Monophyly of the order Rodentia inferred from mitochondrial DNA sequences of the genes for 12S rRNA, 16S rRNA, and tRNA-valine

A recent analysis of amino acid sequence data (Graur et al.) suggested that the mammalian order Rodentia is polyphyletic, in contrast to most morphological data, which support rodent monophyly. At issue is whether the hystricognath rodents, such as the guinea pig, represent an independent evolutionary lineage within mammals, separate from the sciurognath rodents. To resolve this problem, we sequenced a region (2,645 bp) of the mitochondrial genome of the guinea pig containing the complete 12S ribosomal RNA, 16S ribosomal RNA, and transfer RNA(VAL) genes for comparison with the available sciurognath and other mammalian sequences. Several methods of analysis and statistical tests of the data all show strong support for rodent monophyly (91%-98% bootstrap probability, or BP). Calibration with the mammalian fossil record suggests a Cretaceous date (107 mya) for the divergence of sciurognaths and hystricognaths. An older date (38 mya) for the controversial Mus- Rattus divergence also is supported by these data. Our neighbor-joining analyses of all available sequence data (25 genes) confirm that some individual genes support rodent polyphyly but that tandem analysis of all data does not. We propose that the conflicting results are due to several compounding factors. The unique biochemical properties of some hystricognath metabolic proteins, largely responsible for generating this controversy, may have a single explanation: a cascade effect resulting from inactivation of the zinc-binding abilities of insulin. After excluding six genes possibly affected by insulin inactivation, analyses of all available sequence data (7,117 nucleotide sites, 3,099 amino acid sites) resulted in strong support for rodent monophyly (94% BP for DNA sequences, 90% for protein sequences), which lends support to the insulin-cascade hypothesis.      

An ex vivo readout for evaluation of dendritic cell-induced autologous cytotoxic T lymphocyte responses against esophageal cancer

Esophageal cancer is a highly malignant disease that despite surgery and adjuvant therapies has an extremely poor outcome. Dendritic cell (DC) immunotherapy as a novel promising strategy could be an alternative for treating this malignancy. Effective DC-mediated immune responses can be achieved by raising cytotoxic T lymphocyte (CTL) response against multiple antigens through loading DCs with total tumor RNA. However, the efficacy of this strategy first needs to be evaluated in a pre-clinical setting. The aim of the study was to set up an ex vivo autologous human readout assay for assessing the effects of DC-mediated cytotoxic responses, using total tumor RNA as an antigen load. Biopsy specimens of seven esophageal cancer patients were used to establish primary cultures of normal and cancer cells and to obtain autologous RNA for loading DCs. Mature DCs loaded with either normal or tumor RNA were obtained and subsequently used to raise various lymphocytes populations. Apoptosis levels of the autologous cultures were measured before and after incubating the cultures with the different lymphocytes populations. The mean apoptosis levels in the tumor cell cultures, induced by lymphocytes instructed by DCs loaded with tumor RNA, significantly increased with 15.6% ±2.9 SEM (range 3.4–24.5%, t-test, P < 0.05). Incubation of the normal cultures with the lymphocytes populations showed a mean non-significant increase in apoptosis of 0.4% ±3.4 SEM (range −13.9 to 9.8%, t-test, P = 0.7). Here, we introduce a practical, patient-specific autologous readout assay for pre-clinical testing of DC-mediated cytotoxic responses. Additionally, we demonstrated that the use of autologous tumor RNA as a strategy for raising cytotoxic responses against multiple tumor antigens could be effective for treating esophageal cancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tophus burden reduction with pegloticase: results from phase 3 randomized trials and open-label extension in patients with chronic gout refractory to conventional therapy

Introduction

Two replicate randomized, placebo-controlled six-month trials (RCTs) and an open-label treatment extension (OLE) comprised the pegloticase development program in patients with gout refractory to conventional therapy. In the RCTs, approximately 40% of patients treated with the approved dose saw complete response (CR) of at least one tophus. Here we describe the temporal course of tophus resolution, total tophus burden in patients with multiple tophi, tophus size at baseline, and the relationship between tophus response and urate-lowering efficacy.

Methods

Baseline subcutaneous tophi were analyzed quantitatively using computer-assisted digital images in patients receiving pegloticase (8 mg biweekly or monthly) or placebo in the RCTs, and pegloticase in the OLE. Tophus response, a secondary endpoint in the trials, was evaluated two ways. Overall tophus CR was the proportion of patients achieving a best response of CR (without any new/enlarging tophi) and target tophus complete response (TT-CR) was the proportion of all tophi with CR.

Results

Among 212 patients randomized in the RCTs, 155 (73%) had ≥1 tophus and 547 visible tophi were recorded at baseline. Overall tophus CR was recorded in 45% of patients in the biweekly group (P = 0.002 versus placebo), 26% in the monthly group, and 8% in the placebo group after six months of RCT therapy. TT-CR rates at six months were 28%, 19%, and 2% of tophi, respectively. Patients meeting the primary endpoint of sustained urate-lowering response to therapy (responders) were more likely than nonresponders to have an overall tophus CR at six months (54% vs 20%, respectively and 8% with placebo).Both overall tophus CR and TT-CRs increased with treatment duration in the OLE, reaching 70% (39/56) of patients and 55% (132/238) of target tophi after one year of treatment in patients receiving pegloticase during both the RCTs and OLE. At that time point, more tophi had resolved in responders (102/145 or 70% of tophi) than nonresponders (30/93; 32%).

Conclusions

Pegloticase reduced tophus burden in patients with refractory tophaceous gout, especially those achieving sustained urate-lowering. Complete resolution of tophi occurred in some patients by 13 weeks and in others with longer-term therapy.

Trial registrations

{"type":"clinical-trial","attrs":{"text":"NCT00325195","term_id":"NCT00325195"}}NCT00325195, {"type":"clinical-trial","attrs":{"text":"NCT01356498","term_id":"NCT01356498"}}NCT01356498     

T cells expanded in presence of IL-15 exhibit increased antioxidant capacity and innate effector molecules

Persistence of effector cytotoxic T lymphocytes (CTLs) during an immunological response is critical for successfully controlling a viral infection or tumor growth. Various cytokines are known to play an important part in regulating the immune response. The IL-2 family of cytokines that includes IL-2 and IL-15 are known to function as growth and survival factors for antigen-experienced T cells. IL-2 and IL-15 possess similar properties, including the ability to induce T cell proliferation. Whereas long-term IL-2 exposure has been shown to promote apoptosis and limit CD8(+) memory T cell survival and proliferation, it is widely believed that IL-15 can inhibit apoptosis and helps maintain a memory CD8(+) T-cell population. However, mechanisms for superior outcomes for IL-15 as compared to IL-2 are still under investigation. Our data shows that human T cells cultured in the presence of IL-15 exhibit increased expression of anti-oxidant molecules glutathione reductase (GSR), thioredoxin reductase 1 (TXNDR1), peroxiredoxin (PRDX) and superoxide dismutase (SOD). An increased expression of cell-surface thiols, intracellular glutathione, and thioredoxins was also noted in IL-15 cultured T cells. Additionally, IL-15 cultured T cells showed an increase in cytolytic effector molecules. Apart from increased level of Granzyme A and Granzyme B, IL-15 cultured T cells exhibited increased accumulation of reactive oxygen (ROS) and reactive nitrogen species (RNS) as compared to IL-2 cultured T cells. Overall, this study suggests that T cells cultured in IL-15 show increased persistence not only due to levels of anti-apoptotic proteins, but also due to increased anti-oxidant levels, which is complimented by increased cytolytic effector functions.     

Workflow management systems for gene sequence analysis and evolutionary studies – A Review

Post ‘omic’ era has resulted in the development of many primary, secondary and derived databases. Many analytical and visualization bioinformatics tools have been developed to manage and analyze the data available through large sequencing projects. Availability of heterogeneous databases and tools make it difficult for researchers to access information from varied sources and run different bioinformatics tools to get desired analysis done. Building integrated bioinformatics platforms is one of the most challenging tasks that bioinformatics community is facing. Integration of various databases, tools and algorithm is a challenging problem to deal with. This article describes the bioinformatics analysis workflow management systems that are developed in the area of gene sequence analysis and phylogeny. This article will be useful for biotechnologists, molecular biologists, computer scientists and statisticians engaged in computational biology and bioinformatics research.     

Taxol binds to polymerized tubulin in vitro

Taxol, a natural plant product that enhances the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells, was labeled with tritium by catalytic exchange with (3)H(2)O. The binding of [(3)H]taxol to microtubule protein was studied by a sedimentation assay. Microtubules assembled in the presence of [(3)H]taxol bind drug specifically with an apparent binding constant, K(app), of 8.7 x 19(-7) M and binding saturates with a calculated maximal binding ration, B(max), of 0.6 mol taxol bound/mol tubulin dimer. [(3)H]Taxol also binds and assembles phosphocellulose-purified tubulin, and we suggest that taxol stabilizes interactions between dimers that lead to microtubule polymer formation. With both microtubule protein and phosphocellulose- purified tubulin, binding saturation occurs at approximate stoichiometry with the tubulin dimmer concentration. Under assembly conditions, podophyllotoxin and vinblastine inhibit the binding of [(3)H]taxol to microtubule protein in a complex manner which we believe reflects a competition between these drugs, not for a single binding site, but for different forms (dimer and polymer) of tubulin. Steady-state microtubules assembled with GTP or with 5’-guanylyl-α,β-methylene diphosphonate (GPCPP), a GTP analog reported to inhibit microtubule treadmilling (I.V. Sandoval and K. Weber. 1980. J. Biol. Chem. 255:6966-6974), bind [(3)H]taxol with approximately the same stoichiometry as microtubules assembled in the presence of [(3)H]taxol. Such data indicate that a taxol binding site exists on the intact microtubule. Unlabeled taxol competitively displaces [(3)H]taxol from microtubules, while podophyllotoxin, vinblastine, and CaCl(2) do not. Podophyllotoxin and vinblastine, however, reduce the mass of sedimented taxol-stabilized microtubules, but the specific activity of bound [(3)H]taxol in the pellet remains constant. We conclude that taxol binds specifically and reversibly to a polymerized form of tubulin with a stoichiometry approaching unity.     

Phosphorylation and biosynthesis of high molecular weight proteins of tumor nuclear matrix

INTRODUCTIONAsearlyasin1948wehavefr8CtionatedisolatednucleifromnormalandtumorcellsbyextractionwithiMNaCIanddilutealkali[1].Thenuclearresiduewasthenstudiedmorethoroughly[2,3].Lateron,sillillarproteinousnuclearresidueswereisolatedbyotherworkers[46]andasstud…     

The GANC tetraloop: a novel motif in the group IIC intron structure

Tetraloops are a common building block for RNA tertiary structure, and most tetraloops fall into one of three well-characterized classes: GNRA, UNCG, and CUYG. Here, we present the sequence and structure of a fourth highly conserved class of tetraloop that occurs only within the ζ-ζ′ interaction of group IIC introns. This GANC tetraloop was identified, along with an unusual cognate receptor, in the crystal structure of the group IIC intron and through phylogenetic analysis of intron RNA sequence alignments. Unlike conventional tetraloop-receptor interactions, which are stabilized by extensive hydrogen-bonding interactions, the GANC-receptor interaction is limited to a single base stack between the conserved adenosine of the tetraloop and a single purine of the receptor, which consists of a one- to three-nucleotide bulge and does not contain an A-platform. Unlike GNRA tetraloops, the GANC tetraloop forms a sharp angle relative to the adjacent helix, bending by approximately 45° toward the major groove side of the helix. These structural attributes allow GANC tetraloops to fit precisely within the group IIC intron core, thereby demonstrating that structural motifs can adapt to function in a specific niche.