Comparative Genome Analysis of 19 Trueperella pyogenes Strains Originating from Different Animal Species Reveal a Genetically Diverse Open Pan-Genome.

Trueperella pyogenes is a Gram-positive opportunistic pathogen that causes severe cases of mastitis, metritis, and pneumonia in a wide range of animals, resulting in significant economic losses. Although little is known about the virulence factors involved in the disease pathogenesis, a comprehensive comparative genome analysis of T. pyogenes genomes has not been performed till date. Hence, present investigation was carried out to characterize and compare 19 T. pyogenes genomes originating in different geographical origins including the draftgenome of the first Indian origin strain T. pyogenes Bu5. Additionally, candidate virulence determinants that could be crucial for their pathogenesis were also detected and analyzed by using various bioinformatics tools. The pan-genome calculations revealed an open pan-genome of T. pyogenes. In addition, an inventory of virulence related genes, 190 genomic islands, 31 prophage sequences, and 40 antibiotic resistance genes that could play a significant role in organism's pathogenicity were detected. The core-genome based phylogeny of T. pyogenes demonstrates a polyphyletic, host-associated group with a high degree of genomic diversity. The identified core-genome can be further used for screening of drug and vaccine targets. The investigation has provided unique insights into pan-genome, virulome, mobiliome, and resistome of T. pyogenes genomes and laid the foundation for future investigations.

Clinical potential of human-induced pluripotent stem cells : Perspectives of induced pluripotent stem cells.

The recent establishment of induced pluripotent stem (iPS) cells promises the development of autologous cell therapies for degenerative diseases, without the ethical concerns associated with human embryonic stem (ES) cells. Initially, iPS cells were generated by retroviral transduction of somatic cells with core reprogramming genes. To avoid potential genotoxic effects associated with retroviral transfection, more recently, alternative non-viral gene transfer approaches were developed. Before a potential clinical application of iPS cell-derived therapies can be planned, it must be ensured that the reprogramming to pluripotency is not associated with genome mutagenesis or epigenetic aberrations. This may include direct effects of the reprogramming method or "off-target" effects associated with the reprogramming or the culture conditions. Thus, a rigorous safety testing of iPS or iPS-derived cells is imperative, including long-term studies in model animals. This will include not only rodents but also larger mammalian model species to allow for assessing long-term stability of the transplanted cells, functional integration into the host tissue, and freedom from undifferentiated iPS cells. Determination of the necessary cell dose is also critical; it is assumed that a minimum of 1 billion transplantable cells is required to achieve a therapeutic effect. This will request medium to long-term in vitro cultivation and dozens of cell divisions, bearing the risk of accumulating replication errors. Here, we review the clinical potential of human iPS cells and evaluate which are the most suitable approaches to overcome or minimize risks associated with the application of iPS cell-derived cell therapies.

Transposon-based reprogramming to induced pluripotency.

Induced pluripotent stem (iPS) cells represent a recent innovation in the field of stem cells. Commonly, iPS cells are generated by viral transduction of core reprogramming genes, such as Oct4, Sox2, Klf4, c-Myc, Nanog and Lin28. However, integrating viruses, like retro- and lentiviral vectors, may cause insertional mutagenesis and may increase the risk of tumor formation. Therefore, alternative methods which avoid these safety concerns are intensively investigated. Here, we review the current status of transposon-based methods to induce pluripotency. DNA transposons are non-viral elements, which can be effectively integrated into a genome by their corresponding transposase enzyme. The advantages of transposon-based gene transfer are their increased safety, their large cargo capacity, their relatively simple design, and the availability of hyper-active and mutated transposase enzymes. For example, integration-deficient, excision-competent transposase variants allow the complete removal of the reprogramming transposon after successful reprogramming to obtain transposon-free reprogrammed cells. Transposon-based reprogramming will broaden the toolbox for iPS cell production and will advance the establishment of safe, non-viral methods.

Induced pluripotent stem cells: Mechanisms, achievements and perspectives in farm animals.

Pluripotent stem cells are unspecialized cells with unlimited self-renewal, and they can be triggered to differentiate into desired specialized cell types. These features provide the basis for an unlimited cell source for innovative cell therapies. Pluripotent cells also allow to study developmental pathways, and to employ them or their differentiated cell derivatives in pharmaceutical testing and biotechnological applications. Via blastocyst complementation, pluripotent cells are a favoured tool for the generation of genetically modified mice. The recently established technology to generate an induced pluripotency status by ectopic co-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows to extending these applications to farm animal species, for which the derivation of genuine embryonic stem cells was not successful so far. Most induced pluripotent stem (iPS) cells are generated by retroviral or lentiviral transduction of reprogramming factors. Multiple viral integrations into the genome may cause insertional mutagenesis and may increase the risk of tumour formation. Non-integration methods have been reported to overcome the safety concerns associated with retro and lentiviral-derived iPS cells, such as transient expression of the reprogramming factors using episomal plasmids, and direct delivery of reprogramming mRNAs or proteins. In this review, we focus on the mechanisms of cellular reprogramming and current methods used to induce pluripotency. We also highlight problems associated with the generation of iPS cells. An increased understanding of the fundamental mechanisms underlying pluripotency and refining the methodology of iPS cell generation will have a profound impact on future development and application in regenerative medicine and reproductive biotechnology of farm animals.

Sequence and phylogenetic analysis of host-range (E3L, K3L, and C7L) and structural protein (B5R) genes of buffalopox virus isolates from buffalo, cattle, and human in India.

Buffalopox virus (BPXV), a close variant of vaccinia virus (VACV) has emerged as a zoonotic pathogen. The host tropism of poxviruses is governed by host-range genes. Among the host-range genes: E3L, K3L, and C7L are essential for virus replication by preventing interferon resistance, whereas B5R is essential for spread of the virus and evasion from the host's immune response as in VACV. We report sequence analysis of host-range genes: E3L, K3L, C7L, and membrane protein gene (B5R) of BPXVs from buffalo, cattle, and human from recent outbreaks in India-their phylogenetic relationship with reference strain (BP4) and other Orthopoxviruses. BPXVs revealed a sequence homology with VACVs including zoonotic Brazilian VACV-like viruses. The aa sequences of E3L and K3L genes were 100 % similar in buffalo, cattle, and human isolates. However, four significant point mutations (I11K; N12K and S36F in C7L gene and D249G in B5R gene) were observed specific to buffalo isolate only. This signifies that different strains of BPXV were circulated during the outbreak. The mutations in C7L and B5R could play an important role in adaptation of BPXV in human and cattle which needs further functional studies. The strain of BPXV isolated from buffalo may not be adopted in human and cow. Various point mutations were observed in the host-range genes of reference strain (BPXV-BP4) which may be due to several passages of virus in cell culture. The phylogeny constructed based on concatenated gene sequences revealed that BPXVs are not as closely related to vaccine strain (Lister and Lister-derived strain-LC16m8), as hypothesized earlier, rather they are more closely related to reference strain (BPXV-BP4) and other vaccinia and vaccinia-like viruses such as Passatempo and Aracatuba viruses. The availability of information regarding host tropism determinants would allow us to understand molecular mechanism of species tropism of poxviruses which would be useful in unveiling new strategies to control zoonotic poxviral infections.

Efficacy and safety of pemetrexed in elderly cancer patients: results of an integrated analysis.

PURPOSE: To analyze pemetrexed in elderly patients (>or=65 years) based on data collected in three randomized, phase III registration trials. METHODS: Patients who received pemetrexed as monotherapy or in combination with another drug were included in this analysis (N=764). In all studies, pemetrexed 500 mg/m(2) was administered every 21 days. Data from patients receiving pemetrexed were stratified by age +/-65 years. RESULTS: Out of the 764 patients randomized, 271 were >or=65 years of age (35.4%). Of these, 28% had non-small cell lung cancer, 41% pancreatic cancer, and 31% had malignant pleural mesothelioma that was either locally advanced or metastatic. The overall response rate of the integrated database of elderly patients was 21.4%, with complete response in three patients (1.11% in >or=65 years vs. 1.01% in <65 years), partial response in 55 (20.30% vs. 19.68%), and stable disease in 116 (42.80% vs. 43.00%). Median survival time was 8.34 months in both groups, and median time to progressive disease was 4.80 months versus 4.60. Toxicity observed in the elderly group included 70 patients (25.8% vs. 17.0%; p=0.005) with grade 4 toxicity; myelosuppression was the major toxicity, with grade 3/4 neutropenia in 33% versus 22% (p<0.05), and thrombocytopenia in 13% versus 6% (p<0.05). Febrile neutropenia occurred in 4.8% versus 4.7% of patients. Non-hematological grade 3/4 events were fatigue (10.3% vs. 9.5%) and nausea (6.3% vs. 6.5%). CONCLUSIONS: Pemetrexed produced similar treatment effects in older and younger patients, and appeared to be well tolerated in the elderly population. This analysis was limited by the pooling of different disease types and the lack of uniform treatment regimens.