Impact of breath sample collection method and length of storage of breath samples in Tedlar bags on the level of selected volatiles assessed using gas chromatography-ion mobility spectrometry (GC-IMS).

The analysis of volatile organic compounds (VOCs) in exhaled air has attracted the interest of the scientific community because it provides the possibility of monitoring physiological and metabolic processes and non-invasive diagnostics of various diseases. However, this method remains underused in clinical practice as well as in research because of the lack of standardized procedures for the collection, storage and transport of breath samples, which would guarantee good reproducibility and comparability of results. The method of sampling, as well as the storage time of the breath samples in the polymer bags used for sample storage and transport, affect the composition and concentration of VOCs present in the breath samples. The aim of our study was to compare breath samples obtained using two methods with fully disposable equipment: a Haldane sampling tube intended for direct breath collection and breath samples exhaled into a transparent Tedlar bag. The second task was to monitor the stability of selected compounds of real breath samples stored in a Tedlar bag for 6 h. Gas chromatography coupled with ion mobility spectrometry (GC-IMS) implemented in the BreathSpec®device was used to analyse exhaled breath. Our results showed a significant difference in the signal intensity of some volatiles when taking a breath sample with a Haldane tube and a Tedlar bag. Due to its endogenous origin, acetone levels were significantly higher when the Haldane tube sampler was used while elevated levels of 2-propanol and unidentified VOC (designated as VOC 3) in the Tedlar bag samples likely originated from contamination of the Tedlar bags. The VOC stability study revealed compound-specific signal intensity changes of the selected VOCs with storage time in the Tedlar bags, with some volatiles showing increasing signal intensity during storage in Tedlar bags. This limits the use of Tedlar bags only for very limited time and carefully selected purpose. Our results highlight the importance of careful design and implementation of experiments and clinical protocols to obtain relevant and reliable results.

Comparison of pulsed and continuous electromagnetic field generated by WPT system on human dermal and neural cells.

In recent decades, we have seen significant technical progress in the modern world, leading to the widespread use of telecommunications systems, electrical appliances, and wireless technologies. These devices generate electromagnetic radiation (EMR) and electromagnetic fields (EMF) most often in the extremely low frequency or radio-frequency range. Therefore, they were included in the group of environmental risk factors that affect the human body and health on a daily basis. In this study, we tested the effect of exposure EMF generated by a new prototype wireless charging system on four human cell lines (normal cell lines-HDFa, NHA; tumor cell lines-SH-SY5Y, T98G). We tested different operating parameters of the wireless power transfer (WPT) device (87-207 kHz, 1.01-1.05 kW, 1.3-1.7 mT) at different exposure times (pulsed 6 × 10 min; continuous 1 × 60 min). We observed the effect of EMF on cell morphology and cytoskeletal changes, cell viability and mitotic activity, cytotoxicity, genotoxicity, and oxidative stress. The results of our study did not show any negative effect of the generated EMF on either normal cells or tumor cell lines. However, in order to be able to estimate the risk, further population and epidemiological studies are needed, which would reveal the clinical consequences of EMF impact.

In vitro modeling of amyotrophic lateral sclerosis with induced pluripotent stem cell technology-derived cell line ORIONi002-A.

We present here a new iPS cell line for modeling sporadic form of ALS. Cell line was generated by reprogramming skin fibroblasts isolated with explant culture technology from skin biopsy, donated by ALS patient. For reprogramming, polycistronic self-replicating RNA vector was used and derived iPS cells were characterized by immunocytochemistry and FACS (pluripotent factors expression), karyotyping, STR fingerprinting analysis and in vitro differentiation assay. New cell line showed normal (46, XY) karyotype and differentiated in vitro into cells from three germ layers. STR analysis proved the origin and originality of the cell line.

Establishment of PANDA - a new human pancreatic ductal adenocarcinoma cell line with 3D cell culture technology.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive type of malignancy with one of the worst prognoses amongst any type of cancer. Surgery is applicable only to the limited number of patients with locally resectable tumors and currently represents the only curative treatment option. Treatment with chemotherapy and radiotherapy can only extend patient survival. Despite advances in conventional therapies, the five-year survival of PDAC remained largely unchanged. New in vitro and in vivo models are therefore urgently needed to investigate this type of cancer. Here, we present the establishment and characterization of a novel pancreatic cancer cell line, isolated from a patient with PDAC. Cell line abbreviated as PANDA (PANncreatic Ductal Adenocarcinoma) was established with an optimized 3D culture protocol published previously by our group. The new cancer cell line "PANDA" represents a novel in vitro approach for PDAC cancer research and new therapy testing.

Effect of Cold Atmospheric Plasma on Epigenetic Changes, DNA Damage, and Possibilities for Its Use in Synergistic Cancer Therapy.

Cold atmospheric plasma has great potential for use in modern medicine. It has been used in the clinical treatment of skin diseases and chronic wounds, and in laboratory settings it has shown effects on selective decrease in tumour-cell viability, reduced tumour mass in animal models and stem-cell proliferation. Many researchers are currently focusing on its application to internal structures and the use of plasma-activated liquids in tolerated and effective human treatment. There has also been analysis of plasma's beneficial synergy with standard pharmaceuticals to enhance their effect. Cold atmospheric plasma triggers various responses in tumour cells, and this can result in epigenetic changes in both DNA methylation levels and histone modification. The expression and activity of non-coding RNAs with their many important cell regulatory functions can also be altered by cold atmospheric plasma action. Finally, there is ongoing debate whether plasma-produced radicals can directly affect DNA damage in the nucleus or only initiate apoptosis or other forms of cell death. This article therefore summarises accepted knowledge of cold atmospheric plasma's influence on epigenetic changes, the expression and activity of non-coding RNAs, and DNA damage and its effect in synergistic treatment with routinely used pharmaceuticals.

Generation of ORIONi001-A induced pluripotent stem cell line for in vitro modeling of sporadic form of amyotrophic lateral sclerosis.

We generated new in vitro model for sporadic form of amyotrophic lateral sclerosis by reprogramming isolated skin fibroblasts into iPSCs. Fibroblasts were reprogrammed with commercially available synthetic polycistronic, self-replicating RNA vector. As verified by FISH, an early passages of a new iPSC line showed mosaic karyotype (cells with normal and abnormal karyotype 46,XY,t(2;14)(q13;p12) were present), while late passages contained only cells with abnormal karyotype. New iPSCs differentiated into all three germ layers and formed a teratoma in nude mice. Our iPSC line represents a new model for therapy testing and drug development in the field of ALS research.

Cold Atmospheric Plasma: A Powerful Tool for Modern Medicine.

Cold atmospheric plasma use in clinical studies is mainly limited to the treatment of chronic wounds, but its application in a wide range of medical fields is now the goal of many analyses. It is therefore likely that its application spectrum will be expanded in the future. Cold atmospheric plasma has been shown to reduce microbial load without any known significant negative effects on healthy tissues, and this should enhance its possible application to any microbial infection site. It has also been shown to have anti-tumour effects. In addition, it acts proliferatively on stem cells and other cultivated cells, and the highly increased nitric oxide levels have a very important effect on this proliferation. Cold atmospheric plasma use may also have a beneficial effect on immunotherapy in cancer patients. Finally, it is possible that the use of plasma devices will not remain limited to surface structures, because current endeavours to develop sufficiently miniature microplasma devices could very likely lead to its application in subcutaneous and internal structures. This study summarises the available literature on cold plasma action mechanisms and analyses of its current in vivo and in vitro use, primarily in the fields of regenerative and dental medicine and oncology.

A Comparative In Vitro Analysis of the Osteogenic Potential of Human Dental Pulp Stem Cells Using Various Differentiation Conditions.

Dental pulp stem cells (DPSCs) have excellent proliferative properties, mineralization potential and can be easily obtained from third molar teeth. Recently, many studies have focused on isolation and differentiation of DPSCs. In our study, we focused on biological properties of non-differentiated DPSCs in comparison with osteogenic differentiated cells from DPSCs. We analyzed morphology as well as mineralization potential using three varied osteogenic differentiation media. After fifteen days of differentiation, calcium deposit production was observed in all three osteogenic differentiation media. However, only one osteogenic medium, without animal serum supplement, showed rapid and strong calcification-OsteoMAX-XF™ Differentiation Medium. Therefore, we examined specific surface markers, and gene and protein expression of cells differentiated in this osteogenic medium, and compared them to non-differentiated DPSCs. We proved a decrease in expression of CD9 and CD90 mesenchymal stem cell surface markers, as well as downregulation in the expression of pluripotency genes (NANOG and OCT-4) and increased levels of expression in osteogenic genes (ALP, BSP, OCN and RUNX2). Moreover, osteogenic proteins, such as BSP and OCN, were only produced in differentiated cells. Our findings confirm that carefully selected differentiation conditions for stem cells are essential for their translation into future clinical applications.

Liquid Biopsy as a Tool for Differentiation of Leiomyomas and Sarcomas of Corpus Uteri.

Utilization of liquid biopsy in the management of cancerous diseases is becoming more attractive. This method can overcome typical limitations of tissue biopsies, especially invasiveness, no repeatability, and the inability to monitor responses to medication during treatment as well as condition during follow-up. Liquid biopsy also provides greater possibility of early prediction of cancer presence. Corpus uteri mesenchymal tumors are comprised of benign variants, which are mostly leiomyomas, but also a heterogenous group of malignant sarcomas. Pre-surgical differentiation between these tumors is very difficult and the final description of tumor characteristics usually requires excision and histological examination. The leiomyomas and malignant leiomyosarcomas are especially difficult to distinguish and can, therefore, be easily misdiagnosed. Because of the very aggressive character of sarcomas, liquid biopsy based on early diagnosis and differentiation of these tumors would be extremely helpful. Moreover, after excision of the tumor, liquid biopsy can contribute to an increased knowledge of sarcoma behavior at the molecular level, especially on the formation of metastases which is still not well understood. In this review, we summarize the most important knowledge of mesenchymal uterine tumors, the possibilities and benefits of liquid biopsy utilization, the types of molecules and cells that can be analyzed with this approach, and the possibility of their isolation and capture. Finally, we review the typical abnormalities of leiomyomas and sarcomas that can be searched and analyzed in liquid biopsy samples with the final aim to pre-surgically differentiate between benign and malignant mesenchymal tumors.

Molecular regulation of epithelial-to-mesenchymal transition in tumorigenesis (Review).

Numerous studies over the past two decades have focused on the epithelial­to­mesenchymal transition (EMT) and its role in the development of metastasis. Certain studies highlighted the importance of EMT in the dissemination of tumor cells and metastasis of epithelium­derived carcinomas. Tumor metastasis is a multistep process during which tumor cells change their morphology, and start to migrate and invade distant sites. The present review discusses the current understanding of the molecular mechanisms contributing to EMT in embryogenesis, fibrosis and tumorigenesis. Additionally, the signaling pathways that initiate EMT through transcriptional factors responsible for the activation and suppression of various genes associated with cancer cell migration were investigated. Furthermore, the important role of the epigenetic modifications that regulate EMT and the reverse process, mesenchymal­to­epithelial transition (MET) are discussed. MicroRNAs are key regulators of various intracellular processes and current knowledge of EMT has significantly improved due to microRNA characterization. Their effect on signaling pathways and the ensuing events that occur during EMT at the molecular level is becoming increasingly recognized. The current review also highlights the role of circulating tumor cells (CTCs) and CTC clusters, and their ability to form metastases. In addition, the biological properties of different types of circulating cells based on their tumor­forming potential are compared.

The role of statins as therapeutic agents in cancer.

Statins are the inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. This enzyme catalyzes conversion of HMG-CoA to mevalonate, which are the intermediates in cholesterol biosynthetic pathway. Statins also play an important role in carcinogenesis, because they are able to affect the cancer cell metabolism. Their effect has been observed in several cellular processes, such as angiogenesis, metastasis, apoptosis and cell proliferation. However, these effects are highly dependent on type of cancer and individual statins vary in their antitumor potential. This review summarizes the recent epidemiological evidence and preclinical studies that showed effects of all clinically used statins in vitro and in vivo. We also consider the results of different observational and retrospective studies focused on association among statins and cancer risk which are still under open discussion.

DPYD genotype and haplotype analysis and colorectal cancer susceptibility in a case-control study from Slovakia.

Dihydropyrimidine dehydrogenase (DPD) acts as the first-step enzyme catabolizing pyrimidines in vivo. DPYD gene mutations interfere with the breakdown of uracil and thymine. Genetic variations of DPYD can cause an enzyme deficiency state, which results in severe toxicity or other adverse side effects such as DNA damage or RNA damage caused by imbalance of the nucleotide pool. Our case-control study investigates the possible association between seven DPYD gene polymophisms (rs1801267, rs72547602, rs1801160, rs3918290, rs1801159, rs1801158, rs1801265) and risk of colorectal cancer (CRC). The association analysis for DPD was performed on 273 CRC patients and 187 healthy controls. There is significant allele association of SNP rs1801160 with colorectal cancer (p = 0.003, OR = 3.264, 95% CI = 1.425-7.475) in present analysis. Haplotype analysis of four DPYD polymorphisms showed significant difference in the distribution "IISt" haplotype between cases and controls. In comparison to the most common haplotype (VISt), the "IISt" haplotype was associated with increased risk for CRC (p = 0.038, OR = 2.733, 95% CI = 1.019-7.326). The present study suggests that the SNP rs1801160 and the "IISt" haplotype in the DPYD gene may also have a role in colorectal cancer risk.

The Molecular and Cellular Effect of Homocysteine Metabolism Imbalance on Human Health.

Homocysteine (Hcy) is a sulfur-containing non-proteinogenic amino acid derived in methionine metabolism. The increased level of Hcy in plasma, hyperhomocysteinemia, is considered to be an independent risk factor for cardio and cerebrovascular diseases. However, it is still not clear if Hcy is a marker or a causative agent of diseases. More and more research data suggest that Hcy is an important indicator for overall health status. This review represents the current understanding of molecular mechanism of Hcy metabolism and its link to hyperhomocysteinemia-related pathologies in humans. The aberrant Hcy metabolism could lead to the redox imbalance and oxidative stress resulting in elevated protein, nucleic acid and carbohydrate oxidation and lipoperoxidation, products known to be involved in cytotoxicity. Additionally, we examine the role of Hcy in thiolation of proteins, which results in their molecular and functional modifications. We also highlight the relationship between the imbalance in Hcy metabolism and pathogenesis of diseases, such as cardiovascular diseases, neurological and psychiatric disorders, chronic kidney disease, bone tissue damages, gastrointestinal disorders, cancer, and congenital defects.

Benzothiazinones are suicide inhibitors of mycobacterial decaprenylphosphoryl-ß-D-ribofuranose 2'-oxidase DprE1.

Benzothiazinones (BTZs) are antituberculosis drug candidates with nanomolar bactericidal activity against tubercle bacilli. Here we demonstrate that BTZs are suicide substrates of the FAD-dependent decaprenylphosphoryl-ß-D-ribofuranose 2'-oxidase DprE1, an enzyme involved in cell-wall biogenesis. BTZs are reduced by DprE1 to an electrophile, which then reacts in a near-quantitative manner with an active-site cysteine of DprE1, thus providing a rationale for the extraordinary potency of BTZs. Mutant DprE1 enzymes from BTZ-resistant strains reduce BTZs to inert metabolites while avoiding covalent inactivation. Our results explain the basis for drug sensitivity and resistance to an exceptionally potent class of antituberculosis agents.

Investigation of ABC transporter from mycobacterial arabinogalactan biosynthetic cluster.

Two genes from the "mycobacterial arabinogalactan biosynthetic cluster" spanning the region from Rv3779 to Rv3809c in the genome of Mycobacterium tuberculosis H37Rv were annotated as possible components of the ATP-binding cassette transporter. Rv3781 encodes a nucleotide-binding domain and Rv3783 determines production of a membrane-spanning domain. We have examined possible roles of these genes in mycobacterial cell wall biosynthesis through inactivation of their respective orthologs in Mycobacterium smegmatis mc(2)155, phenotypic characterization of the mutant strains via metabolic labeling with [U-(14)C]-glucose, cell-free reactions with UDP-[U-(14)C]-galactose monitoring galactan build-up and transcriptional analysis. Several lines of evidence suggest that this ABC transporter is involved in biosynthesis of arabinogalactan, although more investigation is needed to establish its precise role or the transported substrate.

Biosynthetic origin of the galactosamine substituent of Arabinogalactan in Mycobacterium tuberculosis.

The arabinogalactan (AG) of slow growing pathogenic Mycobacterium spp. is characterized by the presence of galactosamine (GalN) modifying some of the interior branched arabinosyl residues. The biosynthetic origin of this substituent and its role(s) in the physiology and/or pathogenicity of mycobacteria are not known. We report on the discovery of a polyprenyl-phospho-N-acetylgalactosaminyl synthase (PpgS) and the glycosyltransferase Rv3779 from Mycobacterium tuberculosis required, respectively, for providing and transferring the GalN substrate for the modification of AG. Disruption of either ppgS (Rv3631) or Rv3779 totally abolished the synthesis of the GalN substituent of AG in M. tuberculosis H37Rv. Conversely, expression of ppgS in Mycobacterium smegmatis conferred upon this species otherwise devoid of ppgS ortholog and any detectable polyprenyl-phospho-N-acetylgalactosaminyl synthase activity the ability to synthesize polyprenyl-phospho-N-acetylgalactosamine (polyprenyl-P-GalNAc) from polyprenyl-P and UDP-GalNAc. Interestingly, this catalytic activity was increased 40-50-fold by co-expressing Rv3632, the encoding gene of a small membrane protein apparently co-transcribed with ppgS in M. tuberculosis H37Rv. The discovery of this novel lipid-linked sugar donor and the involvement of a the glycosyltransferase C-type glycosyltransferase in its transfer onto its final acceptor suggest that pathogenic mycobacteria modify AG on the periplasmic side of the plasma membrane. The availability of a ppgS knock-out mutant of M. tuberculosis provides unique opportunities to investigate the physiological function of the GalN substituent and the potential impact it may have on host-pathogen interactions.

High content screening identifies decaprenyl-phosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors.

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2' epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.

Identification of a polyprenylphosphomannosyl synthase involved in the synthesis of mycobacterial mannosides.

We report on the identification of a glycosyltransferase (GT) from Mycobacterium tuberculosis H37Rv, Rv3779, of the membranous GT-C superfamily responsible for the direct synthesis of polyprenyl-phospho-mannopyranose and thus indirectly for lipoarabinomannan, lipomannan, and the higher-order phosphatidyl-myo-inositol mannosides.

Chapter 2: Biogenesis of the cell wall and other glycoconjugates of Mycobacterium tuberculosis.

The re-emergence of tuberculosis in its present-day manifestations - single, multiple and extensive drug-resistant forms and as HIV-TB coinfections - has resulted in renewed research on fundamental questions such as the nature of the organism itself, Mycobacterium tuberculosis, the molecular basis of its pathogenesis, definition of the immunological response in animal models and humans, and development of new intervention strategies such as vaccines and drugs. Foremost among these developments has been the precise chemical definition of the complex and distinctive cell wall of M. tuberculosis, elucidation of the relevant pathways and underlying genetics responsible for the synthesis of the hallmark moieties of the tubercle bacillus such as the mycolic acid-arabinogalactan-peptidoglycan complex, the phthiocerol- and trehalose-containing effector lipids, the phosphatidylinositol-containing mannosides, lipomannosides and lipoarabinomannosides, major immunomodulators, and others. In this review, the laboratory personnel who have been the focal point of some to these developments review recent progress towards a comprehensive understanding of the basic physiology and functions of the cell wall of M. tuberculosis.

AftD, a novel essential arabinofuranosyltransferase from mycobacteria.

Arabinogalactan (AG) and lipoarabinomannan (LAM) are the two major cell wall (lipo)polysaccharides of mycobacteria. They share arabinan chains made of linear segments of alpha-1,5-linked D-Araf residues with some alpha-1,3-branching, the biosynthesis of which offers opportunities for new chemotherapeutics. In search of the missing arabinofuranosyltransferases (AraTs) responsible for the formation of the arabinan domains of AG and LAM in Mycobacterium tuberculosis, we identified Rv0236c (AftD) as a putative membrane-associated polyprenyl-dependent glycosyltransferase. AftD is 1400 amino acid-long, making it the largest predicted glycosyltransferase of its class in the M. tuberculosis genome. Assays using cell-free extracts from recombinant Mycobacterium smegmatis and Corynebacterium glutamicum strains expressing different levels of aftD indicated that this gene encodes a functional AraT with alpha-1,3-branching activity on linear alpha-1,5-linked neoglycolipid acceptors in vitro. The disruption of aftD in M. smegmatis resulted in cell death and a decrease in its activity caused defects in cell division, reduced growth, alteration of colonial morphology, and accumulation of trehalose dimycolates in the cell envelope. Overexpression of aftD in M. smegmatis, in contrast, induced the accumulation of two arabinosylated compounds with carbohydrate backbones reminiscent of that of LAM and a degree of arabinosylation dependent on aftD expression levels. Altogether, our results thus indicate that AftD is an essential AraT involved in the synthesis of the arabinan domain of major mycobacterial cell envelope (lipo)polysaccharides.