Antitumor activity of opiorphin,sialorphin and their conjugates with a peptide klaklakklaklak

This is the study on the effect of opiorphin, sialorphin and their analogs on antitumor activity. We demonstrated that conjugation of opiorphin and sialorphin with a proapoptotic, antimicrobial peptide klak (klaklakklaklak) led to compounds (opio‐klak and sialo‐klak) that were cytotoxic against cancer cells (LN18, PC3, A549, HCT116 and B10‐F16) in the MTT test. The conjugated analogs were designed to increase the effectiveness of the peptide. The opio‐klak derivative was the most effective in the in vitro assays and led to a decrease in viability of cancer cells over time as compared with that of untreated controls. In contrast, treatment with either the untargeted klak peptide or opiorphin as a negative control led to a negligible loss in viability. Antitumor effect of the opio‐klak was also observed in vivo in murine melanoma tumor‐bearing mice. Cessation of peptide administration resulted in tumor regrowth. Our results are seemingly valuable for the development of opiorphin analogs with potential clinical applications. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Expression of cagA,virB/D Complex and/or vacA Genes in Helicobacter pylori Strains Originating from Patients with Gastric Diseases

In order to better understand pathogenicity of Helicobacter pylori, particularly in the context of its carcinogenic activity, we analysed expression of virulence genes: cagA, virB/D complex (virB4, virB7, virB8, virB9, virB10, virB11, virD4) and vacA in strains of the pathogen originating from persons with gastric diseases. The studies were conducted on 42 strains of H. pylori isolated from patients with histological diagnosis of non-atrophic gastritis—NAG (group 1, including subgroup 1 containing cagA⁺ isolates and subgroup 2 containing cagA^- strains), multifocal atrophic gastritis—MAG (group 2) and gastric adenocarcinoma—GC (group 3). Expression of H. pylori genes was studied using microarray technology. In group 1, in all strains of H. pylori cagA⁺ (subgroup 1) high expression of the gene as well as of virB/D was disclosed, accompanied by moderate expression of vacA. In strains of subgroup 2 a moderate expression of vacA was detected. All strains in groups 2 and 3 carried cagA gene but they differed in its expression: a high expression was detected in isolates of group 2 and its hyperexpression in strains of group 3 (hypervirulent strains). In both groups high expression of virB/D and vacA was disclosed. Our results indicate that chronic active gastritis may be induced by both cagA⁺ strains of H. pylori, manifesting high expression of virB/D complex but moderate activity of vacA, and cagA^- strains with moderate expression of vacA gene. On the other hand, in progression of gastric pathology and carcinogenesis linked to H. pylori a significant role was played by hypervirulent strains, manifesting a very high expression of cagA and high activity of virB/D and vacA genes.     

Retrospective analysis of idiopathic scoliosis medical records coming from one out-patient clinic for compatibility with Scoliosis Research Society criteria for brace treatment studies

Background

First author attempted to analyse medical records of patients with idiopathic scoliosis for compliance with the Scoliosis Research Society brace studies criteria. A retrospective analysis of medical records of 2705 girls treated from 1989 to 2002 was carried out.

Methods

Age, Cobb, Risser and menarchal status were analyzed for compliance with the Scoliosis Research Society brace studies criteria: a) age ≥10 years, b) Risser 0–2, c) 25–40° Cobb angle, d) no earlier treatment, e) patients before first menses or not more than one year from first menses.

Results

It has been found that 183 girls out of 2705 were ≥10 years old and in the range 25–40° Cobb angle. One hundred two out of 2705 patients revealed eligible for brace effectiveness study according to SRS 2005 criteria. 120 out of 2705 patients revealed eligible for brace brace effectiveness study according to SRS-SOSORT 2014 criteria.

Conclusion

The excluded patients revealed too old or with too significant Cobb angles. This indicates the changing criteria for scoliosis brace treatment over the time. Direct comparison of current results of brace treatment with historical series of cases turns out to be very difficult.

     

Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens

In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD) or performing size selection of the resulting fragments (in the case of single-digest RAD). Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD). In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites that usually causes loci dropout. This should enable the application of hyRAD to analyses at broader evolutionary scales.     

New Amniotic Membrane Based Biocomposite for Future Application in Reconstructive Urology

Objective

Due to the capacity of the amniotic membrane (Am) to support re-epithelisation and inhibit scar formation, Am has a potential to become a considerable asset for reconstructive urology i.e., reconstruction of ureters and urethrae. The application of Am in reconstructive urology is limited due to a poor mechanical characteristic. Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance, without affecting its unique bioactivity profile. This study evaluated biocomposite material composed of Am and nanofibers as a graft for urinary bladder augmentation in a rat model.

Material and Methods

Sandwich-structured biocomposite material was constructed from frozen Am and covered on both sides with two-layered membranes prepared from electrospun poly-(L-lactide-co-E-caprolactone) (PLCL). Wistar rats underwent hemicystectomy and bladder augmentation with the biocomposite material.

Results

Immunohistohemical analysis (hematoxylin and eosin [H&E], anti-smoothelin and Masson’s trichrome staining [TRI]) revealed effective regeneration of the urothelial and smooth muscle layers. Anti-smoothelin staining confirmed the presence of contractile smooth muscle within a new bladder wall. Sandwich-structured biocomposite graft material was designed to regenerate the urinary bladder wall, fulfilling the requirements for normal bladder tension, contraction, elasticity and compliance. Mechanical evaluation of regenerated bladder wall conducted based on Young’s elastic modulus reflected changes in the histological remodeling of the augmented part of the bladder. The structure of the biocomposite material made it possible to deliver an intact Am to the area for regeneration. An unmodified Am surface supported regeneration of the urinary bladder wall and the PLCL membranes did not disturb the regeneration process.

Conclusions

Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance without affecting its unique bioactivity profile.     

The LA Loop as an Important Regulatory Element of the HtrA (DegP) Protease from Escherichia coli: STRUCTURAL AND FUNCTIONAL STUDIES*

Bacterial HtrAs are serine proteases engaged in extracytoplasmic protein quality control and are required for the virulence of several pathogenic species. The proteolytic activity of HtrA (DegP) from Escherichia coli, a model prokaryotic HtrA, is stimulated by stressful conditions; the regulation of this process is mediated by the LA, LD, L1, L2, and L3 loops. The precise mechanism of action of the LA loop is not known due to a lack of data concerning its three-dimensional structure as well as its mode of interaction with other regulatory elements. To address these issues we generated a theoretical model of the three-dimensional structure of the LA loop as per the resting state of HtrA and subsequently verified its correctness experimentally. We identified intra- and intersubunit contacts that formed with the LA loops; these played an important role in maintaining HtrA in its inactive conformation. The most significant proved to be the hydrophobic interactions connecting the LA loops of the hexamer and polar contacts between the LA′ (the LA loop on an opposite subunit) and L1 loops on opposite subunits. Disturbance of these interactions caused the stimulation of HtrA proteolytic activity. We also demonstrated that LA loops contribute to the preservation of the integrity of the HtrA oligomer and to the stability of the monomer. The model presented in this work explains the regulatory role of the LA loop well; it should also be applicable to numerous Enterobacteriaceae pathogenic species as the amino acid sequences of the members of this bacterial family are highly conserved.     

T4 Phage and Its Head Surface Proteins Do Not Stimulate Inflammatory Mediator Production

Viruses are potent activators of the signal pathways leading to increased cytokine or ROS production. The effects exerted on the immune system are usually mediated by viral proteins. Complementary to the progress in phage therapy practice, advancement of knowledge about the influence of bacteriophages on mammalian immunity is necessary. Particularly, the potential ability of phage proteins to act like other viral stimulators of the immune system may have strong practical implications for the safety and efficacy of bacteriophage therapy. Here we present studies on the effect of T4 phage and its head proteins on production of inflammatory mediators and inflammation-related factors: IL-1α, IL-1β, IL-2, IL-6, IL-10, IL-12 p40/p70, IFN-γ, TNF-α, MCP-1, MIG, RANTES, GCSF, GM-CSF and reactive oxygen species (ROS). Plasma cytokine profiles in an in vivo mouse model and in human blood cells treated with gp23*, gp24*, Hoc and Soc were evaluated by cytokine antibody arrays. Cytokine production and expression of CD40, CD80, CD86 and MHC class II molecules were also investigated in mouse bone marrow-derived dendritic cells treated with whole T4 phage particle or the same capsid proteins. The influence of T4 and gp23*, gp24*, Hoc and Soc on reactive oxygen species generation was examined in blood cells using luminol-dependent chemiluminescence assay. In all performed assays, the T4 bacteriophage and its capsid proteins gp23*, gp24*, Hoc and Soc did not affect production of inflammatory-related cytokines or ROS. These observations are of importance for any medical or veterinary application of bacteriophages.