Comparison of Physicochemical, Mechanical, and (Micro-)Biological Properties of Sintered Scaffolds Based on Natural- and Synthetic Hydroxyapatite Supplemented with Selected Dopants.

The specific combinations of materials and dopants presented in this work have not been previously described. The main goal of the presented work was to prepare and compare the different properties of newly developed composite materials manufactured by sintering. The synthetic- (SHAP) or natural- (NHAP) hydroxyapatite serves as a matrix and was doped with: (i) organic: multiwalled carbon nanotubes (MWCNT), fullerenes C60, (ii) inorganic: Cu nanowires. Research undertaken was aimed at seeking novel candidates for bone replacement biomaterials based on hydroxyapatite-the main inorganic component of bone, because bone reconstructive surgery is currently mostly carried out with the use of autografts; titanium or other non-hydroxyapatite -based materials. The physicomechanical properties of the developed biomaterials were tested by Scanning Electron Microscopy (SEM), Dielectric Spectroscopy (BSD), Nuclear Magnetic Resonance (NMR), and Differential Scanning Calorimetry (DSC), as well as microhardness using Vickers method. The results showed that despite obtaining porous sinters. The highest microhardness was achieved for composite materials based on NHAP. Based on NMR spectroscopy, residue organic substances could be observed in NHAP composites, probably due to the organic structures that make up the tooth. Microbiology investigations showed that the selected samples exhibit bacteriostatic properties against Gram-positive reference bacterial strain S. epidermidis (ATCC 12228); however, the property was much less pronounced against Gram-negative reference strain E. coli (ATCC 25922). Both NHAP and SHAP, as well as their doped derivates, displayed in good general compatibility, with the exception of Cu-nanowire doped derivates.

Constitutional variants in POT1, TERF2IP, and ACD genes in patients with melanoma in the Polish population.

Evaluation of the prevalence of POT1, ACD, and TERF2IP mutations among Polish melanoma patients. A cohort of 60 patients from melanoma-prone families, 1500 unselected cases and 1500 controls were genotyped. Methodology included Sanger sequencing, in-silico software predilection, and TaqMan assays. We identified three nonsynonymous variants: POT1 c.903 G>T; TERF2IP c.970 A>G; and ACD c.1544 T>C and a splice site variant ACD c.645 G>A. The c.903 G>T was predicted to be pathogenic according to PolyPhen-2, benign according to Mutation Taster, PROVEAN, AGVGD, and SIFT. The c.645 G>A was defined as disease caused by Mutation Taster and Human Splicing Finder and as variant of unknown significance by ClinVar. The other detected variants were described as benign. The c.903 G>T variant was present in two unselected cases and one control [P = 0.57, odds ratio (OR) = 2.00]; the c.645 G>A variant was not detected among the unselected cases and the controls; the c.970 A>G variant was present in 110 cases and 133 controls (P = 0.14, OR = 0.81); the c.1544 T>C variant was present in 687 cases and 642 controls (P = 0.11, OR = 1.07). We found no loss of heterozygosity of the c.903 G>T, c.970 A>G, and c.645 G>A variants. C.645 G>A variant had no effect on splicing or expression. The changes in POT1 c.903 G>T and ACD c.645 G>A can be classified as rare variants of unknown significance, the other variants appear to be polymorphisms. Germline mutations in POT1, ACD, and TERF2IP are infrequent among Polish melanoma patients.

Reprogramming and transdifferentiation - two key processes for regenerative medicine.

Regenerative medicine based on transplants obtained from donors or foetal and new-born mesenchymal stem cells, encounter important obstacles such as limited availability of organs, ethical issues and immune rejection. The growing demand for therapeutic methods for patients being treated after serious accidents, severe organ dysfunction and an increasing number of cancer surgeries, exceeds the possibilities of the therapies that are currently available. Reprogramming and transdifferentiation provide powerful bioengineering tools. Both procedures are based on the somatic differentiated cells, which are easily and unlimitedly available, like for example: fibroblasts. During the reprogramming procedure mature cells are converted into pluripotent cells - which are capable to differentiate into almost any kind of desired cells. Transdifferentiation directly converts differentiated cells of one type into another differentiated cells type. Both procedures allow to obtained patient's dedicated cells for therapeutic purpose in regenerative medicine. In combination with biomaterials, it is possible to obtain even whole anatomical structures. Those patient's dedicated structures may serve for them upon serious accidents with massive tissue damage but also upon cancer surgeries as a replacement of damaged organ. Detailed information about reprogramming and transdifferentiation procedures as well as the current state of the art are presented in our review.

BRCA1 promoter methylation in peripheral blood is associated with the risk of triple-negative breast cancer.

Methylation of the promoter of the BRCA1 gene in DNA derived from peripheral blood cells is a possible risk factor for breast cancer. It is not clear if this association is restricted to certain types of breast cancer or is a general phenomenon. We evaluated BRCA1 methylation status in peripheral blood cells from 942 breast cancer patients and from 500 controls. We also assessed methylation status in 262 paraffin-embedded breast cancer tissues. Methylation status was assessed using methylation-sensitive high-resolution melting and was categorized as positive or negative. BRCA1 methylation in peripheral blood cells was strongly associated with the risk of triple-negative breast cancer (TNBC) (odds ratio [OR] 4.70; 95% confidence interval [CI]: 3.13-7.07; p < 0.001), but not of estrogen-receptor positive breast cancer (OR 0.80; 95% CI: 0.46-1.42; p = 0.46). Methylation was also overrepresented among patients with high-grade cancers (OR 4.53; 95% CI: 2.91-7.05; p < 0.001) and medullary cancers (OR 3.08; 95% CI: 1.38-6.88; p = 0.006). Moreover, we detected a significant concordance of BRCA1 promoter methylation in peripheral blood and paired tumor tissue (p < 0.001). We found that BRCA1 promoter methylation in peripheral blood cells is associated with approximately five times greater risk of TNBC. We propose that BRCA1 methylation in blood-derived DNA could be a novel biomarker of increased breast cancer susceptibility, in particular for triple-negative tumors.

Aspirin and its pleiotropic application.

Aspirin (acetylsalicylic acid), the oldest synthetic drug, was originally used as an anti-inflammatory medication. Being an irreversible inhibitor of COX (prostaglandin-endoperoxide synthase) enzymes that produce precursors for prostaglandins and thromboxanes, it has gradually found several other applications. Sometimes these applications are unrelated to its original purpose for example its use as an anticoagulant. Applications such as these have opened opportunities for new treatments. In this case, it has been tested in patients with cardiovascular disease to reduce the risk of myocardial infarct. Its function as an anticoagulant has also been explored in the prophylaxis and treatment of pre-eclampsia, where due to its anti-inflammatory properties, aspirin intake may be used to reduce the risk of colorectal cancer. It is important to always consider both the risks and benefits of aspirin's application. This is especially important for proposed use in the prevention and treatment of neurologic ailments like Alzheimer's disease, or in the prophylaxis of myocardial infarct. In such cases, the decision if aspirin should be applied, and at what dose may be guided by specific molecular markers. In this revived paper, the pleiotropic application of aspirin is summarized.

Psoriasis Treatment Changes the Expression Profile of Selected Caspases and their Regulatory MicroRNAs.

BACKGROUND/AIMS: Psoriasis, an autoimmune diseases of the skin, characterized by patches of abnormal/inflammed skin, although not usually life-threatening, it causes severe discomfort, esthetic impairments, and may lead to impaired social functions and social withdrawal. Besides UV-phototherapy, various anti-inflammatory treatments are applied, depending on the severity of symptoms. In 2008, adalimumab (fully humanized human anti-TNF antibody) was launched for the treatment of psoriasis. In the quest to better understand the pathomechanism of adalimumab's therapeutic effects, and the acquired resistance to the drug, we have investigated how its administration affect the regulation of the expression of selected caspases, including those activated by inflammosome. METHODS: The research was initially carried out on normal human dermal fibroblasts (NHDF) treated with adalimumab for 2, 8 and 24 hours in vitro. Then, expression profile of genes encoding caspases and their regulatory micro-RNAs was determined with the use of oligonucleotide microarray. The validation of the microarray results was carried out by qRT-PCR. The in vitro study was followed by ex-vivo investigation of adalimumab's effects on the expression of caspase-6 in blood of the psoriatic patients. The samples were collected before, and 2 hours after adalimumab's administration and the analysis was determined by qRT-PCR. RESULTS: The result of the analysis indicated that introduction of adalimumab to the NHDF culture resulted in the change of the transcription activity of genes encoding caspases and genes encoding miRNAs. The analysis revealed 5 different miRNA molecules regulating the expression of: CASP2, CASP3 and CASP6. There were no statistically significant differences in the expression of gene encoding caspase-6 in the patients' blood before and 2 hours after the anti-TNF drug administration. CONCLUSION: We have found that adalimumab administration affects caspases expression, thus they may be used as molecular markers for monitoring the therapy with the use of an anti-TNF drugs, including adalimumab. It is likely that the mechanisms responsible for changed expression profiles of genes encoding caspase-2,-3, and -6, may be caused by the upregulation of the respective microRNA molecules. Increased expression of genes encoding specific caspases may induce inflammatory processes, as well as trigger apoptosis. Furthermore, the proapoptotic activity of caspases may be enhanced by miRNA molecules, which exhibit proapoptotic function. The overexpression of such miRNAs was observed in our study.

BRCA1 and PARP1 mRNA expression during progression from normal breast to ductal carcinoma in situ and invasive breast cancer: a laser microdissection study.

The contribution of DNA damage repair mechanisms to the progression of normal breast to ductal carcinoma in situ (DCIS) and invasive ductal carcinoma is largely unknown. The purpose of this report was to assess the mRNA expression levels of two important genes associated with DNA repair, BRCA1 and PARP1, in normal breast tissue, DCIS G1, G2 and G3, and co-existing adjacent invasive ductal carcinoma. BRCA1 and PARP1 mRNA expression was assessed in 32 ductal carcinomas in situ of the breast using a laser microdissection and pressure catapulting system and quantitative real-time PCR. The relative expression of BRCA1 mRNA was significantly increased in DCIS G2 and DCIS G3 relative to normal breast tissue (p = 0.02, p = 0.001, respectively). Significant differences in BRCA1 expression were observed between DCIS G1 and G2 (p = 0.02) and between DCIS G1 and G3 (p = 0.0007). No significant differences in BRCA1 expression were observed between normal breast tissue and DCIS G1 and between DCIS component and adjacent invasive ductal carcinoma. No significant differences in the relative expression of PARP1 mRNA were observed between groups. Increased BRCA1 mRNA expression (but not PARP1 mRNA) occurs early in the development of breast cancer, i.e. at the noninvasive (DCIS) stage, suggesting a demand for increased activity of a DNA double-strand break repair by homologous recombination. DCIS G1 and normal breast tissue share highly similar BRCA1 and PARP1 expression level. This finding supports the idea that DCIS G1 belongs to a separate family of precursor lesions with low malignant potential.

Impact of Antibiotics on the Proliferation and Differentiation of Human Adipose-Derived Mesenchymal Stem Cells.

Adipose tissue is a promising source of mesenchymal stem cells. Their potential to differentiate and regenerate other types of tissues may be affected by several factors. This may be due to in vitro cell-culture conditions, especially the supplementation with antibiotics. The aim of our study was to evaluate the effects of a penicillin-streptomycin mixture (PS), amphotericin B (AmB), a complex of AmB with copper (II) ions (AmB-Cu2+) and various combinations of these antibiotics on the proliferation and differentiation of adipose-derived stem cells in vitro. Normal human adipose-derived stem cells (ADSC, Lonza) were routinely maintained in a Dulbecco's Modified Eagle Medium (DMEM) that was either supplemented with selected antibiotics or without antibiotics. The ADSC that were used for the experiment were at the second passage. The effect of antibiotics on proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine-B (SRB) tests. Differentiation was evaluated based on Alizarin Red staining, Oil Red O staining and determination of the expression of ADSC, osteoblast and adipocyte markers by real-time RT-qPCR. The obtained results indicate that the influence of antibiotics on adipose-derived stem cells depends on the duration of exposure and on the combination of applied compounds. We show that antibiotics alter the proliferation of cells and also promote natural osteogenesis, and adipogenesis, and that this effect is also noticeable in stimulated osteogenesis.

BRCA1/2-negative hereditary triple-negative breast cancers exhibit BRCAness.

BRCA1/2-associated breast cancers are sensitive to poly(ADPribose) polymerase (PARP) inhibitors and platinum compounds mainly due to their deficiency in DNA repair via homologous recombination (HR). However, approximately only 15% of triple-negative breast cancers (TNBCs) are BRCA1/2-associated. TNBCs that exhibit BRCAness (a phenotype reflecting impaired HR in BRCA1/2-negative tumors) are also regarded sensitive to PARP inhibitors and platinum compounds. Thus, we hypothesized that hereditary BRCA1/2-negative TNBCs may exhibit BRCAness. To find a subset of hereditary BRCA1/2-negative TNBCs among 360 TNBCs, we first identified a group of 41 hereditary TNBCs by analyzing the family histories of the patients. Next, we tested this group for the presence of germline BRCA1/2 mutations, and finally, we compared the expression levels of 120 genes involved in HR and five other major mechanisms of DNA damage repair between BRCA1/2-associated and BRCA1/2-negative subgroups of hereditary TNBCs using real-time PCR arrays. Approximately 73% of the hereditary TNBCs were BRCA1/2-associated and 27% were BRCA1/2-negative. The expression levels of the analyzed genes showed no significant differences between these two subgroups indicating the BRCAness of the BRCA1/2-negative hereditary TNBCs and thereby distinguishing a novel subset of TNBCs as a potential target for PARP inhibitors or platinum-based therapy. The results show the significance of family history in selecting patients with TNBC for therapies directed at incompetent DNA repair (e.g., PARP inhibitors and/or platinum-based therapies) and indicate that a relatively simple strategy for broadening the target group for these modes of treatment is to identify patients with hereditary TNBCs.

Pathological complete response after cisplatin neoadjuvant therapy is associated with the downregulation of DNA repair genes in BRCA1-associated triple-negative breast cancers.

Pathologic complete response (pCR) after neoadjuvant chemotherapy is considered a suitable surrogate marker of treatment efficacy in patients with triple-negative breast cancers (TNBCs). However, the molecular mechanisms underlying pCR as a result of such treatment remain obscure. Using real-time PCR arrays we compared the expression levels of 120 genes involved in the main mechanisms of DNA repair in 43 pretreatment biopsies of BRCA1-associated TNBCs exhibiting pCR and no pathological complete response (non-pCR) after neoadjuvant chemotherapy with cisplatin. Altogether, 25 genes were significantly differentially expressed between tumors exhibiting pCR and non-pCR, and these genes were downregulated in the pCR group compared to the non-pCR group. A difference in expression level greater than 1.5-fold was detected for nine genes: MGMT, ERCC4, FANCB, UBA1, XRCC5, XPA, XPC, PARP3, and RPA1. The non-homologous end joining and nucleotide excision repair pathways of DNA repair showed the most significant relevance. Expression profile of DNA repair genes associated with pCR was different in the node-positive (20 genes with fold change >1.5) and node-negative (only 3 genes) subgroups. Although BRCA1 germline mutations are the principal defects in BRCA1-associated TNBC, our results indicate that the additional downregulation of other genes engaged in major pathways of DNA repair may play a decisive role in the pathological response of these tumors to cisplatin neoadjuvant chemotherapy. The results suggest that patients with node-positive BRCA1-associated TNBCs that do not exhibit pCR after cisplatin neoadjuvant chemotherapy may be candidates for subsequent therapy with PARP inhibitors, whereas UBA1 may be a potential therapeutic target in node-negative subgroup.

KRAS mutation testing in colorectal cancer as an example of the pathologist's role in personalized targeted therapy: a practical approach.

Identifying targets for personalized targeted therapy is the pathologist's domain and a treasure. For decades, pathologists have had to learn, understand, adopt and implement many new laboratory techniques as they arrived on the scene. Pathologists successfully integrate the results of those tests into final pathology reports that were, and still are, the basis of clinical therapeutic decisions. The molecular methods are different but no more difficult to comprehend in the era of "kit procedures". In recent years, the development of targeted therapies has influenced routine practices in pathology laboratories because the use of molecular techniques is required to include clinically useful predictive information in the pathology report. Pathologists have the knowledge and expertise to identify particular gene mutations using the appropriate molecular tests currently available. This review focuses on the most important recent developments in KRAS mutation testing in metastatic colorectal cancer (CRC), and shows that a pathologist is involved in 10 stages of this procedure. Recent studies have shown that highly sensitive, simple, reliable and rapid assays may significantly improve the identification of CRC patients resistant to anti-EGFR therapy. Thus, direct sequencing does not seem to be an optimal procedure of KRAS testing for clinical purposes. Twelve currently available high-sensitivity diagnostic assays (with the CE-IVD mark) for KRAS mutation testing are briefly described and compared. The suggested pathology report content for somatic mutation tests is described. In conclusion, evidence is presented that sending away paraffin blocks with tumor tissue for KRAS mutation testing may not be in the best interest of patients. Instead, an evidence-based approach indicates that KRAS mutation testing should be performed in pathology departments, only with the use of CE-IVD/FDA-approved KRAS tests, and with the obligatory, periodic participation in the KRAS EQA scheme organized by the European Society of Pathology as an independent international body.

Colorectal cancers differ in respect of PARP-1 protein expression.

Recent findings raise the possibility of PARP inhibitor therapy in colorectal cancers (CRCs). However, the extent of PARP-1 protein expression in clinical specimens of CRC is not known. Using immunohistochemistry we assessed PARP-1 protein expression in tissue microarrays of 151 CRCs and its association with the patient's age, sex, Astler-Coller stage, grade and site of the tumor. High PARP nuclear immunoreactivity was found in 68.2% (103/151) of all cases. In turn, 31.8% (48/151) of tumors showed low PARP expression, including 9 (6%) PARP-1 negative CRCs. There was a significant association of PARP-1 expression with the site of CRC and Astler-Coller stage. A high PARP expression was noted in 79.1% of colon vs. 53.9% of rectal tumors (p = 0.001). The mean PARP-1 score was 1.27 times higher in colon vs. rectal cancers (p = 0.009) and it was higher in stage B2 vs. stage C of CRCs (p = 0.018). In conclusion, the level of PARP-1 protein nuclear expression is associated with the tumor site and heterogeneous across clinical specimens of CRC, with the majority of CRCs expressing a high level but minority - low or no PARP-1 expression. These findings may have a clinical significance because the assessment of PARP-1 expression in tumor samples may improve selection of patients with CRC for PARP inhibitor therapy.

Potassium channels in barley: cloning, functional characterization and expression analyses in relation to leaf growth and development.

It is not known how the uptake and retention of the key osmolyte K(+) in cells are mediated in growing leaf tissue. In the present study on the growing leaf 3 of barley, we have cloned the full-length coding sequence of three genes which encode putative K(+) channels (HvAKT1, HvAKT2, HvKCO1/HvTPK1), and of one gene which encodes a putative K(+) transporter (HvHAK4). The functionality of the gene products of HvAKT1 and HvAKT2 was tested through expression in Xenopus laevis oocytes. Both are inward-rectifying K(+) channels which are inhibited by Cs(+). Function of HvAKT1 in oocytes requires co-expression of a calcineurin-interacting protein kinase (AtCIPK23) and a calcineurin B-like protein (AtCBL9) from Arabidopsis, showing cross-species complementation of function. In planta, HvAKT1 is expressed primarily in roots, but is also expressed in leaf tissue. HvAKT2 is expressed particularly in leaf tissue, and HvHAK4 is expressed particularly in growing leaf tissue. Within leaves, HvAKT1 and HvAKT2 are expressed predominantly in mesophyll. Expression of genes changes little in response to low external K(+) or salinity, despite major changes in K(+) concentrations and osmolality of cells. Possible contributions of HvAKT1, HvAKT2, HvKCO1 and HvHAK4 to regulation of K(+) relations of growing barley leaf cells are discussed.