Carboplatin-induced upregulation of pan ß-tubulin and class III ß-tubulin is implicated in acquired resistance and cross-resistance of ovarian cancer.

Resistance to platinum- and taxane-based chemotherapy represents a major obstacle to long-term survival in ovarian cancer (OC) patients. Here, we studied the interplay between acquired carboplatin (CBP) resistance using two OC cell models, MES-OV CBP and SK-OV-3 CBP, and non-P-glycoprotein-mediated cross-resistance to paclitaxel (TAX) observed only in MES-OV CBP cells. Decreased platination, mesenchymal-like phenotype, and increased expression of α- and γ-tubulin were observed in both drug-resistant variants compared with parental cells. Both variants revealed increased protein expression of class III ß-tubulin (TUBB3) but differences in TUBB3 branching and nuclear morphology. Transient silencing of TUBB3 sensitized MES-OV CBP cells to TAX, and surprisingly also to CBP. This phenomenon was not observed in the SK-OV-3 CBP variant, probably due to the compensation by other ß-tubulin isotypes. Reduced TUBB3 levels in MES-OV CBP cells affected DNA repair protein trafficking and increased whole-cell platination level. Furthermore, TUBB3 depletion augmented therapeutic efficiency in additional OC cells, showing vice versa drug-resistant pattern, lacking ß-tubulin isotype compensation visible at the level of total ß-tubulin (TUBB) in vitro and ex vivo. In summary, the level of TUBB in OC should be considered together with TUBB3 in therapy response prediction.

Transcriptome analysis of newly established carboplatin-resistant ovarian cancer cell model reveals genes shared by drug resistance and drug-induced EMT.

BACKGROUND: In ovarian cancer (OC) therapy, even initially responsive patients develop drug resistance. METHODS: Here, we present an OC cell model composed of variants with differing degrees of acquired resistance to carboplatin (CBP), cross-resistance to paclitaxel, and CBP-induced metastatic properties (migration and invasion). Transcriptome data were analysed by two approaches identifying differentially expressed genes and CBP sensitivity-correlating genes. The impact of selected genes and signalling pathways on drug resistance and metastatic potential, along with their clinical relevance, was examined by in vitro and in silico approaches. RESULTS: TMEM200A and PRKAR1B were recognised as potentially involved in both phenomena, also having high predictive and prognostic values for OC patients. CBP-resistant MES-OV CBP8 cells were more sensitive to PI3K/Akt/mTOR pathway inhibitors Rapamycin, Wortmannin, SB216763, and transcription inhibitor Triptolide compared with parental MES-OV cells. When combined with CBP, Rapamycin decreased the sensitivity of parental cells while Triptolide sensitised drug-resistant cells to CBP. Four PI3K/Akt/mTOR inhibitors reduced migration in both cell lines. CONCLUSIONS: A newly established research model and two distinct transcriptome analysis approaches identified novel candidate genes enrolled in CBP resistance development and/or CBP-induced EMT and implied that one-gene targeting could be a better approach than signalling pathway inhibition for influencing both phenomena.

Variable recovery of cryopreserved hematopoietic stem cells and leukocyte subpopulations in leukapheresis products.

INTRODUCTION: Due to the expansion of cell therapy using not only haematopoietic stem cells (HSC) but also other leukocyte subpopulations, the loss of these cells in cryopreserved apheresis products needs to be evaluated. Various factors that could negatively affect post-thaw recovery, such as leukapheresis product characteristics, storage time and cryopreservation protocols have been identified. METHODS: The post-thaw recovery of HSCs, lymphocytes, NK cells and monocytes, as well as the factors that could adversely affect it were analysed in autologous and allogeneic leukapheresis products. RESULTS: The lowest post-thaw recovery was observed in autologous and allogeneic CD34+ cells, with the median of 73.7% and 68.1%, respectively. In leukocyte subpopulation, the lowest post-thaw recovery was observed for CD14+ cells, both autologous and allogeneic. The highest post-thaw recovery was observed for CD3+/CD8+ cells in autologous, and for CD19+ cells in allogeneic samples. The statistically significant difference was observed between autologous and allogeneic PBSC products for CD3+ cell recovery (P = 0.031) and CD3+/CD8+ cell recovery (P = 0.009). The evaluation of factors that could adversely affect the post-thaw recovery in autologous samples showed weak negative correlations between platelet concentration and CD3+ recovery, as well as between storage time and CD3+CD8+ recovery. In allogeneic samples, a strong negative correlation was observed only between the percentage of granulocytes and CD3+, CD3+/CD8+ and CD3+/CD4+ cell recoveries. CONCLUSION: Since various post-thaw recoveries of leukocyte subpopulations were observed, the cell therapy manufacturing centers should evaluate how their cryopreservation method and other factors affect the recovery of cell population of interest in their settings.

Extra Virgin Olive Oil Secoiridoids Modulate the Metabolic Activity of Dacarbazine Pre-Treated and Treatment-Naive Melanoma Cells.

Nowadays, many individuals, whether healthy or diagnosed with disease, tend to expose themselves to various easily accessible natural products in hopes of benefiting their health and well-being. Mediterranean populations have traditionally used olive oil not only in nutrition but also in cosmetics, including skincare. In this study, the phenolic profile-composed of twelve compounds altogether, including the secoiridoids oleocanthal (OCAL) and oleacein (OCEIN)-of extra virgin olive oil (EVOO) from autochthonous cultivars from Croatia was determined using 1H qNMR spectroscopy and HPLC-DAD analysis, and its biological activity was investigated in melanoma cell lines. The EVOO with the highest OCEIN content had the strongest anti-cancer activity in A375 melanoma cells and the least toxic effect on the non-cancerous keratocyte cell line (HaCaT). On the other hand, pure OCAL was shown to be more effective and safer than pure OCEIN. Post-treatment with any of the EVOO phenolic extracts (EVOO-PEs) enhanced the anti-cancer effect of the anti-cancerous drug dacarbazine (DTIC) applied in pre-treatment, while they did not compromise the viability of non-cancerous cells. The metastatic melanoma A375M cell line was almost unresponsive to the EVOO-PEs themselves, as well as to pure OCEIN and OCAL. Our results demonstrate that olive oils and/or their compounds may have a potentially beneficial effect on melanoma treatment. However, their usage can be detrimental or futile, especially in healthy cells, due to inadequately applied concentrations/combinations or the presence of resistant cells.

Sex affects the response of Wistar rats to polyvinyl pyrrolidone (PVP)-coated silver nanoparticles in an oral 28 days repeated dose toxicity study.

BACKGROUND: Silver nanoparticles (AgNPs) are widely used in biomedicine due to their strong antimicrobial, antifungal, and antiviral activities. Concerns about their possible negative impacts on human and environmental health directed many researchers towards the assessment of the safety and toxicity of AgNPs in both in vitro and in vivo settings. A growing body of scientific information confirms that the biodistribution of AgNPs and their toxic effects vary depending on the particle size, coating, and dose as well as on the route of administration and duration of exposure. This study aimed to clarify the sex-related differences in the outcomes of oral 28 days repeated dose exposure to AgNPs. METHODS: Wistar rats of both sexes were gavaged daily using low doses (0.1 and 1 mg Ag/kg b.w.) of polyvinylpyrrolidone (PVP)-coated small-sized (10 nm) AgNPs. After exposure, blood and organs of all rats were analysed through biodistribution and accumulation of Ag, whereas the state of the liver and kidneys was evaluated by the levels of reactive oxygen species (ROS) and glutathione (GSH), catalase (CAT) activity, superoxide dismutase (SOD) and glutathione peroxidase (GPx), expression of metallothionein (Mt) genes and levels of Mt proteins. RESULTS: In all animals, changes in oxidative stress markers and blood parameters were observed indicating the toxicity of AgNPs applied orally even at low doses. Sex-related differences were noticed in all assessed parameters. While female rats eliminated AgNPs from the liver and kidneys more efficiently than males when treated with low doses, the opposite was observed for animals treated with higher doses of AgNPs. Female Wistar rats exposed to 1 mg PVP-coated AgNPs/kg b.w. accumulated two to three times more silver in the blood, liver, kidney and hearth than males, while the accumulation in most organs of digestive tract was more than ten times higher compared to males. Oxidative stress responses in the organs of males, except the liver of males treated with high doses, were less intense than in the organs of females. However, both Mt genes and Mt protein expression were significantly reduced after treatment in the liver and kidneys of males, while they remained unchanged in females. CONCLUSIONS: Observed toxicity effects of AgNPs in Wistar rats revealed sex-related differences in response to an oral 28 days repeated exposure.

Extracellular Hsp70 modulates the inflammatory response of cigarette smoke extract in NCI-H292 cells.

NEW FINDINGS: What is the central question of this study? Does extracellular heat shock protein 70 (eHsp70) alter cigarette smoke extract (CSE)-induced inflammatory responses in NCI-H292 bronchial epithelial cells? What is the main finding and its importance? eHsp70 modulates inflammatory responses and TLR2, TLR4 and Hsp70 gene expression, and protects NCI-H292 cells against CSE-induced cytotoxicity. eHsp70 might be implicated in development of inflammatory diseases affected by cigarette smoke, such as COPD. ABSTRACT: One of the major risk factors for development of chronic obstructive pulmonary disease (COPD) is cigarette smoke. Extracellular Hsp70 (eHsp70) is increased in sera of COPD patients, and can act as damage-associated molecular pattern (DAMP). In this study, we explored inflammatory parameters (cytokine concentrations, Toll-like receptor (TLR) 2 and 4 and Hsp70 expression, mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) activation, and cytotoxicity) after exposure of bronchial-epithelial NCI-H292 cells to cigarette smoke extract (CSE) alone (2.5 and 15%) and in combinations with recombinant human (rh) Hsp70 (0.3, 1 and 3 µg ml-1 ). We applied specific MAPKs, NF-κB and Hsp70 inhibitors to elucidate rhHsp70 inflammation-associated responses. CSE alone and combinations of 15% CSE with rhHsp70 stimulated IL-1α, IL-6 and IL-8 release. However, rhHsp70 applied with 2.5% CSE decreased secretion of cytokines indicating antagonistic effects. Individual and combined treatments with 2.5% CSE suppressed TLR2 expression. CSE at 15% induced TLR2 and TLR4 gene expression, whereas rhHsp70 abolished that effect. rhHsp70 and 15% CSE alone reduced, while their combination increased, intracellular Hsp70 mRNA level. CSE alone and in combination with rhHsp70 activated extracellular signal-regulated kinase and p38 MAPKs, while inhibition of MAPKs, NF-κB and Hsp70 attenuated IL-6 and IL-8 secretion. CSE at 15% reduced cell viability and induced apoptosis, as shown by MTS and caspases-3/7 assays. CSE at 2.5% alone stimulated lactate dehydrogenase release, but cellular membrane integrity remained intact in co-treatments with rhHsp70. rhHsp70 might modulate the inflammatory response of CSE and could also protect NCI-H292 cells against CSE cytotoxicity. Those effects are implemented via MAPK and NF-κB signalling pathways.

Quality Assessment of Cryopreserved Peripheral Blood Stem Cell Products: Evaluation of Two Methods for Flow Cytometric Viability Testing.

INTRODUCTION: The standard flow cytometry method for viability testing using 7-aminoactinomycin D (7-AAD) determines cells in necrosis and late apoptosis. The colony-forming unit (CFU) assay, which evaluates the proliferation ability of HSCs, is also used in graft quality assessment despite known deficiencies that make this assay impractical in routine clinical settings. The aim was to compare the effectiveness of the flow cytometry 7-AAD/annexin V method with the 7-AAD method in assessing the quality of HSCs in autologous and allogeneic peripheral blood stem cell (PBSC) products. METHODS: Thirty autologous and 30 allogeneic fresh and thawed cryopreserved PBSC products were included in this study. The viability of HSCs was determined using the 7-AAD method and 7-AAD/annexin V method on a flow cytometer, while their clonogenic capacity was assessed by CFU assay. RESULTS: There was an excellent correlation for CD34+ cell viability between the 7-AAD and the 7-AAD/annexin V method for fresh samples (Rs = 0.930, p < 0.001) and a good correlation for thawed PBSC samples (Rs = 0.739, p < 0.001). Excellent correlation was observed for post-thaw CD34+ cell recovery between the two methods for viability (Rs = 0.980, p < 0.001). Statistical analysis showed a weak correlation between CFU-GM recovery and CD34+ cell recovery, regardless of which viability testing method was used (7-AAD method p = 0.021, Rs = 0.298; 7-AAD/annexin V method p = 0.029, Rs = 0.282). CONCLUSIONS: Results of this study showed that in the quality assessment of cryopreserved PBSC product viability, the 7-AAD/annexin V method had no added value compared to the 7-AAD method, which was suitable enough for routine quality control of cryopreserved autologous and allogeneic PBSC samples.

Citrinin Provoke DNA Damage and Cell-Cycle Arrest Related to Chk2 and FANCD2 Checkpoint Proteins in Hepatocellular and Adenocarcinoma Cell Lines.

Citrinin (CIT), a polyketide mycotoxin produced by Penicillium, Aspergillus, and Monascus species, is a contaminant that has been found in various food commodities and was also detected in house dust. Several studies showed that CIT can impair the kidney, liver, heart, immune, and reproductive systems in animals by mechanisms so far not completely elucidated. In this study, we investigated the CIT mode of action on two human tumor cell lines, HepG2 (hepatocellular carcinoma) and A549 (lung adenocarcinoma). Cytotoxic concentrations were determined using an MTT proliferation assay. The genotoxic effect of sub-IC50 concentrations was investigated using the alkaline comet assay and the impact on the cell cycle using flow cytometry. Additionally, the CIT effect on the total amount and phosphorylation of two cell-cycle-checkpoint proteins, the serine/threonine kinase Chk2 and Fanconi anemia (FA) group D2 (FANCD2), was determined by the cell-based ELISA. The data were analyzed using GraphPad Prism statistical software. The CIT IC50 for HepG2 was 107.3 µM, and for A549, it was >250 µM. The results showed that sensitivity to CIT is cell-type dependent and that CIT in sub-IC50 and near IC50 induces significant DNA damage and cell-cycle arrest in the G2/M phase, which is related to the increase in total and phosphorylated Chk2 and FANCD2 checkpoint proteins in HepG2 and A549 cells.

Galectin-1 and galectin-3 expression profiles in classically and alternatively activated human macrophages.

BACKGROUND: Galectins have been identified as modulators of many monocyte/macrophage functions. In the response to a wide range of environmental cues macrophages may exhibit different biochemical and biological characteristics, but two main subtypes, classically (M1) and alternatively (M2) activated macrophages have been recognized. To contribute to elucidation of role and regulation of galectin-1 and galectin-3 in differently activated macrophages we explored their expression profiles in these cells. METHODS: Human monocytes obtained from blood donors were differentiated into classically (M1) and alternatively (M2a/M2c) activated macrophages. Gene and protein expression levels of intra- and extracellular galectins were investigated by qRT-PCR, Western-blot, flow cytometry, and ELISA while cytokine and surface receptor expression profiling was performed by flow cytometry. RESULTS: Differentiation/polarization of human monocytes into classically (M1) and alternatively (M2a/M2c) activated macrophages was followed by profound changes of galectin-3 expression and its proteolytic cleavage. Expression and secretion of Gal-3 was tightly regulated and significantly differed among classically (M1) and alternatively (M2a/M2c) activated macrophages, while the differences of galectin-1 expression profiles were not as pronounced. Human monocytes exhibited high amount of free galectin-3 receptors, while on both types of activated macrophages were fully saturated. CONCLUSIONS: Galectin-3 is more distinctive descriptor of macrophages differentiation/activation than galectin-1. Its specific expression and secretion pattern in M1 vs. M2a/M2c macrophages contributes to better understanding of its role and regulation in these cells. GENERAL SIGNIFICANCE: Recognition of distinct galectin-1 and galectin-3 expression profiles in differently activated macrophages provides a new insight on biological characteristics of these cells and sheds a new light of galectin-3 as a modulator of individual macrophage subset. This article is part of a Special Issue entitled Glycoproteomics.

GT microsatellite repeats in the heme oxygenase-1 gene promoter associated with abdominal aortic aneurysm in Croatian patients.

Abdominal aortic aneurysm (AAA) is a complex genetic disorder caused by the interplay of genetic and environmental risk factors. The number of (GT)(n) repeats in the heme oxygenase-1 (HO-1) gene promoter modulates transcription of this enzyme, which might have anti-inflammatory, antioxidant, antiapoptotic, and antiproliferative effect. The distribution of alleles and genotypes in Croatian individuals genotyped for the (GT)(n) HO-1 polymorphism was similar to that in other European populations. Frequency of the short (S) alleles (GT < 25) was higher in AAA patients (41.9%) than in non-AAA individuals (28.2%, p = 0.0026) because there were more SL heterozygotes among the AAA patients. The SL genotype appeared to increase the risk for AAA, but the increase was not statistically significant after adjustment for age, sex, smoking, hypertension, and hyperlipidemia (OR = 1.53, 95% CI 0.90-3.09, p = 0.062). These findings contradict those of the only other study performed so far on the association of (GT)(n) HO-1 polymorphism and AAA.

The impact of cryoprotectant exposure time on post-thaw viability of autologous and allogeneic hematopoietic stem cells and leukocyte subpopulations.

Although the use of cryoprotectant dimethyl sulfoxide (DMSO) is the gold standard in cryopreservation of hematopoietic stem cells, it is well known that it has a negative effect on cell viability. The aim of this prospective study was to examine how the length of post-thaw exposure to DMSO affects the cell viability and stability of peripheral blood stem cell (PBSC) samples. Additionally, the effects of donor type and pre-cryopreservation storage time on post-thaw viability during the stability study were evaluated. In 30 autologous and 30 allogeneic PBSC samples viable CD34+, CD14+, CD19+, CD16+/56+, and CD3+ cells were determined immediately after thawing, and one-and three-hours post-thaw. Analysis of the absolute count of viable cells in thawed samples showed a significant difference between all measurement points for CD34+ (p < 0.001), CD14+ (p < 0.001), and CD19+ cells (p < 0.001). No significant differences were observed for post-thaw stability of allogeneic samples analysed between products stored before cryopreservation ≥ 24 hours (N = 20), and those stored < 24 hours (N = 10), except for viable CD3+/CD4+ cells after three hours post-thaw (p = 0.028). In conclusion, DMSO had different effects on leukocyte subpopulations in cryopre-served PBSC samples. The type of donors and the length of storage before cryopreservation did not affect the post-thaw stability of cryopreserved PBSC samples.

Oleuropein in olive leaf, branch, and stem extracts: stability and biological activity in human cervical carcinoma and melanoma cells.

Olive leaves as a main byproduct of olive oil and fruit industry are a valuable source of phytochemicals such as polyphenols, with multiple biomedical effects. Apart from leaves, olive branches and stems make up a significant amount of olive waste. It is well known that the drying process and long-term storage affect the stability and concentration of polyphenols present in raw materials. For that matter, two different means of storing olive waste, at room temperature and +4 °C, were compared by determining the content of the polyphenol oleuropein (OLE) in olive leaf, branch, and stem extracts (LE, BE, and SE) by HPLC-DAD method. Total phenols (TPC), o-diphenols (o-DPC), and total flavonoids (TFC) content in extracts were assessed by UV-Vis measurements. LE prepared from leaves stored at +4 °C had the highest OLE content, 30.7 mg g-1 of dry extract (DE). SE from stems stored at +4 °C was the richest in TPC and TFC (193 mg GAE/g DE and 82.9 mg CE/g DE, respectively), due to the higher purity of the extract. The biological activity of extracts was determined on cervical cancer (HeLa), melanoma (A375), metastatic melanoma (A375M) tumor cell lines, and on spontaneously immortalized cell line of keratinocytes (HaCaT), using the MTT assay. The data show that all extracts had a similar dose-dependent effect on cell viability in HeLa cells, while the effect of LE on melanoma A375 and A375M, and HaCaT cells was cell-line dependent.

Employment of single-strand conformation polymorphism analysis in screening for α-1,3 glucosyltransferase gene mutation A333V in Croatian population.

Congenital disorder of glycosylation type Ic (CDG-Ic) is caused by mutations in hALG6 gene encoding α-1,3 glucosyltransferase (NP_037471.2), an enzyme that catalyzes the addition of the first glucose residue to the growing lipid-linked oligosaccharide precursor in N-glycosylation process. The most frequent mutation in hALG6 gene causing CDG-Ic is c.998C>T that results in p.A333V substitution. Up-to-date, no CDG-Ic patient has been detected in Croatia. However, as a part of the comprehensive project undertaken with the aim to estimate the frequencies of the carriers for specific mutations and polymorphisms related to particular CDGs in Croatian population, we screened genomic DNA samples obtained from 600 healthy nonconsanguineous Croatian residents to determine the frequency of the A333V mutation. For that purpose, we established the conditions for polymerase chain reaction-based single-strand conformation polymorphism analysis that is suitable for primary screening and in population studies, especially when the initial sample volume is small or DNA quantity is limited. None of the analyzed samples carried this mutation, indicating that the frequency of the patients carrying this homozygous mutation in Croatian population would be <1 in 1.4×10(6).

Sterigmatocystin, 5-Methoxysterigmatocistin, and Their Combinations Are Cytotoxic and Genotoxic to A549 and Hepg2 Cells and Provoke Phosphorylation of Chk2, but Not Fancd2 Checkpoint Proteins.

Sterigmatocystin (STC) and 5-methoxysterigmatocystin (5-M-STC) are structurally related mycotoxins with cytotoxic and genotoxic properties. In the present study, we hypothesized that DNA damage induced by non-cytotoxic concentrations of single and combined mycotoxins could alter the phosphorylation of the checkpoint proteins Chk2 and FANCD2 (ELISA) in HepG2 and A549 cells. The cytotoxic potential (MTT test) of single and combined STC and 5-M-STC, the nature of their interaction (additivity, antagonism, or synergy) and DNA damage level (alkaline comet assay) in HepG2 and A549 cells were also investigated. All experiments were performed after 24 h of mycotoxin treatment. 5-M-STC was 10-folds more cytotoxic than STC to both HepG2 and A549 cells. Both mycotoxins are genotoxic to HepG2 and A549 cells by inducing both double and single DNA strand breaks that activate Chk2 (especially in HepG2 cells) but not the FANCD2 protein. STC exerted higher genotoxic potential than 5-M-STC in HepG2 and A549 cells when both toxins were applied individually at the same concentration. Dual combinations of non-cytotoxic mycotoxin concentrations showed additive to antagonizing cytotoxic and genotoxic effects. The absence and low activation of checkpoint proteins during prolonged exposure to non-cytotoxic concentrations of STC and 5-M-STC could support cell proliferation and carcinogenesis.

Association of fibrinogen and plasmin inhibitor, but not coagulation factor XIII gene polymorphisms with coronary artery disease.

BACKGROUND: In the final phase of clot formation, fibrinogen constitutes frame, whereas factor XIII (FXIII) active form is responsible for the covalent cross-linking of fibrin fibres and plasmin inhibitor (PI), thus contributing to clot stability. It could be expected that any change of coagulation factors' structure affects the clot formation and modulates the atherothrombotic risk. The aim was to determine the frequency of four single nucleotide polymorphisms: (i) A > G in codon 312 of the fibrinogen α-chain gene (rs6050, Thr312AlaFGA), (ii) C > T at position 10034 of the 3 - untranslated region in the fibrinogen γ-chain gene (rs2066865, 10034C > T FGG), (iii) C > T in codon 564 of the FXIII-A subunit gene (rs5982, Pro564LeuFXIII-A), and (iv) C > T in codon 6 of the plasmin inhibitor gene (rs2070863, Arg6TrpPI) in Croatian patients and their association with coronary artery disease (CAD). METHODS: We performed the unrelated case-control association study on the consecutive sample of patients 18 years old, who had undergone coronary angiography for investigation of chest pain and suspected CAD. The cases were patients with confirmed CAD (N=201), and the controls were the subjects with no CAD (N=119). Samples were genotyped using PCR-RFLP analysis. RESULTS: Observed frequencies of the rare alleles of Thr312Ala FGA, 10034C > T FGG, Leu564Pro FXIII-A and Arg6Trp PI polymorphisms were 21%, 17%, 14%, 20%, respectively. Patients with 10034C > T FGG CC genotype had 3.5 times (95% CI 1.02-12.03) higher adjusted odds for CAD than patients with 10034C > T FGG TT genotype. Patients with Arg6Trp PI CC genotype had 3.86 times (95% CI 1.23-12.12) higher odds for CAD than patients with Arg6Trp PI TT genotype. It seems that those genotype-related higher odds are also male-gender related. No difference was observed regarding any other investigated polymorphism. CONCLUSIONS: Our finding suggests that 10034C > T FGG and Arg6Trp PI are associated with CAD.

Increased HSP70 and TLR2 Gene Expression and Association of HSP70 rs6457452 Single Nucleotide Polymorphism with the Risk of Chronic Obstructive Pulmonary Disease in the Croatian Population.

Heat shock protein 70 (Hsp70) engages Toll-like receptors (TLR) 2 and 4 when found in the extracellular compartment and contributes to inflammation in chronic obstructive pulmonary disease (COPD). Since there is growing evidence for the genetic risk factors for COPD, the gene expression of HSP70, TLR2 and TLR4 was determined, as well as the association between HSP70, TLR2 and TLR4 single nucleotide polymorphisms, (SNPs) and COPD. The gene expression was assessed in peripheral blood cells of 137 COPD patients and 95 controls by a quantitative polymerase chain reaction (qPCR), while a total of nine SNPs were genotyped by TaqMan allelic discrimination real-time PCR. HSP70 and TLR2 gene expression was increased in COPD patients compared to the controls, regardless of the disease severity and smoking status of participants. The rs6457452 SNP of HSP70 was associated with COPD, indicating the protective role of the T allele (OR = 0.46, 95% CI = 0.24-0.89, p = 0.022). Furthermore, COPD C/T heterozygotes showed a decreased HSP70 mRNA level compared to COPD C/C homozygotes. In conclusion, HSP70 and TLR2 may have a role in the pathogenesis of COPD, and the HSP70 rs6457452 variant might influence the genetic susceptibility to COPD in the Croatian population.

Interaction of Differently Sized, Shaped, and Functionalized Silver and Gold Nanoparticles with Glycosylated versus Nonglycosylated Transferrin.

Exposure of nanomaterials (NMs) to biological medium results in their direct interaction with biomolecules and the formation of a dynamic biomolecular layer known as the biomolecular corona. Despite numerous published data on nano-biointeractions, the role of protein glycosylation in the formation, characteristics, and fate of such nano-biocomplexes has been almost completely neglected, although most serum proteins are glycosylated. This study aimed to systematically investigate the differences in interaction of metallic NPs with glycosylated vs nonglycosylated transferrin. To reach this aim, we compared interaction mechanisms between differently sized, shaped, and surface-functionalized silver NMs and gold NMs to commercially available human transferrin (TRF), a glycosylated protein, and to its nonglycosylated recombinant form (ngTRF). Bovine serum albumin (BSA) was also included in the study for comparative purposes. Characterization of NMs was performed using transmission electron microscopy and dynamic and electrophoretic light scattering techniques. Fluorescence quenching and circular dichroism methods were used to evaluate protein binding constants on the nanosurface and conformational changes after the protein-NM interactions, respectively. Competitive binding of TRF, ngTRF, and BSA to the surface of different NMs was evaluated by separating them after extraction from protein corona by gel electrophoresis following quantification with a commercial protein assay. The results showed that the binding strength between NMs and transferrin and the changes in the secondary protein structure largely depend not only on NM physicochemical properties but also on the protein glycosylation status. Data gained by this study highlight the relevance of protein glycosylation for all future design, development, and efficacy and safety assessment of NMs.

Teratoma Growth Retardation by HDACi Treatment of the Tumor Embryonal Source.

Among testicular germ cell tumors, teratomas may often be very aggressive and therapy-resistant. Our aim was to investigate the impact of histone deacetylase inhibitors (HDACi) on the in vitro growth of experimental mouse teratoma by treating their embryonic source, the embryo-proper, composed only of the three germ layers. The growth of teratomas was measured for seven days, and histopathological analysis, IHC/morphometry quantification, gene enrichment analysis, and qPCR analysis on a selected panel of pluripotency and early differentiation genes followed. For the first time, within teratomas, we histopathologically assessed the undifferentiated component containing cancer stem cell-like cells (CSCLCs) and differentiated components containing numerous lymphocytes. Mitotic indices were higher than apoptotic indices in both components. Both HDACi treatments of the embryos-proper significantly reduced teratoma growth, although this could be related neither to apoptosis nor proliferation. Trichostatin A increased the amount of CSCLCs, and upregulated the mRNA expression of pluripotency/stemness genes as well as differentiation genes, e.g., T and Eomes. Valproate decreased the amount of CSCLCs, and downregulated the expressions of pluripotency/stemness and differentiation genes. In conclusion, both HDACi treatments diminished the inherent tumorigenic growth potential of the tumor embryonal source, although Trichostatin A did not diminish the potentially dangerous expression of cancer-related genes and the amount of CSCLC.

Effect of scuba diving on the oxidant/antioxidant status, SIRT1 and SIRT3 expression in recreational divers after a winter nondive period.

The aim of this study was to examine the effects of scuba diving on oxidative damage markers in erythrocytes and plasma, antioxidant system in peripheral blood mononuclear cells (PBMCs), as well as sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3) gene expressions in recreational divers after a winter nondive period (at least 5 months). For that purpose, 17 male recreational divers performed an immersion at a depth of 30 m for 30 min. Blood samples were collected immediately before and after diving, 3 and 6 h after diving. Erythrocyte lipid peroxidation measured by thiobarbituric-reactive substances (TBARS) method was significantly increased immediately after diving, but returned to the baseline 6 h after diving, while no significant change was found for plasma TBARS and protein carbonyl derivates in both plasma and erythrocytes. Diving-induced catalase (CAT), superoxide dismutase 2 (SOD2), and consequently total superoxide dismutase (SOD) activities in the PBMC samples (significantly increased immediately after diving, reached the maximum activities 3 h after diving, while 6 h after diving only CAT activity remained significantly increased). No significant change was observed for SOD1 activity and gene expression, as well as SOD2 expression, while CAT and SIRT1 expressions were slightly decreased immediately after diving and 3 h after diving. Interestingly, SIRT3 expression was significantly increased 6 h after diving. In conclusion, after the first dive to 30 m after a nondive season, activation of antioxidant defence was not sufficient to prevent oxidative damage, while SIRT3 upregulation could be a step towards an adaptive response to the diving.

Galectin-3 in macrophage-like cells exposed to immunomodulatory drugs.

During the last few decades, the effects of immunomodulatory drugs on numerous molecules and biological processes have been widely studied. Nevertheless, the relationship between immunomodulatory drugs and lectin expression/function is still to be elucidated. In this study, we used THP-1-derived macrophages to investigate the effects of non-steroidal anti-inflammatory drugs (aspirin and indomethacin) and glucocorticoids (hydrocortisone and dexamethasone) on galectin-3, a multifunctional beta-galactoside binding lectin, which in general acts as a strong pro-inflammatory signal. The results showed that all immunomodulatory drugs applied in clinically relevant doses affect both the gene (LGALS3) and protein expression level of galectin-3. The provoked changes on protein level are qualitatively and quantitatively different comparing to the effects on galectin-3 mRNA level, and depend on the differentiation state of the cell, drug type and applied concentration as well as on time of the exposure. Our data revealed galectin-3 as a new target molecule of immunomodulatory drugs, thus suggesting an additional pathway of their action on immune response.