Isolation and in vitro characterization of novel S. epidermidis phages for therapeutic applications.

S. epidermidis is an important opportunistic pathogen causing chronic prosthetic joint infections associated with biofilm growth. Increased tolerance to antibiotic therapy often requires prolonged treatment or revision surgery. Phage therapy is currently used as compassionate use therapy and continues to be evaluated for its viability as adjunctive therapy to antibiotic treatment or as an alternative treatment for infections caused by S. epidermidis to prevent relapses. In the present study, we report the isolation and in vitro characterization of three novel lytic S. epidermidis phages. Their genome content analysis indicated the absence of antibiotic resistance genes and virulence factors. Detailed investigation of the phage preparation indicated the absence of any prophage-related contamination and demonstrated the importance of selecting appropriate hosts for phage development from the outset. The isolated phages infect a high proportion of clinically relevant S. epidermidis strains and several other coagulase-negative species growing both in planktonic culture and as a biofilm. Clinical strains differing in their biofilm phenotype and antibiotic resistance profile were selected to further identify possible mechanisms behind increased tolerance to isolated phages.

Supplementation with >Your< Iron Syrup Corrects Iron Status in a Mouse Model of Diet-Induced Iron Deficiency.

The objective of this study was to compare the effects of >Your< Iron Syrup, a novel oral liquid iron-containing food supplement, with the commonly prescribed iron sulphate (Fe-sulphate) in a mouse model of diet-induced iron deficiency. Standard inbred BALB/cOlaHsd mice were fed low-iron diet for 11 weeks to induce significant decrease in blood haemoglobin and haematocrit and were then supplemented by gavage with either >Your< Iron Syrup or Fe-sulphate for two weeks. In >Your< Iron Syrup group, several markers of iron deficiency, such as serum iron concentration, transferrin saturation and ferritin level were significantly improved in both female and male mice. Fe-sulphate induced similar responses, except that it did not significantly increase iron serum in females and serum ferritin in both sexes. Fe-sulphate significantly increased liver-iron content which >Your< Iron Syrup did not. Transcription of Hamp and selected inflammatory genes in the liver was comparable between the two supplementation groups and with the Control diet group. Some sex-specific effects were noted, which were more pronounced and less variable in males. In conclusion, >Your< Iron Syrup was efficient, comparable and in some parameters superior to Fe-sulphate in improving iron-related parameters without inducing a response of selected liver inflammation markers in a mouse model of diet-induced iron deficiency.

Transgenic Mouse Models in Cancer Research.

The use of existing mouse models in cancer research is of utmost importance as they aim to explore the casual link between candidate cancer genes and carcinogenesis as well as to provide models to develop and test new therapies. However, faster progress in translating mouse cancer model research into the clinic has been hampered due to the limitations of these models to better reflect the complexities of human tumors. Traditionally, immunocompetent and immunodeficient mice with syngeneic and xenografted tumors transplanted subcutaneously or orthotopically have been used. These models are still being widely employed for many different types of studies, in part due to their widespread availability and low cost. Other types of mouse models used in cancer research comprise transgenic mice in which oncogenes can be constitutively or conditionally expressed and tumor-suppressor genes silenced using conventional methods, such as retroviral infection, microinjection of DNA constructs, and the so-called "gene-targeted transgene" approach. These traditional transgenic models have been very important in studies of carcinogenesis and tumor pathogenesis, as well as in studies evaluating the development of resistance to therapy. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing approach has revolutionized the field of mouse cancer models and has had a profound and rapid impact on the development of more effective systems to study human cancers. The CRISPR/Cas9-based transgenic models have the capacity to engineer a wide spectrum of mutations found in human cancers and provide solutions to problems that were previously unsolvable. Recently, humanized mouse xenograft models that accept patient-derived xenografts and CD34+ cells were developed to better mimic tumor heterogeneity, the tumor microenvironment, and cross-talk between the tumor and stromal/immune cells. These features make them extremely valuable models for the evaluation of investigational cancer therapies, specifically new immunotherapies. Taken together, improvements in both the CRISPR/Cas9 system producing more valid mouse models and in the humanized mouse xenograft models resembling complex interactions between the tumor and its environment might represent one of the successful pathways to precise individualized cancer therapy, leading to improved cancer patient survival and quality of life.

The decalog of long non-coding RNA involvement in cancer diagnosis and monitoring.

Long non-coding RNAs (lncRNAs) are transcripts without protein-coding capacity; initially regarded as "transcriptional noise", lately they have emerged as essential factors in both cell biology and mechanisms of disease. In this article, we present basic knowledge of lncRNA molecular mechanisms, associated physiological processes and cancer association, as well as their diagnostic and therapeutic value in the form of a decalog: (1) Non-coding RNAs (ncRNAs) are transcripts without protein-coding capacity divided by size (short and long ncRNAs), function (housekeeping RNA and regulatory RNA) and direction of transcription (sense/antisense, bidirectional, intronic and intergenic), containing a broad range of molecules with diverse properties and functions, such as messenger RNA, transfer RNA, microRNA and long non-coding RNAs. (2) Long non-coding RNAs are implicated in many molecular mechanisms, such as transcriptional regulation, post-transcriptional regulation and processing of other short ncRNAs. (3) Long non-coding RNAs play an important role in many physiological processes such as X-chromosome inactivation, cell differentiation, immune response and apoptosis. (4) Long non-coding RNAs have been linked to hallmarks of cancer: (a) sustaining proliferative signaling; (b) evading growth suppressors; (c) enabling replicative immortality; (d) activating invasion and metastasis; (e) inducing angiogenesis; (f) resisting cell death; and (g) reprogramming energy metabolism. (5) Regarding their impact on cancer cells, lncRNAs are divided into two groups: oncogenic and tumor-suppressor lncRNAs. (6) Studies of lncRNA involvement in cancer usually analyze deregulated expression patterns at the RNA level as well as the effects of single nucleotide polymorphisms and copy number variations at the DNA level. (7) Long non-coding RNAs have potential as novel biomarkers due to tissue-specific expression patterns, efficient detection in body fluids and high stability. (8) LncRNAs serve as novel biomarkers for diagnostic, prognostic and monitoring purposes. (9) Tissue specificity of lncRNAs enables the development of selective therapeutic options. (10) Long non-coding RNAs are emerging as commercial biomarkers and therapeutic agents.

Conformationally constrained lipid A mimetics for exploration of structural basis of TLR4/MD-2 activation by lipopolysaccharide.

Recognition of the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, by the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD-2) complex is essential for the control of bacterial infection. A pro-inflammatory signaling cascade is initiated upon binding of membrane-associated portion of LPS, a glycophospholipid Lipid A, by a coreceptor protein MD-2, which results in a protective host innate immune response. However, activation of TLR4 signaling by LPS may lead to the dysregulated immune response resulting in a variety of inflammatory conditions including sepsis syndrome. Understanding of structural requirements for Lipid A endotoxicity would ensure the development of effective anti-inflammatory medications. Herein, we report on design, synthesis, and biological activities of a series of conformationally confined Lipid A mimetics based on ß,α-trehalose-type scaffold. Replacement of the flexible three-bond ß(1→6) linkage in diglucosamine backbone of Lipid A by a two-bond ß,α(1↔1) glycosidic linkage afforded novel potent TLR4 antagonists. Synthetic tetraacylated bisphosphorylated Lipid A mimetics based on a ß-GlcN(1↔1)α-GlcN scaffold selectively block the LPS binding site on both human and murine MD-2 and completely abolish lipopolysaccharide-induced pro-inflammatory signaling, thereby serving as antisepsis drug candidates. In contrast to their natural counterpart lipid IVa, conformationally constrained Lipid A mimetics do not activate mouse TLR4. The structural basis for high antagonistic activity of novel Lipid A mimetics was confirmed by molecular dynamics simulation. Our findings suggest that besides the chemical structure, also the three-dimensional arrangement of the diglucosamine backbone of MD-2-bound Lipid A determines endotoxic effects on TLR4.

Chimeric flagellin as the self-adjuvanting antigen for the activation of immune response against Helicobacter pylori.

Helicobacter pylori infection can cause gastritis, peptic ulcer and can lead to gastric cancer. Lengthy antibiotic therapy does not protect the host against reinfection. H. pylori evolved to evade the recognition of the immune response by modifying several of its components whose orthologous proteins from other bacteria activate the innate immune response. Flagella are essential for the H. pylori effective colonization of human duodenum and stomach. TLR5, a member of the Toll-like receptor family, recognizes flagellin of most bacteria, such as Escherichia coli, but does not recognize the flagellin FlaA of H. pylori. We restored the ability of FlaA for the recognition by TLR5 by engineering a chimeric flagellin, in which both terminal segments of H. pylori flagellin were replaced by the corresponding segments from TLR5-activating E. coli flagellin. Recombinant chimeric flagellin folded correctly and was able to activate TLR5. Significantly increased serum IgG and IgA antibody responses were determined in mice vaccinated with chimeric flagellin in comparison to mice vaccinated with a control protein (FlaA) or negative control. Antibody titers remained high even 8 months after the last immunization. Antibodies were able to bind native flagellin from H. pylori lysate. Vaccination with chimeric flagellin provided mice with significant protection against H. pylori. The approach of chimeric flagellin can therefore generate effective immunogens that enable activation of innate and adaptive immune response and can be used to construct efficient vaccines against H. pylori or other flagellated bacteria that evade TLR5 recognition.

Cross talk between microRNA and coding cancer genes.

MicroRNAs (miRNAs) are a class of noncoding RNAs (ncRNAs) and posttranscriptional gene regulators shown to be involved in pathogenesis of all types of human cancers. Their aberrant expression as tumor suppressors can lead to cancerogenesis by inhibiting malignant potential, or when acting as oncogenes, by activating malignant potential. Differential expression of miRNA genes in tumorous tissues can occur owing to several factors including positional effects when mapping to cancer-associated genomic regions, epigenetic mechanisms, and malfunctioning of the miRNA processing machinery, all of which can contribute to a complex miRNA-mediated gene network misregulation. They may increase or decrease expression of protein-coding genes, can target 3'-UTR or other genic regions (5'-UTR, promoter, coding sequences), and can function in various subcellular compartments, developmental, and metabolic processes. Because expanding research on miRNA-cancer associations has already produced large amounts of data, our main objective here was to summarize main findings and critically examine the intricate network connecting the miRNAs and coding genes in regulatory mechanisms and their function and phenotypic consequences for cancer. By examining such interactions, we aimed to gain insights for the development of new diagnostic markers as well as identification of potential venues for more selective tumor therapy. To enable efficient examination of the main past and current miRNA discoveries, we developed a Web-based miRNA timeline tool that will be regularly updated (http://www.integratomics-time.com/miRNA_timeline). Further development of this tool will be directed at providing additional analyses to clarify complex network interactions between miRNAs, other classes of ncRNAs, and protein-coding genes and their involvement in development of diseases including cancer. This tool therefore provides curated relevant information about the miRNA basic research and therapeutic application all at hand on one site to help researchers and clinicians in making informed decision about their miRNA cancer-related research or clinical practice.

Catalog of microRNA seed polymorphisms in vertebrates.

MicroRNAs (miRNAs) are a class of non-coding RNA that plays an important role in posttranscriptional regulation of mRNA. Evidence has shown that miRNA gene variability might interfere with its function resulting in phenotypic variation and disease susceptibility. A major role in miRNA target recognition is ascribed to complementarity with the miRNA seed region that can be affected by polymorphisms. In the present study, we developed an online tool for the detection of miRNA polymorphisms (miRNA SNiPer) in vertebrates (http://www.integratomics-time.com/miRNA-SNiPer) and generated a catalog of miRNA seed region polymorphisms (miR-seed-SNPs) consisting of 149 SNPs in six species. Although a majority of detected polymorphisms were due to point mutations, two consecutive nucleotide substitutions (double nucleotide polymorphisms, DNPs) were also identified in nine miRNAs. We determined that miR-SNPs are frequently located within the quantitative trait loci (QTL), chromosome fragile sites, and cancer susceptibility loci, indicating their potential role in the genetic control of various complex traits. To test this further, we performed an association analysis between the mmu-miR-717 seed SNP rs30372501, which is polymorphic in a large number of standard inbred strains, and all phenotypic traits in these strains deposited in the Mouse Phenome Database. Analysis showed a significant association between the mmu-miR-717 seed SNP and a diverse array of traits including behavior, blood-clinical chemistry, body weight size and growth, and immune system suggesting that seed SNPs can indeed have major pleiotropic effects. The bioinformatics analyses, data and tools developed in the present study can serve researchers as a starting point in testing more targeted hypotheses and designing experiments using optimal species or strains for further mechanistic studies.

The role of intermediary domain of MyD88 in cell activation and therapeutic inhibition of TLRs.

Adaptor MyD88 has a pivotal role in TLR and IL-1R signaling and is involved in mediating excessive inflammation. MyD88 is composed of a death domain and a Toll/IL-1R domain connected by an intermediary domain (INT). The alternatively spliced form of MyD88 lacking the INT prevents signaling through MyD88-dependent TLRs. We designed a peptide from the INT and showed that it inhibits TLR4 activation by LPS when linked to a cell-penetrating peptide. As a new approach for the delivery of signaling-inhibitory peptides, INT peptide acylation also provided efficient cell translocation and inhibition of activation. We determined that INT peptide targets IL-1R-associated kinase 4. Furthermore, MyD88 mutant and molecular modeling refines the MyD88- IL-1R-associated kinase 4 interaction model based on the Myddosome structure. In addition to TLR4, INT peptide also inhibited TLR5, TLR2, TLR9, and IL-1R signaling but not TLR3, which uses Toll/IL-1R domain-containing adapter inducing IFN-ß signaling adaptor. Inhibition of signaling in murine and human cells was observed by decreased NF-κB activation, cytokine mRNA synthesis, and phosphorylation of downstream kinases. In the endotoxemic mouse model, INT peptide suppressed production of inflammatory cytokines and improved survival, supporting therapeutic application of INT peptides for the suppression of inflammatory conditions mediated by MyD88.

Epigenetic regulation of microRNAs in cancer: an integrated review of literature.

MicroRNAs (miRNAs) belong to the heterogeneous class of non-coding RNAs (ncRNAs) that regulate the translation and degradation of target mRNAs, and control approximately 30% of human genes. MiRNA genes might be silenced in human tumors (oncomiRs) by aberrant hypermethylation of CpG islands that encompass or lie adjacent to miRNA genes and/or by histone modifications. We performed literature search for research articles describing epigenetically regulated miRNAs in cancer and identified 45 studies that were published between 2006 and 7/2010. The data from those papers are fragmented and methodologically heterogeneous and our work represents first systematic review towards to integration of diverse sets of information. We reviewed the methods used for detection of miRNA epigenetic regulation, which comprise bisulfite genomic sequencing PCR (BSP), bisulfite pyrosequencing, methylation specific PCR (MSP), combined bisulfite restriction analysis (COBRA), methylation sensitive single nucleotide primer extension (Ms-SNuPE), MassARRAY technique and some modifications of those methods. This integrative study revealed 122 miRNAs that were reported to be epigenetically regulated in 23 cancer types. Compared to protein coding genes, human oncomiRs showed an order of magnitude higher methylation frequency (11.6%; 122/1048 known miRNAs). Nearly half, (45%; 55/122) epigenetically regulated miRNAs were associated with different cancer types, but other 55% (67/122) miRNAs were present in only one cancer type and therefore representing cancer-specific biomarker potential. The data integration revealed miRNA epigenomic hot spots on the chromosomes 1q, 7q, 11q, 14q and 19q. CpG island analysis of corresponding miRNA precursors (pre-miRNAs) revealed that 20% (26/133) of epigenetically regulated miRNAs had a CpG island within the range of 5kb upstream, among them 14% (19/133) of miRNAs resided within the CpG island. Our integrative survey and analyses revealed candidate cancer-specific miRNA epigenetic signatures which provide the basis for new therapeutic strategies in cancer by targeting the epigenetic regulation of miRNAs.

Defects in cholesterol synthesis genes in mouse and in humans: lessons for drug development and safer treatments.

This review describes the mouse knockout models of cholesterol synthesis, together with human malformations and drugs that target cholesterogenic enzymes. Generally, the sooner a gene acts in cholesterol synthesis, the earlier the phenotype occurs. Humans with loss of function of early cholesterogenic enzymes have not yet been described, and in the mouse, loss of Hmgcr is preimplantation lethal. Together, these results indicate that the widely prescribed cholesterol-lowering statins are potentially teratogenic. The Mvk knockout is early embryonic lethal in the mouse, the absence of Fdft1 is lethal at E9.5-12.5 dpc, while the Cyp51 knockouts die at 15.0 dpc. Fungal CYP51 inhibitor azoles are teratogenic in humans, potentially leading to symptoms of Antley-Bixler syndrome. The X-linked mutations in Nsdhl and Ebp are embryonic lethal in male mice, while heterozygous females are also affected. Consequently, the anticancer drugs, tamoxifen and toremifene, inhibiting human EBP, may be harmful in early pregnancy. The Dhcr7 and Dhcr24 knockout mice die shortly after birth, while humans survive with Smith-Lemli-Opitz syndrome or desmosterolosis. Since cholesterol is essential for hedgehog signaling, disturbance of this pathway by antipsychotics and -depressants explains some drug side effects. In conclusion, defects in cholesterol synthesis are generally lethal in mice, while humans with impaired later steps of the pathway can survive with severe malformations. Evidence shows that drugs targeting or, by coincidence, inhibiting human cholesterol synthesis are better avoided in early pregnancy. Since some drugs with teratogenic potential still stay on the market, this should be avoided in new cholesterol-related drug development.

Divergent physical activity and novel alternative responses to high fat feeding in polygenic fat and lean mice.

We determined whether altered physical activity levels might underlie the contrasting adiposity of a divergently selected polygenic murine model of metabolic syndrome (Fat; F) and leanness (Lean; L) mice. We measured physical activity with a long term running wheel experiment and performed an additional high fat diet intervention. Further, we measured posture allocation by visual monitoring within the home cage as a non-exercise correlate of 'normal' physical activity. Whilst initially similar, running wheel activity of the F line declined with age, while the activity of the L line increased. Food intake was higher in the L line and increased with wheel exposure. Vertical rearing measured by video quantification in the home cage, without the stimulus of a running wheel was also significantly higher in the L line. The two lines developed novel alternate strategies to defend their body weight when exposed to high fat diets with a running wheel. F mice increased their running wheel activity, and despite unaltered food intake, still gained weight. L mice reduced their food intake and maintained activity levels without a significant change in body weight. Phenotypic selection for divergence in body fat content has co-segregated with a genetic predisposition for divergent physical activity levels and different strategies for coping with exposure to high fat diets that will facilitate the discovery of the genes underlying these important obesity related traits.