Epoetin alpha,epoetin beta and darbepoetin alfa: two-dimensional gel electrophoresis isoforms characterization and mass spectrometry analysis

Since 1989 recombinant human erythropoietin (rhEPO) has been used as a drug for the correction of anemia, but the misuse of rhEPO as an ergogenic agent among athletes is a widespread doping practice. As a consequence there is a need for developing reference methods for the detection of rhEPO in biological fluids, and to be able to differentiate the recombinant from the natural protein. Recombinant human erythropoietin differs from its natural counterpart in the glycidic part of the molecule. Three different commercial recombinant products Epoetin alpha (Eprex, Janssen Cilag), Epoetin beta (Neorecormon, Roche) and Darbepoetin alfa (Nespo, Dompè) have been used to evaluate the performance of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) for the separation of isoforms and the identification of the proteins respectively. All the compounds studied were well separated by means of 2-DE: Epoetin alpha and beta focused in the same isoelectric point region giving rise to six and eight spots respectively, whereas Darbepoetin alfa was found in a more acidic zone with two spots. Results obtained with micro high-performance liquid chromatography-electrospray ionization-time of flight (TOF) MS and matrix-assisted laser desorption/ionization-time of flight MS for the three rhEPOs are reported. These preliminary results suggest that by means of 2-DE and MS it should be possible to reveal the presence of rhEPOs for antidoping purposes.     

Analysis of hepatitis C virus hypervariable region 1 sequence from cryoglobulinemic patients and associated controls

Chronic hepatitis C virus (HCV) infection is frequently associated with extrahepatic manifestations, including nonmalignant and malignant B-cell lymphoproliferative disorders. It has been reported that specific changes or recurring motifs in the amino acid sequence of the HCV hypervariable region 1 (HVR1) may be associated with cryoglobulinemia. We searched for specific insertions/deletions and/or amino acid motifs within HVR1 in samples from 80 symptomatic and asymptomatic patients with and 33 patients without detectable cryoglobulins, all with chronic HCV infection. At variance with the results of a previous study which reported a high frequency of insertions at position 385 of HVR1 from cryoglobulinemic patients, we found a 6.2% prevalence of insertions in samples from patients with and a 9.1% prevalence in those without cryoglobulinemia. Moreover, statistical and bioinformatics approaches including Fisher's exact test, k-means clustering, Tree determinant-residue identification, correlation of mutations, principal component analysis, and phylogenetic analysis failed to show statistically significant differences between sequences from cryoglobulin-negative and -positive patients. Our findings suggest that cryoglobulinemia may arise by virtue of as-yet-unidentified host- rather than virus-specific factors. Specific changes in HCV envelope sequence distribution are unlikely to be directly involved in the establishment of pathological B-cell monoclonal proliferation.     

Genome mining for natural product biosynthetic gene clusters in the Subsection V cyanobacteria

Background

Cyanobacteria are well known for the production of a range of secondary metabolites. Whilst recent genome sequencing projects has led to an increase in the number of publically available cyanobacterial genomes, the secondary metabolite potential of many of these organisms remains elusive. Our study focused on the 11 publically available Subsection V cyanobacterial genomes, together with the draft genomes of Westiella intricata UH strain HT-29-1 and Hapalosiphon welwitschii UH strain IC-52-3, for their genetic potential to produce secondary metabolites. The Subsection V cyanobacterial genomes analysed in this study are reported to produce a diverse range of natural products, including the hapalindole-family of compounds, microcystin, hapalosin, mycosporine-like amino acids and hydrocarbons.

Results

A putative gene cluster for the cyclic depsipeptide hapalosin, known to reverse P-glycoprotein multiple drug resistance, was identified within three Subsection V cyanobacterial genomes, including the producing cyanobacterium H. welwitschii UH strain IC-52-3. A number of orphan NRPS/PKS gene clusters and ribosomally-synthesised and post translationally-modified peptide gene clusters (including cyanobactin, microviridin and bacteriocin gene clusters) were identified. Furthermore, gene clusters encoding the biosynthesis of mycosporine-like amino acids, scytonemin, hydrocarbons and terpenes were also identified and compared.

Conclusions

Genome mining has revealed the diversity, abundance and complex nature of the secondary metabolite potential of the Subsection V cyanobacteria. This bioinformatic study has identified novel biosynthetic enzymes which have not been associated with gene clusters of known classes of natural products, suggesting that these cyanobacteria potentially produce structurally novel secondary metabolites.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1855-z) contains supplementary material, which is available to authorized users.     

Scoring predictive models using a reduced representation of proteins: model and energy definition

Background  

Reduced representations of proteins have been playing a keyrole in the study of protein folding. Many such models are available, with different representation detail. Although the usefulness of many such models for structural bioinformatics applications has been demonstrated in recent years, there are few intermediate resolution models endowed with an energy model capable, for instance, of detecting native or native-like structures among decoy sets. The aim of the present work is to provide a discrete empirical potential for a reduced protein model termed here PC2CA, because it employs a PseudoCovalent structure with only 2 Centers of interactions per Amino acid, suitable for protein model quality assessment.     

Early angiogenic response to shock waves in a three-dimensional model of human microvascular endothelial cell culture (HMEC-1)

The exact nature of shock wave (SW) action is not, as yet, fully understood, although a possible hypothesis may be that shock waves induce neoangiogenesis. To test this hypothesis, a three-dimensional (3D) culture model on Matrigel was developed employing a human microvascular endothelial cell line (HMEC-1) which was stimulated with low energy soft- focused SW generated by an SW lithotripter. After 12 hours we observed a statistically significant increase in capillary connections subsequent to shock-wave treatment in respect to the control group and a marked 3-hour down-regulation in genes involved in the apoptotic processes (BAX, BCL2LI, GADD45A, PRKCA), in cell cycle (CDKN2C, CEBPB, HK2, IRF1, PRKCA), oncogenes (JUN, WNT1), cell adhesion (ICAM-1), and proteolytic systems (CTSD, KLK2, MMP10). Our preliminary results indicate that microvascular endothelial cells in vitro quickly respond to SW, proliferating and forming vessel-like structures, depending on the energy level employed and the number of shocks released. The early decreased expression in the analysed genes could be interpreted as the first reactive response of the endothelial cells to the external stimuli and the prelude to the events characterizing the neo-angiogenic sequence.     

The benzomorphan-based LP1 ligand is a suitable MOR/DOR agonist for chronic pain treatment

AimsPowerful analgesics relieve pain primarily through activating mu opioid receptor (MOR), but the long-term use of MOR agonists, such as morphine, is limited by the rapid development of tolerance. Recently, it has been observed that simultaneous stimulation of the delta opioid receptor (DOR) and MOR limits the incidence of tolerance induced by MOR agonists. 3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-phenylpropanamide (LP1) is a centrally acting agent with antinociceptive activity comparable to morphine and is able to bind and activate MOR and DOR. The aim of this work was to evaluate and compare the induction of tolerance to antinociceptive effects from treatment with LP1 and morphine.Main methodsHere, we evaluated the pharmacological effects of LP1 administered at a dose of 4 mg/kg subcutaneously (s.c.) twice per day for 9 days to male Sprague–Dawley rats. In addition, the LP1 mechanism of action was assessed by measurement of LP1-induced [³⁵S]GTPγS binding to the MOR and DOR.Key findingsData obtained from the radiant heat tail flick test showed that LP1 maintained its antinociceptive profile until the ninth day, while tolerance to morphine (10 mg/kg s.c. twice per day) was observed on day 3. Moreover, LP1 significantly enhanced [³⁵S]GTPγS binding in the membranes of HEK293 cells expressing either the MOR or the DOR.SignificanceLP1 is a novel analgesic agent for chronic pain treatment, and its low tolerance-inducing capability may be correlated with its ability to bind both the MOR and DOR.     

Liver metastases and SBRT: A new paradigm?

BackgroundThe outstanding innovations made by early diagnosis, novel surgical techniques, effective chemotherapy regimens and conformal radiotherapy, have significantly improved patients overall survival and quality of life. Multidisciplinary approach to cancer has also led to an increased prevalence of patients with few, organ-confined metastases, who can experience long-term survival even if their disease is no longer localized. Liver is one of the most common site for metastatic disease from several cancers, and when metastatic disease is confined to liver, given the ability of this organ to regenerate almost to its optimal volume, surgical resection represents the standard of care because is associated with a better prognosis. Approximately 70–90% of liver metastases, however, are unresectable and a safe, effective alternative therapeutic option is necessary for these patients.Materials and methodsA review of the current literature was performed to analyze the role of SBRT in treating liver metastases from different cancers. A literature search using the terms “SBRT” and “liver metastases” was carried out in PUBMED.ResultsStereotactic body radiation therapy has shown to provide promising results in the treatment of liver metastases, thanks to the ability of this procedure to deliver a conformal high dose of radiation to the target lesion and a minimal dose to surrounding critical tissues.ConclusionStereotactic body radiation therapy is a non-invasive, well-tolerated and effective treatment for patients with liver metastases not suitable for surgical resection.     

Bacterial Communities Involved in Soil Formation and Plant Establishment Triggered by Pyrite Bioweathering on Arctic Moraines

In arctic glacier moraines, bioweathering primed by microbial iron oxidizers creates fertility gradients that accelerate soil development and plant establishment. With the aim of investigating the change of bacterial diversity in a pyrite-weathered gradient, we analyzed the composition of the bacterial communities involved in the process by sequencing 16S rRNA gene libraries from different biological soil crusts (BSC). Bacterial communities in three BSC of different morphology, located within 1 m distance downstream a pyritic conglomerate rock, were significantly diverse. The glacier moraine surrounding the weathered site showed wide phylogenetic diversity and high evenness with 15 represented bacterial classes, dominated by Alphaproteobacteria and pioneer Cyanobacteria colonizers. The bioweathered area showed the lowest diversity indexes and only nine bacterial families, largely dominated by Acidobacteriaceae and Acetobacteraceae typical of acidic environments, in accordance with the low pH of the BSC. In the weathered BSC, iron-oxidizing bacteria were cultivated, with counts decreasing along with the increase of distance from the rock, and nutrient release from the rock was revealed by environmental scanning electron microscopy-energy dispersive X-ray analyses. The vegetated area showed the presence of Actinomycetales, Verrucomicrobiales, Gemmatimonadales, Burkholderiales, and Rhizobiales, denoting a bacterial community typical of developed soils and indicating that the lithoid substrate of the bare moraine was here subjected to an accelerated colonization, driven by iron-oxidizing activity.