The Long and Winding Road of Molecular Data in Phylogenetic Analysis

The use of molecules and reactions as evidence, markers and/or traits for evolutionary processes has a history more than a century long. Molecules have been used in studies of intra-specific variation and studies of similarity among species that do not necessarily result in the analysis of phylogenetic relations. Promoters of the use of molecular data have sustained the need for quantification as the main argument to make use of them. Moreover, quantification has allowed intensive statistical analysis, as a condition and a product of increasing automation. All of these analyses are subject to the methodological anxiety characteristic of a community in search of objectivity (Suárez-Díaz and Anaya-Muñoz, Stud Hist Philos Biol Biomed Sci 39:451–458, 2008). It is in this context that scientists compared and evaluated protein and nucleic acid sequence data with other types of molecular data – including immunological, electrophoretic and hybridization data. This paper argues that by looking at long-term historical processes, such as the use of molecular evidence in evolutionary biology, we gain valuable insights into the history of science. In that sense, it accompanies a growing concern among historians for big-pictures of science that incorporate the fruitful historical research on local cases of the last decades.     

Successful β cells islet regeneration in streptozotocin-induced diabetic baboons using ultrasound-targeted microbubble gene therapy with cyclinD2/CDK4/GLP1

Both major forms of diabetes mellitus (DM) involve β-cell destruction and dysfunction. New treatment strategies have focused on replenishing the deficiency of β-cell mass common to both major forms of diabetes by islet transplantation or β-cell regeneration. The pancreas, not the liver, is the ideal organ for islet regeneration, because it is the natural milieu for islets. Since islet mass is known to increase during obesity and pregnancy, the concept of stimulating pancreatic islet regeneration in vivo is both rational and physiologic. This paper proposes a novel approach in which non-viral gene therapy is targeted to pancreatic islets using ultrasound targeted microbubble destruction (UTMD) in a non-human primate model (NHP), the baboon. Treated baboons received a gene cocktail comprised of cyclinD2, CDK, and GLP1, which in rats results in robust and durable islet regeneration with normalization of blood glucose, insulin, and C-peptide levels. We were able to generate important preliminary data indicating that gene therapy by UTMD can achieve in vivo normalization of the intravenous (IV) glucose tolerance test (IVGTT) curves in STZ hyperglycemic-induced conscious tethered baboons. Immunohistochemistry clearly demonstrated evidence of islet regeneration and restoration of β-cell mass.     

Transforming Growth Factor β Signaling Overcomes Dasatinib Resistance in Lung Cancer

Lung cancer is the second most common cancer and the leading cause of cancer-related deaths. Despite recent advances in the development of targeted therapies, patients with advanced disease remain incurable, mostly because metastatic non-small cell lung carcinomas (NSCLC) eventually become resistant to tyrosine kinase inhibitors (TKIs). Kinase inhibitors have the potential for target promiscuity because the kinase super family is the largest family of druggable genes that binds to a common substrate (ATP). As a result, TKIs often developed for a specific purpose have been found to act on other targets. Drug affinity chromatography has been used to show that dasatinib interacts with the TGFβ type I receptor (TβR-I), a serine-threonine kinase. To determine the potential biological relevance of this association, we studied the combined effects of dasatinib and TGFβ on lung cancer cell lines. We found that dasatinib treatment alone had very little effect; however, when NSCLC cell lines were treated with a combination of TGFβ and dasatinib, apoptosis was induced. Combined TGFβ-1 + dasatinib treatment had no effect on the activity of Smad2 or other non-canonical TGFβ intracellular mediators. Interestingly, combined TGFβ and dasatinib treatment resulted in a transient increase in p-Smad3 (seen after 3 hours). In addition, when NSCLC cells were treated with this combination, the pro-apoptotic protein BIM was up-regulated. Knockdown of the expression of Smad3 using Smad3 siRNA also resulted in a decrease in BIM protein, suggesting that TGFβ-1 + dasatinib-induced apoptosis is mediated by Smad3 regulation of BIM. Dasatinib is only effective in killing EGFR mutant cells, which is shown in only 10% of NSCLCs. Therefore, the observation that wild-type EGFR lung cancers can be manipulated to render them sensitive to killing by dasatinib could have important implications for devising innovative and potentially more efficacious treatment strategies for this disease.     

Ubiquinone synthesis and its regulation in Pneumocystis carinii

The opportunistic pathogen Pneumocystis causes a type of pneumonia in individuals with defective immune systems such as AIDS patients. Atovaquone, an analog of ubiquinone (coenzyme Q [CoQ]), is effective in clearing mild to moderate cases of the infection. Rat-derived Pneumocystis carinii was the first organism in which CoQ synthesis was clearly demonstrated to occur in both mitochondrial and microsomal subcellular fractions. Atovaquone inhibits microsomal CoQ synthesis with no effect on mitochondrial CoQ synthesis. We here report on additional studies evaluating CoQ synthesis and its regulation in the organism. Buparvaquone also inhibited CoQ synthesis and it reduced the synthesis of all four CoQ homologs in the microsomal but not the mitochondrial fraction. Glyphosate, which inhibits a reaction in the de novo synthesis of the benzoquinone moiety of CoQ reduced cellular ATP levels. Bacterial and plant quinones, and several chemically synthesized phenolics, flavanoids, and naphthoquinones that inhibit electron transport in other organisms were shown to reduce CoQ synthesis in P. carinii. The inhibitory action of naphthoquinone compounds appeared to depend on their molecular size and structural flexibility rather than redox potential. Results of experiments examining the synthesis of the polyprenyl chain of CoQ were consistent with negative feedback control of CoQ synthesis. These studies on P. carinii suggest that cellular sites and the control of CoQ synthesis in different organisms and cell types might be more diverse than previously thought.     

Water-soluble contrast agents targeted at the estrogen receptor for molecular magnetic resonance imaging

Novel estrogen-conjugated pyridine-containing Gd(III) and Eu(III) contrast agents (EPTA-Gd/Eu) were designed and effectively synthesized. Convenient to administration and MRI experiments, both EPTA-Gd and EPTA-Eu are soluble in water. The EPTA-Gd selectively binds with a micromolar affinity to the estrogen receptor and induces proliferation of human breast cancer cells. The EPTA-Gd is not lethal and does not cause any adverse effects when administrated intravenously. It enhances T1 and T2 nuclear relaxation rates of water and serves as a selective contrast agent for localizing the estrogen receptor by MRI.     

Morphological events during the Trypanosoma cruzi cell cycle

The replication and segregation of organelles producing two identical daughter cells must be precisely controlled during the cell cycle progression of eukaryotes. In kinetoplastid flagellated protozoa, this includes the duplication of the single mitochondrion containing a network of DNA, known as the kinetoplast, and a flagellum that grows from a cytoplasmic basal body through the flagellar pocket compartment before emerging from the cell. Here, we show the morphological events and the timing of these events during the cell cycle of the epimastigote form of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease. DNA staining, flagellum labeling, bromodeoxyuridine incorporation, and ultra-thin serial sections show that nuclear replication takes 10% of the whole cell cycle time. In the middle of the G2 stage, the new flagellum emerges from the flagellar pocket and grows unattached to the cell body. While the new flagellum is still short, the kinetoplast segregates and mitosis occurs. The new flagellum reaches its final size during cytokinesis when a new cell body is formed. These precisely coordinated cell cycle events conserve the epimastigote morphology with a single nucleus, a single kinetoplast, and a single flagellum status of the interphasic cell.     

Therapeutic Intervention in Multiple Sclerosis with Alpha B-Crystallin: A Randomized Controlled Phase IIa Trial

As a molecular chaperone and activator of Toll-like receptor 2-mediated protective responses by microglia and macrophages, the small heat shock protein alpha B-crystallin (HspB5) exerts therapeutic effects in different animal models for neuroinflammation, including the model for multiple sclerosis (MS). Yet, HspB5 can also stimulate human antigen-specific memory T cells to release IFN-γ, a cytokine with well-documented detrimental effects during MS. In this study, we explored in a Phase IIa randomized clinical trial the therapeutic application of HspB5 in relapsing-remitting MS (RR-MS), using intravenous doses sufficient to support its protective effects, but too low to trigger pathogenic memory T-cell responses. These sub-immunogenic doses were selected based on in vitro analysis of the dose-response profile of human T cells and macrophages to HspB5, and on the immunological effects of HspB5 in healthy humans as established in a preparatory Phase I study. In a 48-week randomized, placebo-controlled, double-blind Phase IIa trial, three bimonthly intravenous injections of 7.5, 12.5 or 17.5 mg HspB5 were found to be safe and well tolerated in RR-MS patients. While predefined clinical endpoints did not differ significantly between the relatively small groups of MS patients treated with either HspB5 or placebo, repeated administration especially of the lower doses of HspB5 led to a progressive decline in MS lesion activity as monitored by magnetic resonance imaging (MRI), which was not seen in the placebo group. Exploratory linear regression analysis revealed this decline to be significant in the combined group receiving either of the two lower doses, and to result in a 76% reduction in both number and total volumes of active MRI lesions at 9 months into the study. These data provide the first indication for clinical benefit resulting from intervention in RR-MS with HspB5. Trial Registration: ClinicalTrials.gov Phase I: NCT02442557; Phase IIa: NCT02442570