementation studies using yeast mutants showed that both copies of the SvMT3 gene were functional. Moreover, we examined the expression of MT3 gene(s) in selected ecotypes under different copper treatments to show the tissue-specific expression response to copper stress. We demonstrated that higher copper concentrations specifically affected MT3 expression among ecotypes. Our analysis shows that MT3a has similar expression pattern in cupricolous ecotypes while MT3b has common expression features shared by all metallophyte S. vulgaris ecotypes. Our data indicate that down-regulation of MT3b root expression in higher copper concentrations is associated with copper stress. We propose that there might be a specific regulation of SvMT3s transcription depending on the type of heavy metal tolerance.