Multiple Delivery of siRNA against Endoglin into Murine Mammary Adenocarcinoma Prevents Angiogenesis and Delays Tumor Growth

Endoglin is a transforming growth factor-β (TGF- β) co-receptor that participates in the activation of a signaling pathway that mediates endothelial cell proliferation and migration in angiogenic tumor vasculature. Therefore, silencing of endoglin expression is an attractive approach for antiangiogenic therapy of tumors. The aim of our study was to evaluate the therapeutic potential of small interfering RNA (siRNA) molecules against endoglin in vitro and in vivo. Therapeutic potential in vitro was assessed in human and murine endothelial cells (HMEC-1, 2H11) by determining endoglin expression level, cell proliferation and tube formation. In vivo, the therapeutic potential of siRNA molecules was evaluated in TS/A mammary adenocarcinoma growing in BALB/c mice. Results of our study showed that siRNA molecules against endoglin have a good antiangiogenic therapeutic potential in vitro, as expression of endoglin mRNA and protein levels in mouse and human microvascular endothelial cells after lipofection were efficiently reduced, which resulted in the inhibition of endothelial cell proliferation and tube formation. In vivo, silencing of endoglin with triple electrotransfer of siRNA molecules into TS/A mammary adenocarcinoma also significantly reduced the mRNA levels, number of tumor blood vessels and the growth of tumors. The obtained results demonstrate that silencing of endoglin is a promising antiangiogenic therapy of tumors that could not be used as single treatment, but as an adjunct to the established cytotoxic treatment approaches.     

TRPV1 antagonist with high analgesic efficacy: 2-Thio pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides

A series of 2-thio pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. Among them, compound 24S showed stereospecific and excellent TRPV1 antagonism of capsaicin-induced activation. Further, it demonstrated strong anti-allodynic in a rat neuropathic pain model. Consistent with its action in vitro being through TRPV1, compound 24S blocked capsaicin-induced hypothermia in mice. Docking analysis of 24S with our hTRPV1 homology model was performed to identify its binding mode.     

Small within-day increases in temperature affects boldness and alters personality in coral reef fish

Consistent individual differences in behaviour, termed personality, are common in animal populations and can constrain their responses to ecological and environmental variation, such as temperature. Here, we show for the first time that normal within-daytime fluctuations in temperature of less than 3°C have large effects on personality for two species of juvenile coral reef fish in both observational and manipulative experiments. On average, individual scores on three personality traits (PTs), activity, boldness and aggressiveness, increased from 2.5- to sixfold as a function of temperature. However, whereas most individuals became more active, aggressive and bold across temperature contexts (were plastic), others did not; this changed the individual rank order across temperatures and thus altered personality. In addition, correlations between PTs were consistent across temperature contexts, e.g. fish that were active at a given temperature also tended to be both bold and aggressive. These results (i) highlight the importance of very carefully controlling for temperature when studying behavioural variation among and within individuals and (ii) suggest that individual differences in energy metabolism may contribute to animal personality, given that temperature has large direct effects on metabolic rates in ectotherms.     

Deletion 17q12 is a recurrent copy number variant that confers high risk of autism and schizophrenia

Autism spectrum disorders (ASD) and schizophrenia are neurodevelopmental disorders for which recent evidence indicates an important etiologic role for rare copy number variants (CNVs) and suggests common genetic mechanisms. We performed cytogenomic array analysis in a discovery sample of patients with neurodevelopmental disorders referred for clinical testing. We detected a recurrent 1.4 Mb deletion at 17q12, which harbors HNF1B, the gene responsible for renal cysts and diabetes syndrome (RCAD), in 18/15,749 patients, including several with ASD, but 0/4,519 controls. We identified additional shared phenotypic features among nine patients available for clinical assessment, including macrocephaly, characteristic facial features, renal anomalies, and neurocognitive impairments. In a large follow-up sample, the same deletion was identified in 2/1,182 ASD/neurocognitive impairment and in 4/6,340 schizophrenia patients, but in 0/47,929 controls (corrected p = 7.37 × 10⁻⁵). These data demonstrate that deletion 17q12 is a recurrent, pathogenic CNV that confers a very high risk for ASD and schizophrenia and show that one or more of the 15 genes in the deleted interval is dosage sensitive and essential for normal brain development and function. In addition, the phenotypic features of patients with this CNV are consistent with a contiguous gene syndrome that extends beyond RCAD, which is caused by HNF1B mutations only.     

Generation of bioactive peptides by biological libraries

Biological libraries are powerful tools for discovery of new ligands as well as for identification of cellular interaction partners. Since the first development of the first biological libraries in form of phage displays, numerous biological libraries have been developed. For the development of new ligands, the usage of synthetic oligonucleotides is the method of choice. Generation of random oligonucleotides has been refined and various strategies for random oligonucleotide design were developed. We trace the progress and design of new strategies for the generation of random oligonucleotides, and include a look at arising diversity biases. On the other hand, genomic libraries are widely employed for investigation of cellular protein-protein interactions and targeted search of proteomic binding partners. Expression of random peptides and proteins in a linear form or integrated in a scaffold can be facilitated both in vitro and in vivo. A typical in vitro system, ribosome display, provides the largest available library size. In vivo methods comprise smaller libraries, the size of which depends on their transformation efficiency. Libraries in different hosts such as phage, bacteria, yeast, insect cells, mammalian cells exhibit higher biosynthetic capabilities. The latest library systems are compared and their strengths and limitations are reviewed.     

Contribution of Copper Ion Resistance to Survival of Escherichia coli on Metallic Copper Surfaces

Bacterial contamination of touch surfaces poses a serious threat for public health. The use of bactericidal surface materials, such as copper and its alloys, might constitute a way to aid the use of antibiotics and disinfectants, thus minimizing the risk of emergence and spread of multiresistant germs. The survival of Escherichia coli on metallic copper surfaces has been studied previously; however, the mechanisms underlying bacterial inactivation on copper surfaces have not been elucidated. Data presented in this study suggest that bacteria are killed rapidly on dry copper surfaces. Several factors, such as copper ion toxicity, copper chelators, cold, osmotic stress, and reactive oxygen species, but not anaerobiosis, influenced killing rates. Strains deleted in copper detoxification systems were slightly more sensitive than was the wild type. Preadaptation to copper enhanced survival rates upon copper surface exposure. This study constitutes a first step toward understanding the reasons for metallic copper surface-mediated killing of bacteria.     

Thrombin inhibitors with novel P1 binding pocket functionality: free energy of binding analysis

The high incidence of thrombembolic diseases justifies the development of new antithrombotics. The search for a direct inhibitor has resulted in the synthesis of a considerable number of low molecular weight molecules that inhibit human α-thrombin potently. However, efforts to develop an orally active drug remain in progress as the most active inhibitors with a highly basic P1 moiety exhibit an unsatisfactory bioavailability profile. In our previous work we solved several X-ray structures of human α-thrombin in complexes with (1) novel bicyclic arginine mimetics attached to the glycylproline amide and pyridinone acetamide scaffold and (2) inhibitors with a novel aza scaffold and with charged or neutral P1 moieties. In the present contribution, we correlate the structures of the complex between these inhibitors and the protein with the calculated free energy of binding. The energy of solvation was calculated using the Poisson–Boltzmann approach. In particular, the requirements for successful recognition of an inhibitor at the protein’s active site pocket S1 are discussed. Figure We report here on free energy of binding analysis of thrombin inhibitors with novel aza scaffold and novel bicyclic arginine mimetics in S1 pocket of thrombin     

Development of experimental genetic tools for Campylobacter fetus

Molecular analysis of the virulence mechanisms of the emerging pathogen Campylobacter fetus has been hampered by the lack of genetic tools. We report the development and functional analysis of Escherichia coli-Campylobacter shuttle vectors that are appropriate for C. fetus. Some vectors were constructed based on the known Campylobacter coli plasmid pIP1455 replicon, which confers a wide host range in Campylobacter spp. Versatility in directing gene expression was achieved by introducing a strong C. fetus promoter. The constructions carry features necessary and sufficient to detect the expression of phenotypic markers, including molecular reporter genes in both subspecies of C. fetus, while retaining function in C. jejuni. The capacity to express several gene products from different vectors in a single host can be advantageous but requires distinct plasmid replicons. To this end, replication features derived from a cryptic plasmid of C. fetus subsp. venerealis strain 4111/108, designated pCFV108, were adapted for a compatible series of constructions. The substitution of the C. coli replication elements reduced vector size while apparently limiting the host range to C. fetus. The complementation of a ciprofloxacin-resistant mutant phenotype via vector-driven gyrA expression was verified. Cocultivation demonstrated that shuttle vectors based on the pCFV108 replicon were compatible with pIP1455 replication functions, and the stable maintenance of two plasmids in a C. fetus subsp. venerealis host over several months was observed. The application of both vector types will facilitate the investigation of the genetics and cellular interactions of the emerging pathogen C. fetus.     

The impact of a boosting immunogen on the differentiation of secondary memory CD8+ T cells

While recent studies have demonstrated that secondary CD8⁺ T cells develop into effector-memory cells, the impact of particular vaccine regimens on the elicitation of these cells remains poorly defined. In the present study we evaluated the effect of three different immunogens—recombinant vaccinia, recombinant adenovirus, and plasmid DNA—on the generation of memory cellular immune responses. We found that vectors that induce the rapid movement of CD8⁺ T cells into the memory compartment during a primary immune response also drive a rapid differentiation of these cells into effector-memory CD8⁺ T cells following a secondary immunization. In contrast, the functional profiles of both CD8⁺ and CD4⁺ T cells, assessed by measuring antigen-stimulated gamma interferon and interleukin-2 production, were not predominantly shaped by the boosting immunogen. We also demonstrated that the in vivo expression of antigen by recombinant vectors was brief following boosting immunization, suggesting that antigen persistence has a minimal impact on the differentiation of secondary CD8⁺ T cells. When used in heterologous or in homologous prime-boost combinations, these three vectors generated antigen-specific CD8⁺ T cells with different phenotypic profiles. Expression of the memory-associated molecule CD27 on effector CD8⁺ T cells decreased following heterologous but not homologous boosting, resulting in a phenotypic profile similar to that seen on primary CD8⁺ T cells. These data therefore suggest that the phenotype of secondary CD8⁺ T cells is determined predominantly by the boosting immunogen whereas the cytokine profile of these cells is shaped by both the priming and boosting immunogens.     

Biological Flora of Central Europe: Baldellia ranunculoides (Alismataceae)

Baldellia ranunculoides (L.) Parl. (Alismataceae) is a taxonomically problematic aquatic plant with an historically ill-defined distribution and global conservation status. This paper finds morphological, ecological and molecular evidence for two distinct taxa, probably best described as subspecies: (1) B. ranunculoides subsp. ranunculoides and (2) B. ranunculoides subsp. repens and provides detailed distribution data on their overlapping range, in different habitats, across the cool, high rainfall areas of western Europe and west Mediterranean. The two subspecies are amongst the relatively large number of threatened European and north Africa aquatic plants and this paper provides a systematic review of their relative conservation pressures and management needs, with particular emphasis on the status of both taxa in central Europe. Other observations indicate probable evolutionary relationships within B. ranunculoides s.l. and its associated taxa and the review points out where these and other research topics could potentially be pursued.