Myocardial improvement with human embryonic stem cell-derived cardiomyocytes enriched by p38MAPK inhibition

Background aimsWe have shown previously that inhibition of the p38 mitogen-activated protein kinase (p38MAPK) directs the differentiation of human embryonic stem cell (hESC)-derived cardiomyocytes (hCM). We investigated the therapeutic benefits of intramyocardial injection of hCM differentiated from hESC by p38MAPK inhibition using closed-chest ultrasound-guided injection at a clinically relevant time post-myocardial infarction (MI) in a mouse model.MethodsMI was induced in mice and the animals treated at day 3 with: (a) hCM, (b) human fetal fibroblasts (hFF) as cell control, or (c) medium control (n = 10 animals/group). Left ventricular ejection fraction (LVEF) was evaluated post-MI prior to therapy, and at days 28 and 60 post-cell therapy. Hearts were analyzed at day 60 for infarct size, angiogenesis, cell fate and teratoma formation.ResultsLVEF was improved in the hCM-treated animals compared with both hFF and medium control-treated animals at day 28 (39.03 ± 1.79% versus 27.89 ± 1.27%, P < 0.05, versus 32.90 ± 1.46%, P < 0.05, respectively), with sustained benefit until day 60. hCM therapy resulted in significantly smaller scar size, increased capillary bed area, increased number of arterioles, less native cardiomyocyte (CM) apoptosis, and increased CM proliferation compared with the other two groups. These benefits were achieved despite a very low retention rate of the injected cells at day 60, as assessed by immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR). Therapy with hCM did not result in intramyocardial teratoma formation at day 60.ConclusionsThis study demonstrates that hCM derived from p38MAPK-treated hESC have encouraging therapeutic potential.     

Nickel and copper complexes with few amide-based macrocyclic and open-chain ligands

The present work shows three new amide-based ligands H₂L¹, H₂L² and H₂L³ and their nickel and copper complexes. The X-ray structural analysis substantiate that the ligands constitute a square-based basal plane around the metal center. The crystal structures also show interesting solid state packing due to hydrogen-bonding and various weak C?H interactions. The solution-based spectral studies support the solid-state geometry observed for these complexes. The electrochemical results show that the Ni^3+/2+ and Cu^3+/2+ redox couple primarily depends on the N₄ donors composed of N_amide and N_amine atoms. It was observed that the ligands H₂L¹ and H₂L² are better suited to stabilize the Cu(III) species whereas ligand H₂L³ is ideal for the stabilization of Ni(III) species. On the basis of electrochemical findings, transient Ni³⁺ species were generated and characterized by the absorption spectroscopy.     

Interactive effect of arsenic and fluoride on cardio-respiratory disorders in male rats: possible role of reactive oxygen species

Epidemiological evidence demonstrates positive correlation between environmental and occupational arsenic or fluoride exposure and risk to various cardio-respiratory disorders. Arsenic-exposure has been associated with atherosclerosis, hypertension, cerebrovascular diseases, ischemic heart disease, and peripheral vascular disorders, whereas Fluoride-exposure manifests cardiac irregularities and low blood pressure (BP). Present study aims to study the combined effects of these toxicants on various cardio-respiratory variables in male rats. Single intravenous (i.v.) dose of arsenic (1, 5, 10 mg/kg) or fluoride (5, 10, 20, 36.5 mg/kg) either alone or in combination were administered. Individual exposure to arsenic or fluoride led to a significant depletion of mean arterial pressure, heart rate (HR), respiration rate and neuromuscular (NM) transmission in a dose-dependent manner. These changes were accompanied by increased levels of blood reactive oxygen species (ROS) and decreased glutathione (GSH) concentrations. An increase in the blood acetyl cholinesterase (AChE) activity was observed in both arsenic or fluoride exposed rats. These changes were significantly more pronounced in arsenic-exposed animals than in fluoride. During combined exposure to arsenic (5 mg/kg) + fluoride (20 mg/kg) or arsenic (10 mg/kg) + fluoride (36.5 mg/kg) the toxic effects were more pronounced compared to individual toxicities of arsenic or fluoride alone. However, combined exposure to arsenic (5 mg/kg) + fluoride (36.5 mg/kg) resulted in antagonistic effects on variables suggestive of altered cardio-respiratory function and oxidative stress. The results from the present study suggest that arsenic or fluoride individually demonstrate cardio-respiratory failure at all doses whereas during combination exposure these toxins show variable toxicities; both synergistic and antagonistic effects depending upon the dose. Moreover, it may be concluded that arsenic and/or fluoride cardio-respiratory toxicity may be mediated via oxidative stress. However, these results are new in the discipline thus requires further exploration.     

Destruction complex function in the Wnt signaling pathway of Drosophila requires multiple interactions between Adenomatous polyposis coli 2 and Armadillo

The tumor suppressor Adenomatous polyposis coli (APC) negatively regulates Wnt signaling through its activity in the destruction complex. APC binds directly to the main effector of the pathway, β-catenin (βcat, Drosophila Armadillo), and helps to target it for degradation. In vitro studies demonstrated that a nonphosphorylated 20-amino-acid repeat (20R) of APC binds to βcat through the N-terminal extended region of a 20R. When phosphorylated, the phospho-region of an APC 20R also binds βcat and the affinity is significantly increased. These distinct APC-βcat interactions suggest different models for the sequential steps of destruction complex activity. However, the in vivo role of 20R phosphorylation and extended region interactions has not been rigorously tested. Here we investigated the functional role of these molecular interactions by making targeted mutations in Drosophila melanogaster APC2 that disrupt phosphorylation and extended region interactions and deletion mutants missing the Armadillo binding repeats. We tested the ability of these mutants to regulate Wnt signaling in APC2 null and in APC2 APC1 double-null embryos. Overall, our in vivo data support the role of phosphorylation and extended region interactions in APC2's destruction complex function, but suggest that the extended region plays a more significant functional role. Furthermore, we show that the Drosophila 20Rs with homology to the vertebrate APC repeats that have the highest affinity for βcat are functionally dispensable, contrary to biochemical predictions. Finally, for some mutants, destruction complex function was dependent on APC1, suggesting that APC2 and APC1 may act cooperatively in the destruction complex.     

Predicting response to platinum and non-platinum drugs through bioluminescence resonance energy transfer (BRET) based bio-molecular interactions in platinum resistant epithelial ovarian cancer

Therapy induced rewiring of signalling networks often lead to acquirement of platinum-resistance, thereby necessitating the use of non-platinum agents as second-line treatment particularly for epithelial ovarian cancer (EOC). A prior subject-specific assessment can guide the choice of optimal non-platinum agent/s and possible targeted therapeutic/s. Assessment of protein-protein interactions are superior to simple cytotoxicity assays to determine therapeutic efficacy and associated molecular responses. Utilizing improved PIP3-AKT and ERK1/2 activation Bioluminescence Resonance Energy Transfer (BRET) sensors, we report chemotherapy-induced ERK1/2 activation predominantly in cisplatin-paclitaxel resistant EOC cells and increased activation of both ERK1/2 and AKT in malignant ascites derived cancer cells from platinum-resistant patients but not from treatment-naive or platinum-sensitive relapse patients. Further, majority of the non-platinum drugs except irinotecan increased ERK1/2 activation in platinum-taxol resistant cells as observed by live-cell BRET assessment which were associated with p90RSK1/2 and BAD activation along with upregulation of multidrug transporter gene ABCC1 and cell survival genes like cyclin D1 and Bcl2. Interestingly, only irinotecan was able to sensitize these resistant cells. Altogether, this first report of BRET based sensing of molecular pathway activations in platinum resistant cell lines and patient's derived cancer cells highlight the clinical potential of BRET sensors in management of therapy resistant cancer.     

An allogenic therapeutic strategy for canine spinal cord injury using mesenchymal stem cells

This study was conducted to characterize canine bone marrow-derived mesenchymal stem cells (BMSCs); in vivo tracking in mice, and therapeutic evaluation in canine clinical paraplegia cases. Canine BMSCs were isolated, cultured, and characterized in vitro as per International Society for Cellular Therapy criteria, and successfully differentiated to chondrogenic, osteogenic, and adipogenic lineages. To demonstrate the homing property, the pGL4.51 vector that contained luciferase reporter gene was used to transfect BMSCs. Successfully transfected cells were injected around the skin wound in mice and in vivo imaging was done at 6, 12 and 24 hr post MSCs delivery. In vivo imaging revealed that transfected BMSCs migrated and concentrated predominantly toward the center of the wound. BMSCs were further evaluated for allogenic therapeutic potential in 44 clinical cases of spinal cord injuries (SCI) and compared with conventional therapy (control). Therapeutic potential as evaluated by different body reflexes and recovery score depicted significantly better results in stem cell-treated group compared to control group. In conclusion, allogenic canine BMSCs can serve as potent therapeutic candidate in cell-based therapies, especially for diseases like SCI, where the conventional medication is not so promising.     

VAPC, an human endogenous inhibitor for hepatitis C virus (HCV) infection, is intrinsically unstructured but forms a "fuzzy complex" with HCV NS5B

Nearly 200 million people are infected by hepatitis C virus (HCV) worldwide. For replicating the HCV genome, the membrane-associated machinery needs to be formed by both HCV non-structural proteins (including NS5B) and human host factors such as VAPB. Recently, the 99-residue VAPC, a splicing variant of VAPB, was demonstrated to inhibit HCV replication via binding to NS5B, thus acting as an endogenous inhibitor of HCV infection. So far, the structure of VAPC remains unknown, and its interaction with NS5B has not been biophysically characterized. In this study, we conducted extensive CD and NMR investigations on VAPC which led to several striking findings: 1) although the N-terminal 70 residues are identical in VAPC and VAPB, they constitute the characteristic β-barrel MSP fold in VAPB, while VAPC is entirely unstructured in solution, only with helical-like conformations weakly populated. 2) VAPC is indeed capable of binding to NS5B, with an average dissociation constant (Kd) of ～20 μM. Intriguingly, VAPC remains dynamic even in the complex, suggesting that the VAPC-NS5B is a "fuzzy complex". 3) NMR mapping revealed that the major binding region for NS5B is located over the C-terminal half of VAPC, which is composed of three discrete clusters, of which only the first contains the region identical in VAPC and VAPB. The second region containing ～12 residues appears to play a key role in binding since mutation of 4 residues within this region leads to almost complete loss of the binding activity. 4) A 14-residue mimetic, VAPC-14 containing the second region, only has a ～3-fold reduction of the affinity. Our study not only provides critical insights into how a human factor mediates the formation of the HCV replication machinery, but also leads to design of VAPC-14 which may be further used to explore the function of VAPC and to develop anti-HCV molecules.     

Behavioral Response of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Lepidoptera: Noctuidae) to Cotton with and Without Expression of the CrylAc δ-Endotoxin Protein of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Berliner

Behavioral responses of larvae and adults of cotton bollworm, Helicoverpa armigera to cotton with (GK-12) and without (SI-3) expression of the CrylAc -endotoxin protein of Bacillus thuringiensis (Bt) Berliner were observed during 2001 and 2002. Our results showed that 8.3% individuals fed with flowers and bolls of GK-12 could develop from neonate to pupa; however, pupal weight decreased by 48.6% and duration of development was delayed by 7.6 days compared with those fed with flowers–bolls of SI-3. Deterrence index (DI) of larvae decreased in later instars, which indicated that the Bt toxin decreased with age. Feeding frequency of 4th-instar larvae on GK-12 leaves decreased by 38.8%, but movement frequency increased by 37.1%. Larvae moved at least one plant away by the age of 10 days in both pure and mixed plantings of SI-3 and GK-12 in the field. Adults preferred to lay eggs on SI-3. The total number of eggs deposited on SI-3 plants in 3 days were about 232 and 95% greater than that on GK-12 plants at bud–flower stage and flower–boll stage, respectively. Based on the behavior of larva and adults in response to the transgenic cotton, the potential effect of refuge strategy in resistance management of H. armigera is discussed.     

Cell growth and death in malignant lymphomas. A quantitative analysis

OBJECTIVE: To review the value of biopathologic factors in single lymphomatous patients across the boundaries of histologic classification. STUDY DESIGN: In a series of previous studies, based on a large collection of biopsy samples, the value of the above biopathologic characteristics in individual lymphomatous patients was quantitatively evaluated. RESULTS: The relationships between apoptotic index and growth fraction, in light of the expression of oncogenes, which regulate cell birth and death, were of particular value in determining the growth pattern of different lymphoma cases across the boundaries of histologic classification. CONCLUSION: The study of mechanisms that regulate cell proliferation and death might have therapeutic implications as the proper therapeutic approach should be based on detailed knowledge of the kinetic and molecular characteristics of each tumor.