Vijaya R. Dirisala

Circulating miRNA markers show promise as new prognosticators for multiple myeloma

1Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA 2Myeloma Unit, Division of Hematology, University of Torino, Azienda Ospedaliera Città della Salute e della Scienza di Torino, Torino, Italy 3Molecular Virology, Comprehensive Cancer Center, The Ohio State University, Columbus, OHIO, USA 4Lymphoma & Genomics Research Program, Institute of Oncology Research IOR, Via Vela 6, 6500 Bellinzona, Switzerland 5Department of Clinical and Biological Sciences, Division of Geriatric, S. Luigi Gonzaga Hospital, University of Torino, Italy 6Centro di Riferimento Oncologico della Basilicata, Rionero in Vulture (PZ), Italy 7Italian Multiple Myeloma Network, GIMEMA, Italy 8Abramson Cancer Center, Division of Hematology-Oncology, University of Pennsylvania, Philadelphia, PA, USA

An Update on Clinical Burden, Diagnostic Tools, and Therapeutic Options of Staphylococcus aureus:

Staphylococcus aureus is an important pathogen responsible for a variety of diseases ranging from mild skin and soft tissue infections, food poisoning to highly serious diseases such as osteomyelitis, endocarditis, and toxic shock syndrome. Proper diagnosis of pathogen and virulence factors is important for providing timely intervention in the therapy. Owing to the invasive nature of infections and the limited treatment options due to rampant spread of antibiotic-resistant strains, the trend for development of vaccines and antibody therapy is increasing at rapid rate than development of new antibiotics. In this article, we have discussed elaborately about the host-pathogen interactions, clinical burden due to S aureus infections, status of diagnostic tools, and treatment options in terms of prophylaxis and therapy.

Cloning of guinea pig IL-4: Reduced IL-4 mRNA after vaccination or Mycobacterium tuberculosis infection

Interleukin-4 (IL-4), a pleiotropic cytokine produced by T-helper type 2 (Th2) cells, is involved in promoting humoral immune responses, allergic reactions and asthma. Previous studies suggested an important role for IL-4 in susceptibility to pulmonary tuberculosis; however, the role of IL-4 has not been studied in the guinea pig, a highly relevant model for this disease. In the present study, we cloned a cDNA for guinea pig IL-4 and examined, for the first time, mRNA expression by real-time RT-PCR in cultured guinea pig cells. High levels of IL-4 mRNA expression were detected in spleen T cells of naïve animals after in vitro stimulation with PMA plus ionomycin for 4-24 h. The expression of IL-4 mRNA was low in spleen and lymph node cells immunized with ovalbumin (OVA) plus Complete Freunds Adjuvant (CFA) in response to OVA (Th1), but significantly higher in the guinea pigs immunized with OVA plus alum (Th2). BCG vaccination reduced the expression of IL-4 mRNA in both spleen and lung digest cells compared to naïve guinea pigs, while levels of IFN-γ were similar in both groups. Furthermore, lung cells from Mycobacterium tuberculosis-infected guinea pigs stimulated in vitro with PPD or MPT64 showed low levels of IL-4 mRNA expression. Thus, BCG vaccination or M. tuberculosis infection modulates IL-4 mRNA expression in the guinea pig. Cloning of guinea pig IL-4 will allow us to address the role of IL-4 in vaccine-induced resistance to pulmonary TB in a highly relevant animal model.

Molecular cloning and expression of the IL-10 gene from guinea pigs.

The Guinea pig (Cavia porcellus) is one of the most relevant small animals for modeling human tuberculosis (TB) in terms of susceptibility to low dose aerosol infection, the organization of granulomas, extrapulmonary dissemination and vaccine-induced protection. It is also considered to be a gold standard for a number of other infectious and non-infectious diseases; however, this animal model has a major disadvantage due to the lack of readily available immunological reagents. In the present study, we successfully cloned a cDNA for the critical Th2 cytokine, interleukin-10 (IL-10), from inbred Strain 2 guinea pigs using the DNA sequence information provided by the genome project. The complete open reading frame (ORF) consists of 537 base pairs which encodes a protein of 179 amino acids. This cDNA sequence exhibited 87% homology with human IL-10. Surprisingly, it showed only 84% homology with the previously published IL-10 sequence from the C4-deficient (C4D) guinea pig, leading us to clone IL-10 cDNA from the Hartley strain of guinea pig. The IL-10 gene from the Hartley strain showed 100% homology with the IL-10 sequence of Strain 2 guinea pigs. In order to validate the only published IL-10 sequence existing in Genbank reported from C4D guinea pigs, genomic DNA was isolated from tissues of C4D guinea pigs. Amplification with various sets of primers showed that the IL-10 sequence reported from C4D guinea pigs contained numerous errors. Hence the IL-10 sequence that is being reported by us replaces the earlier sequence making our IL-10 sequence to be the first one accurate from guinea pig. Recombinant guinea pig IL-10 proteins were subsequently expressed in both prokaryotic and eukaryotic cells, purified and were confirmed by N-terminal sequencing. Polyclonal anti-IL-10 antibodies were generated in rabbits using the recombinant IL-10 protein expressed in this study. Taken together, our results indicate that the DNA sequence information provided by the genome project is useful to directly clone much needed cDNAs necessary to study TB in the guinea pig. The newly cloned guinea pig IL-10 cDNA and recombinant proteins will serve as valuable resources for immunological studies in the guinea pig model of TB and other diseases.

Prokaryotic expression and in vitro functional analysis of IL-1β and MCP-1 from guinea pig.

The Guinea pig (Cavia porcellus) is an excellent animal model for studying human tuberculosis (TB) and also for a number of other infectious and non-infectious diseases. One of the major roadblocks in effective utilization of this animal model is the lack of readily available immunological reagents. In order to address this issue, guinea pig interleukin 1 beta (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were efficiently cloned and expressed in a prokaryotic expression vector, and the expressed proteins in soluble form from both the genes were confirmed by N-terminal sequencing. The biological activity of recombinant guinea pig IL-1β was demonstrated by its ability to drive proliferation in thymocytes, and the recombinant guinea pig MCP-1 exhibited chemotactic activity for guinea pig resident peritoneal macrophages. These biologically active recombinant guinea pig proteins will facilitate an in-depth understanding of the role they play in the immune responses of the guinea pig to TB and other diseases.

Molecular characterization of the human ABO blood group orthologus system in pigs

The selection and use of animals with blood group 0 in the process of transplanting pig organs or tissues into humans can positively contribute to the control of acute immune rejection due to differences in blood groups. Exon-specific PCRs for the porcine blood group A transferase gene against genomic DNA from either blood group A or 0 animals resulted in the amplification failure of the A0 blood group gene exon 8 from blood group 0 animals. To characterize the genetic abnormality in the genome of blood group 0 animals, we screened bacterial artificial chromosome (BAC) clones from a Korean native pig BAC library which had the blood group 0 allele, and carried out shotgun sequencing. The analysis showed that the 0 allele has a large deletion between exon 7 of the A0 blood group gene and the neighbouring SURF6. We also showed that the ABO blood group antigens in humans and the A0 blood group antigens in pigs are coded by mutations within the orthologous glycosyltransferase gene. In addition, we developed a multiplex genotyping method for the porcine A0 blood group gene.

Discovery of cSNPs in pig using full-length enriched cDNA libraries of the Korean native pig as a source of genetic diversity

Clones from full-length enriched cDNA libraries serve as valuable resources for functional genomic studies. We analyzed 3.210 chromatograms obtained from sequencing the 5′-ends of brainstem, liver, neocortex, and spleen clones derived from full-length enriched cDNA libraries of Korean native pigs. In addition, 50,000 pig EST sequence trace files were obtained from Genbank and combined with our sequencing information for SNP identificationin silico. For the SNP analysis, neocortex, and liver libraries were newly constructed, whereas the sequencing results from brainstem and spleen libraries were from previously constructed libraries. The putative SNPs from thein silico analysis were confirmed by genomic PCR from a group of 20 pigs of four different breeds. Using this approach, 86% of cSNPs identifiedin silico were confirmed and the SNP detection frequency was 1 SNP per 338 bp. Interestingly, we found a valine deletion at amino acid position 126 of the neuronal and endocrine protein gene in the Korean native pig. We confirmed that this deletion was caused by alternative splicing at the NAGNAG acceptors. Our study shows that large-scale EST sequencing of Korean native pigs can be effectively employed for natural polymorphism-based pig genome analysis.

Recombinant pharmaceutical protein production in plants: unraveling the therapeutic potential of molecular pharming

There is an increasing demand for the generation of recombinant pharmaceutical proteins for a wide array of therapeutic applications. In comparison to bacterial, yeast and animal cells, the production of recombinant proteins in plants with economic and therapeutic importance has only started recently. The most important prerequisite of any expression systems is that it should be simple and inexpensive. In this regard, plant-based expression has emerged an as accepted alternative to conventional expression platforms due to economic feasibility, rapid scalability, higher stability of recombinant proteins, safety due to lack of harmful substances (human, animal pathogens and pyrogens) and capability of producing proteins with desired secondary modifications. Heterologous expression using plants has played a pivotal role in the development of a myriad of recombinant proteins, including neutraceuticals and monoclonal antibodies being utilized in various therapeutic approaches. This paper presents an overview about the current status, various strategies and advantages of pharmaceutical protein production in plant expression systems. We also present a summary of expression of therapeutic monoclonal antibodies, vaccines, clinical trials and the regulatory aspects of plant-based expression. Furthermore, the challenges encountered in plant expression such as costs associated with existing purification strategies are discussed.

Evolution of Synonymous Codon Usage in the Mitogenomes of Certain Species of Bilaterian Lineage with Special Reference to Chaetognatha

Chaetognatha is a minor phylum, comprising transparent marine invertebrates varying in size from 0.5 to 12 cm. The exact phylogenetic position of Chaetognatha in Metazoa has not been deciphered as some embryological characteristics place chaetognaths among deuterostomes and some morphological characteristics place these among protostomes. In this study, the major factors that drive synonymous codon usage bias (SCUB) in the mitogenomes of representative species of Chaetognatha and chosen species of other closely related phyla were analyzed. Spearmans rank correlation analyses of nucleotide contents suggested that mutational pressure and selection were acting in all examined mitogenomes but with varying intensities. The quantification of SCUB using effective number of codons vs. GC composition at the third codon position (GC3) plot suggested that mutational pressure due to GC compositional constraints might be one of the major influencing forces driving the SCUB in all chaetognaths except Sagitta enflata. However, neutrality plots revealed no significant correlation between GC3 and cumulative GC content at first and second codon positions (GC12) in all other species, except in Daphnia pulex. The parity rule 2 bias plot showed that significant compositional differences existed between C and G, as well as between A and T, contents in most of the protein-coding genes (PCGs) and, comparatively, A and T contents were used more proportionally than C and G contents in all chosen mitogenomes. Chi-square analysis revealed the presence of putative optimal codons in all species, except in S. enflata. The correspondence analysis identified that mutational pressure and selection act on the mitogenomes of the selected chaetognaths and other phyla with varying intensities. The cluster analysis based on relative synonymous codon usage (RSCU) values revealed that RSCU variations in the PCGs of mitogenomes of chaetognaths are more comparable with those of protostomes. Apart from mutational pressure and selection, certain unknown selective forces might be acting on the PCGs in the analyzed mitogenomes as the phenomenon of SCUB could not be explained by mutational pressure, by selection, or by both.

Deciphering the effect of novel bacterial exopolysaccharide-based nanoparticle cream against Propionibacterium acnes

Acne vulgaris (acne) is a chronic inflammatory disease prevalent among adolescents and adults, with significant psychological effects. The aetiology of acne is multifactorial. Several pathophysiological associations have been identified in which Propionibacterium acnes plays a major role. This bacteria primarily affects areas containing oil glands including the face, back and trunk, where it causes the formation of seborrhoea and inflammatory lesions. The treatment methods currently in place have side effects. A novel alternative method with no side effects is hence required. In this study, we report the synthesis of an exopolysaccharide (EPS)-producing bacterial-based nanoparticle as a stable biocompatible material for drug delivery. We then evaluated the effectiveness of EPS-based nanoparticle cream against P. acnes. Our results demonstrate that EPS nanoparticles have great potential as a safe and effective topical treatment for acne vulgaris and other associated infections.