Amyloids, amorphous aggregates and assemblies of peptides - Assessing aggregation.

Amyloid and amorphous aggregates represent the two major categories of aggregates associated with diseases, and although exhibiting distinct features, researchers often treat them as equivalent, which demonstrates the need for more thorough characterization. Here, we compare amyloid and amorphous aggregates based on their biochemical properties, kinetics, and morphological features. To further decipher this issue, we propose the use of peptide self-assemblies as minimalistic models for understanding the aggregation process. Peptide building blocks are significantly smaller than proteins that participate in aggregation, however, they make a plausible means to bridge the gap in discerning the aggregation process at the more complex, protein level. Additionally, we explore the potential use of peptide-inspired models to research the liquid-liquid phase separation as a feasible mechanism preceding amyloid formation. Connecting these concepts can help clarify our understanding of aggregation-related disorders and potentially provide novel drug targets to impede and reverse these serious illnesses.

The Virus-Induced Upregulation of the miR-183/96/182 Cluster and the FoxO Family Protein Members Are Not Required for Efficient Replication of HSV-1.

Herpes simplex virus 1 (HSV-1) expresses a large number of miRNAs, and their function is still not completely understood. In addition, HSV-1 has been found to deregulate host miRNAs, which adds to the complexity of the regulation of efficient virus replication. In this study, we comprehensively addressed the deregulation of host miRNAs by massive-parallel sequencing. We found that only miRNAs expressed from a single cluster, miR-183/96/182, are reproducibly deregulated during productive infection. These miRNAs are predicted to regulate a great number of potential targets involved in different cellular processes and have only 33 shared targets. Among these, members of the FoxO family of proteins were identified as potential targets for all three miRNAs. However, our study shows that the upregulated miRNAs do not affect the expression of FoxO proteins, moreover, these proteins were upregulated in HSV-1 infection. Furthermore, we show that the individual FoxO proteins are not required for efficient HSV-1 replication. Taken together, our results indicate a complex and redundant response of infected cells to the virus infection that is efficiently inhibited by the virus.

Targeting Microenvironment of Melanoma and Head and Neck Cancers in Photodynamic Therapy.

BACKGROUND: Photodynamic therapy (PDT), in comparison to other skin cancers, is still far less effective for melanoma, due to the strong absorbance and the role of melanin in cytoprotection. The tumour microenvironment (TME) has a significant role in tumour progression, and the hypoxic TME is one of the main reasons for melanoma progression to metastasis and its resistance to PDT. Hypoxia is also a feature of solid tumours in the head and neck region that indicates negative prognosis. OBJECTIVE: The aim of this study was to individuate and describe systematically the main strategies in targeting the TME, especially hypoxia, in PDT against melanoma and head and neck cancers (HNC), and assess the current success in their application. METHODS: PubMed was used for searching, in MEDLINE and other databases, for the most recent publications on PDT against melanoma and HNC in combination with the TME targeting and hypoxia. RESULTS: In PDT for melanoma and HNC, it is very important to control hypoxia levels, and amongst the different approaches, oxygen self-supply systems are often applied. Vascular targeting is promising, but to improve it, optimal drug-light interval, and formulation to increase the accumulation of the photosensitiser in the tumour vasculature, have to be established. On the other side, the use of angiogenesis inhibitors, such as those interfering with VEGF signalling, is somewhat less successful than expected and needs to be further investigated. CONCLUSION: The combination of PDT with immunotherapy by using multifunctional nanoparticles continues to develop and seems to be the most promising for achieving a complete and lasting antitumour effect.

Evaluation of the Antioxidant Capacity, Antimicrobial and Antiproliferative Potential of Fir (Abies alba Mill.) Honeydew Honey Collected from Gorski kotar (Croatia).

The paper examines the antiproliferative, antimicrobial and antioxidative effects of fir (Abies alba Mill.) honeydew honey from mountain region of Croatia (Gorski kotar) as a potential replacement for standard antibiotics and chemotherapeutic agents. Cell viability, annexin V assay and flow cytometry analysis served to analyse the antiproliferative effect on, apoptosis induction in and cell death of cancer cell lines: HeLa, MCF-7, SW620, CFPAC-1, MIA PaCa-2 and normal diploid human fibroblasts (BJ). Antimicrobial activity was tested against Staphylococcus and Acinetobacter strains by agar well diffusion and microdilution assays. The DPPH˙ assay determined the radical scavenging activity, while mathematical models helped to evaluate the kinetic data of DPPH˙ inhibition. Antiproliferative effect on all tested cell lines and the prominent effect on normal diploid human fibroblasts (BJ), colorectal adenocarcinoma (SW620, metastatic) and breast epithelial adenocarcinoma (MCF-7, metastatic) was observable. The mechanisms of antiproliferative effect included accumulation of cells in the sub-G1 phase in all tested cells and induction of apoptosis in SW620 and MCF-7 cells predominantly. The antibacterial assays showed that antibiotic-resistant strains of both bacteria, including multi-resistant strain A. baumannii ATCC® BAA-1605™, were sensitive to all tested honey samples. Radical scavenging assay suggests that antioxidants present in the honey possess different radical suppressing abilities and that they react at different rates with radicals, thereby causing two steps of reaction. The results of the study indicate that Croatian fir honeydew honey has a therapeutic potential due to the strong biological activity and can serve to protect human health.

Inflammatory Gene Expression Upon TGF-ß1-Induced p38 Activation in Primary Dupuytren's Disease Fibroblasts.

OBJECTIVES: Inflammation is an underlying mechanism behind fibrotic processes and differentiation of cells into myofibroblasts. Presented study therefore provides new data on activation of autoimmune and inflammatory immune response genes that accompany activation of p38 and cell differentiation in primary cells derived from Dupuytren's disease (DD) patients. METHODS: Primary non-Dupuytren's disease cells (ND) were isolated from macroscopically unaffected palmar fascia adjacent to diseased tissue obtained from patients diagnosed with the last stage of DD and cultured in vitro. Gene expression, collagen gel contraction assay and analysis of secreted proteins were performed in ND cells treated with TGF-ß1 and/or inhibitor of p38 phosphorylation. RESULTS: During differentiation of ND fibroblasts, increased expression of immune response genes PAI-1, TIMP-1, CCL11, and IL-6 was found. These changes were accompanied by increased cell contractility and activation of p38 and its target kinase MK2. Inhibition of p38 phosphorylation reversed these processes in vitro. CONCLUSIONS: TGF-ß1 induced p38 phosphorylation in ND cells grown from macroscopically unaffected palmar fascia adjacent to diseased tissue from DD patients. This was accompanied by activation of the cytokine genes CCL-11 and IL-6 and secretion of extracellular matrix regulatory proteins PAI-1 and TIMP-1. A combined approach directed toward inflammation and p38 MAPK-mediated processes in DD might be considered for improving management of DD patients and prevention of recurrence.

70-kDa heat shock cognate protein expression as a putative biomarker of heavy-metal burden in Mytilus galloprovincialis.

BACKGROUND: Heavy metals naturally occur in the marine environment and ecosystems. Due to anthropogenic influence they became common waters and coastal regions pollutants in particular where their concentrations remain hazardously high. We therefore tested a protocol for combined analysis of 6 heavy metal (Pb, Cd, Cr, Zn, Fe and Hg) concentrations in mussels Mytilus galloprovincialis collected from a coastal industrial zone (shipyard locality) and mariculture facilities in combination with expression analysis of multi xenobiotic resistance related genes and stress-related gene (HSC-70). FINDINGS: In this paper we tested a protocol for heavy-metal levels assessment by use of a highly sensitive analytical method, ICP-OES, combined with expression analysis of multi xenobiotic resistance related genes, including the stress-related gene encoding 70-kDa heat shock cognate protein on mussels (Mytillus Galloprovincialis). Mussels from the shipyard locality had higher heavy metal concentrations, except Fe. Higher metal concentrations did not influence expression of multi xenobiotic resistance related genes with exception of stress-related gene (HSC-70) encoding 70-kDa heat shock cognate protein. CONCLUSIONS: Our results indicate that mussels sampled in the industrial area have increased metal concentrations in comparison with the aquaculture locality, that are accompanied by increased transcript levels of HSC-70.

Association of TGFB1 29C/T and IL6 -572G/C polymorphisms with developmental hip dysplasia: a case-control study in adults with severe osteoarthritis.

PURPOSE: Developmental dysplasia of the hip (DDH) increases the risk of severe adult hip osteoarthritis (OA). Transforming growth factor-ß1 (TGF-beta1) and interleukin-6 (IL-6) are included in pathogenesis of OA, as well as in development of the musculoskeletal system. We investigated the association of single nucleotide polymorphisms (SNPs) known to reflect on the circulating levels of the two cytokines, specifically, 29 T → C transition in the TGFB1 signal sequence (rs1800470) and -572G → C transversion in the IL6 promoter (rs1800796), with DDH. METHODS: We conducted a case-control study in consecutive unrelated adults with severe hip OA scheduled for total hip arthroplasty. Cases, patients with OA secondary to DDH (n = 68) and controls, patients with OA unrelated to DDH (n = 152) were genotyped at the two loci. RESULTS: With adjustment for age, sex and genotype at the concurrent locus, cases were more likely (OR = 2.42, 95%CI 1.08-5.43; p = 0.032) to be transition homozygous at TGFB1 locus 29, and also more likely (OR = 6.36, 95%CI 2.57-15.7; p < 0.001) to be transversion homozygous at IL6 locus -572 than controls. Cases were also more likely (OR = 11.3, 95%CI 4.25-29.8; p < 0.001) than controls to carry one of the three genotypes combining transition/transversion homozygosity at both loci, or transition/transversion homozygosity at one and heterozygosity at the concurrent locus. CONCLUSIONS: Data suggest association between TGFB1 29 T → C transition (rs1800470) and IL6 -572G → C transversion (rs1800796) with DDH, and also a possibility of TGF-beta1 and IL-6 interaction in DDH pathogenesis.

New anticancer active and selective phenylene-bisbenzothiazoles: synthesis, antiproliferative evaluation and DNA binding.

Novel amidino-derivatives of phenylene-bisbenzothiazoles were synthesized and tested for their antiproliferative activity against several human cancer cell lines, as well as DNA-binding properties. The synthetic approach used for preparation of isomeric amidino substituted-phenylene-bis-benzothyazoles 3a-3f was achieved by condensation reaction of isophthaloyl dichloride 1a and terephthaloyl dichloride 1b or with phthalic acid 1c with 5-amidinium-2-aminobenzothiolate 2a and 5-(imidazolinium-2-yl)-2-aminobenzothiolate 2b in good yields. The targeted compounds were converted in the desired water soluble dihydrochloride salts by reaction of appropriate free base with concd HCl in ethanol or acetic acid. All tested compounds (3a-3f) showed antiproliferative effects on tumour cells in a concentration-dependant manner. The strongest activity and cytotoxicity was observed for diimidazolinyl substituted phenylene-bisbenzothiazole compound 3b. These effects were shown to be related to DNA-binding properties, topoisomerase I and II poisoning effects and apoptosis induction. The highest tested selectivity towards tumour cells was observed for the imidazolyl substituted phenylene-benzothiazole 3d that showed no cytotoxic effects on normal fibroblasts making it an excellent candidate for further chemical optimization and preclinical evaluation.

Antiproliferative potency of novel benzofuran-2-carboxamides on tumour cell lines: cell death mechanisms and determination of crystal structure.

In this manuscript the synthesis and biological activity of novel heterocyclic derivatives of benzofuran-2-carboxamides 3a-j and 6a-f is presented. Biological evaluation in vitro revealed that only few compounds exerted concentration-depended antiproliferative effects on tumour cell lines at micromolar concentrations. In particular, 2-imidazolynyl substituted compound 6f showed selectivity on SK-BR-3 cell line while 2-N-acetamidopyridyl substituted 3h and 2-imidazolynyl substituted amide 3i showed selective concentration-dependent antiproliferative effects on SW620 cell line. Compounds 3h and 6f induced apoptosis while compound 3i acted through a cell death mechanism other than apoptosis.

Microarray analysis of Dupuytren's disease cells: the profibrogenic role of the TGF-ß inducible p38 MAPK pathway.

BACKGROUND: Dupuytren's disease (DD) is a nodular palmar fibromatosis that causes irreversible permanent contracture of fingers and results in the loss of hand function. Surgery still remains the only available solution for DD patients but cannot permanently cure the disease nor reduce high recurrence rates. With this rationale, we designed a study aimed at an improved understanding of the molecular mechanisms underlying DD. Our major focus was an analysis of the global gene expression profile and signalling pathways in DD cells with the aim of identifying novel biomarkers and/or therapeutic targets. METHODS: Primary cells were cultured from surgically removed diseased and healthy tissue. Microarray expression analysis (HG-U133A array, Affymetrix) and qPCR was performed with total RNA isolated from primary DD cells. Mechanistic studies involving inhibition of p38 phosphorylation were performed on normal human fibroblasts' and primary DD cells' in vitro models. Expression of stem cell markers in primary fibroblasts/myofibroblasts was assessed as well. RESULTS: We identified 3 p38MAPK signalling pathway regulatory genes, THBS1, GADD45α and NUAK1, all involved in cellular proliferation and production of the extracellular matrix proteins. Inhibition of the p38MAPK signalling pathway induced down-regulation of myofibroblast markers, α-smooth muscle actin and palladin. A stem-cell like subpopulation positive for CD90 marker was identified among primary DD cells. CONCLUSION: The study reveals involvement of the p38 MAPK pathway as a possible signalling cascade in the pathogenesis of Dupuytren's disease. Moreover, a particular stem cell-like CD90(+) subpopulation was identified that might contribute to DD development.

Synthesis and antiproliferative evaluation of some new amidino-substituted bis-benzothiazolyl-pyridines and pyrazine.

Novel diamidino substituted conformationally restricted derivatives of bis-benzothiazolyl-pyridines and pyrazine were synthesized and their antiproliferative activity against several human cancer cell lines were determinated. The synthetic approach used for preparation of isomeric amidinobenzotiazolyl disubstituted pyridines 3a-3k and pyrazine 3l was achieved by condenzation reaction of commercially available pyridine and pyrazine dicarboxylic acids with amidino- 2a and 2-imidazolinyl-substituted 2-aminothiophenol 2b in polyphosphoric acid in moderate to good yield. The condenzation reaction was greatly optimized. The targeted compounds were converted in the desired water soluble dihydrochloride salts by reaction of appropriate free base with concd HCl in ethanol or acetic acid. Antiproliferative assays revealed significant differences in antiproliferative activities of diamidino- and diimidazolinyl-derivatives, the latter exerting stronger concentration-dependent antiproliferative effects on tested tumor cell lines and thus being a prominent compound class for further chemical optimization and biological studies. Biological studies on SW620 cell line and BJ fibroblasts performed for the diimidazolinyl-derivative 3b revealed oxidative stress as a possible mechanism of antiproliferative action and predicted antineoplastic properties for this class of compounds.

In vivo assessment of DNA damage induced in oral mucosa cells by fixed and removable metal prosthodontic appliances.

Given long-term effect on oral tissues due to contact with dental appliances, the biocompatibility studies of casting alloys are of great importance. It has been previously documented that metal dental appliances, due to corrosion, might induce genotoxic and mutagenic effects in cells. Therefore, the aim of presented study was to examine the genotoxicity of two dental casting alloys (Co-Cr-Mo and Ni-Cr) commonly used in fixed and removable prosthodontic appliances that are in contact with the oral epithelium for 5 years or more. For that purpose, 55 age-matched subjects were included in the study; 30 wearers of prosthodontic appliances and 25 controls. Buccal cells of oral mucosa were collected and processed for further analysis. The cell viability has been assessed by trypan blue exclusion test, while genotoxic effect of metal ions on DNA in oral mucosa cells was studied by use of alkaline comet assay. Results have shown significantly higher comet assay parameters (tail length and percentage DNA in the tail) in the group wearing metal appliances. Both subjects with Co-Cr-Mo alloy and Ni-Cr alloy showed significantly higher comet assay parameters when compared with controls. It has been confirmed that metal ions released by the two base metal dental casting alloys examined in this study, might be responsible for DNA damage of oral mucosa cells. Therefore, the results of this study emphasize the importance of the in vivo evaluation of dental materials with respect to their genotoxicity, which is of major importance to ensure long-term biocompatibility.

In vivo toxicity study of N-1-sulfonylcytosine derivatives and their mechanisms of action in cervical carcinoma cell line.

New N-1-sulfonylpyrimidines showed potent growth inhibitory activity against human and mouse tumour cells of different origin. 1-(p-toluenesulfonyl)cytosine (TsC) and 1-(p-toluenesulfonyl)cytosine hydrochloride (TsC × HCl) inhibited the growth of human cervical carcinoma cells (HeLa), and had no significant cytotoxic effects on normal human foreskin fibroblasts (BJ). TsC and TsC × HCl interfered with the HeLa cell cycle progression bringing about the accumulation of G1 phase cells and the induction of apoptosis. Antiproliferative effects of TsC and TsC × HCl were additionally confirmed by investigating de novo synthesis of RNA, DNA and proteins in HeLa cells. Monitoring gene expression using DNA Chip Analysis and quantitative PCR showed that TsC × HCl affects the expression of several cell-cycle regulating genes implying that cell cycle arrest and DNA damage-induced apoptosis might account for the observed cellular effects. In vivo experiments revealed low toxicity of TsC × HCl, as demonstrated by unaltered haematological and metabolic blood parameters. In conclusion, potent antitumour efficacy and low toxicity of new compounds in comparison with the common chemotherapy drug 5-FU make them promising anticancer agents. Additional pre-clinical and clinical studies are warranted to illuminate the mode of action of these newly synthesized compounds in vivo, which would lay the groundwork for their further optimization.

Novel imidazo[4,5-b]pyridine and triaza-benzo[c]fluorene derivatives: synthesis, antiproliferative activity and DNA binding studies.

In the present paper, we have described the synthesis and biological activity of the novel derivatives of imidazo[4,5-b]pyridines and triaza-benzo[c]fluorenes (7-21, 24-26, 28-29). A preponderance of these compounds exerted strong cytostatic effects on the panel of seven human tumour cell lines in a dose-dependent manner. In particular, imidazo[4,5-b]pyridines and triaza-benzo[c]fluorenes including 2-imidazolinyl derivatives showed the most potent antitumour activity. Similarly, triaza-benzo[c]fluorenes 18 and 20 induced strong growth inhibition of tested tumour cell lines, and showed low cytotoxicity in normal human fibroblasts. DNA interaction studies of these compounds demonstrated that N-methylated 16 and 2-imidazolinyl 28 triaza-benzo[c]fluorenes bind to DNA in an intercalative mode.

Integrated gene networks in breast cancer development.

Breast cancer is a complex and heterogenous disease. Classical molecular medical approaches cannot fully understand and comprehend its pathogenesis. In this review, the development of new biological markers for the early detection and creation of guided and specific therapy of breast cancer are discussed in light of the rapid advances in the "omics". Results of cancer research in combination with large-scale methods that examine the expression status of genes and proteins have identified a large number of new biomarkers as well as confirmed the human growth hormone as an important player in the pathogenesis of this disease through its autocrine regulation where it influences the activation of Pax5 and HOXA1 gene networks.

Novel diamidino-substituted derivatives of phenyl benzothiazolyl and dibenzothiazolyl furans and thiophenes: synthesis, antiproliferative and DNA binding properties.

A series of new diamidino-, diisopropylamidino-, and diimidazolinyl-substituted derivatives of phenyl benzothiazolyl and dibenzothiazolyl furans and thiophenes were successfully prepared and evaluated for their antiproliferative activity on tumor cell lines in vitro, DNA binding propensity, and sequence selectivity as well as cellular distribution. A strong antiproliferative effect of the tested compounds was observed on all tested cell lines in a concentration-dependent response pattern. In general, imidazolinyl-substituted derivatives and/or the thiophene core were in correlation with increased antiproliferative activity. Two compounds (2b and 3b) were chosen for biological studies due to their differential antiproliferative properties. The DNA binding properties of this new series of compounds were assessed and evidenced their efficient minor groove binding properties with preferential interaction at AT-rich sites. Both compounds also present nuclear subcellular localization, suggesting that their cellular mode of action implies localization in the DNA compartment and direct inhibition of DNA replication and induction of apoptosis.