A Novel 3670-Base Pair Mitochondrial DNA Deletion Resulting in Multi-systemic Manifestations in a Child

Mitochondrial DNA (mtDNA) deletion is a rare occurrence that results in defects to oxidative phosphorylation. The common clinical presentations of mtDNA deletion vary but include mitochondrial myopathy, Pearson syndrome, Kearns-Sayre syndrome, and progressive external ophthalmoplegia. Here, we report the case of a 10-year-old boy who presented with progressive deterioration of his clinical status (which included hypoglycemia, short stature, sensorineural hearing loss, retinitis pigmentosa, and chronic gastrointestinal dysmotility) that progressed to acute deterioration with pancreatitis, Fanconi syndrome, lactic acidosis, and acute encephalopathy. Following treatment, the patient was stabilized and his neurological condition improved. Through a combination of histological examinations and biochemical and molecular analyses, mitochondrial disease was confirmed. A novel 3670-base pair deletion (deletion of mtDNA nt 7,628-11,297) was identified in the muscle tissue. A direct repeat of CTACT at the breakpoints was also detected.     

Fabrication of tunable micropatterned substrates for cell patterning via microcontact printing of polydopamine with poly(ethylene imine)-grafted copolymers

Cell patterning is an important tool for biomedical research. In this work, we modified a technique combining mussel-inspired surface chemistry and microcontact printing (μCP) to modulate surface chemistry for cell patterning. Polymerized dopamine on poly(dimethylsiloxane) stamps was transferred to several cell-unfavorable substrates via μCP. Since cells only attached to the polydopamine (PDA)-imprinted areas, cell patterns were formed on a variety of cell-unfavorable surfaces. The stability of PDA imprints was proved under several harsh conditions. The cell affinity of PDA was modulated by co-deposition with several poly(ethylene imine) (PEI)-based copolymers, such as PEI, PEI-g-PEG (poly(ethylene glycol)) and PEI-g-galactose. The imprints of PDA/PEI-g-PEG provide the formation of cell patterns on cell-favorable substrates. Neuronal PC12 cells were patterned via imprinting of PDA/PEI, while HepG2/C3A cells were arranged on the imprint of PDA/PEI-g-galactose. Finally, co-culture of HepG2/C3A cells and L929 fibroblasts was accomplished by our micropatterning approach. This study demonstrated this simple and economic technique provides a powerful tool for development of functional patterned substrates for cell patterning. This technique should profit the preparation of cell patterns to study fundamental cell biology and to apply to biomedical engineering such as cell-based biosensors, diagnostic devices and tissue engineering.     

Effects of hyperbaric oxygenation on oxidative stress in acute transient focal cerebral ischemic rats

The aim of this study was to investigate the effects of hyperbaric oxygenation (HBO) after brain ischemia. Middle cerebral artery occlusion (MCAO) procedure was used to induce the brain ischemia. Rats were assigned to control or HBO group after brain ischemia. In order to examine the role of glutathione after HBO treatment, another group of brain ischemic rats were included to receive the glutathione synthesis inhibitor and HBO treatment. HBO was administered at a pressure of 3 atmospheres absolute for 1 h with 100% oxygen, starting at 3 h post brain ischemia in HBO groups. Animals in control group were placed in their home cage and exposed to normobaric room air. The infarct volume (IV), activation of astrocyte, and level of total glutathione and lipid peroxidation (LP) were assessed 24 h post-reperfusion. Significant reduction in IV was noted in HBO group when compared with control group. The activation of astrocyte was significantly increased in the right cerebral cortex and right striatum in the HBO group when compared with those of the control group. The glutathione level was higher with lower LP level in right cortex and right striatum after HBO as compared with those of the control. However, such effects of HBO treatment were markedly reduced by glutathione synthesis inhibitor administration. These results show that inhibiting glutathione synthesis dramatically reduces the effectiveness of HBO in acute transient focal cerebral ischemia.     

How Do Heme-Protein Sensors Exclude Oxygen? Lessons Learned from Cytochrome c', Nostoc puntiforme Heme Nitric Oxide/Oxygen-Binding Domain, and Soluble Guanylyl Cyclase

Abstract Significance: Ligand selectivity for dioxygen (O(2)), carbon monoxide (CO), and nitric oxide (NO) is critical for signal transduction and is tailored specifically for each heme-protein sensor. Key NO sensors, such as soluble guanylyl cyclase (sGC), specifically recognized low levels of NO and achieve a total O(2) exclusion. Several mechanisms have been proposed to explain the O(2) insensitivity, including lack of a hydrogen bond donor and negative electrostatic fields to selectively destabilize bound O(2), distal steric hindrance of all bound ligands to the heme iron, and restriction of in-plane movements of the iron atom. Recent Advances: Crystallographic structures of the gas sensors, Thermoanaerobacter tengcongensis heme-nitric oxide/oxygen-binding domain (Tt H-NOX(1)) or Nostoc puntiforme (Ns) H-NOX, and measurements of O(2) binding to site-specific mutants of Tt H-NOX and the truncated β subunit of sGC suggest the need for a H-bonding donor to facilitate O(2) binding. Critical Issues: However, the O(2) insensitivity of full length sGC with a site-specific replacement of isoleucine by a tyrosine on residue 145 and the very slow autooxidation of Ns H-NOX and cytochrome c' suggest that more complex mechanisms have evolved to exclude O(2) but retain high affinity NO binding. A combined graphical analysis of ligand binding data for libraries of heme sensors, globins, and model heme shows that the NO sensors dramatically inhibit the formation of six-coordinated NO, CO, and O(2) complexes by direct distal steric hindrance (cyt c'), proximal constraints of in-plane iron movement (sGC), or combinations of both following a sliding scale rule. High affinity NO binding in H-NOX proteins is achieved by multiple NO binding steps that produce a high affinity five-coordinate NO complex, a mechanism that also prevents NO dioxygenation. Future Directions: Knowledge advanced by further extensive test of this "sliding scale rule" hypothesis should be valuable in guiding novel designs for heme based sensors. Antioxid. Redox Signal. 17, 1246-1263.     

Zanthoxylum avicennae extracts inhibit cell proliferation through protein phosphatase 2A activation in HA22T human hepatocellular carcinoma cells in vitro and in vivo

Hepatocellular carcinoma is a common type of cancer that is usually associated with poor prognosis. In this study, we examined the in vitro and in vivo mechanisms of the traditional Vietnamese herb Zanthoxylum avicennae on the inhibition of HA22T human hepatocellular carcinoma cell proliferation. HA22T cells were treated with different concentrations of Zanthoxylum avicennae extracts (YBBEs) and analyzed with the MTT assay, western blot analysis, flow cytometry, siRNA transfection assays and co-immunoprecipitation assay. Additionally, the HA22T-implanted xenograft nude mouse model was applied to confirm the cellular effects. YBBEs showed a strong inhibition of HA22T cell viability in a dose-dependent manner and significantly reduced cell proliferation-related proteins as well as induced cell cycle arrest in the G2/M phase. Protein phosphatase 2A (PP2A) siRNA or okadaic acid totally blocked YBBE-mediated cell proliferation inhibition. In addition, an HA22T-implanted nude mouse model further confirmed that YBBEs inhibit HA22T tumor cell growth and downregulate the survival and cell cycle regulating proteins, as well as activate the PP2A protein. Our findings indicate that the inhibition of HA22T cell proliferation by YBBEs is mediated through PP2A activation.     

Immunotherapy for SV40 T/t antigen-induced breast cancer by recombinant adeno-associated virus serotype 2 carrying interleukin-15 in mice

Human interleukin-15 (hIL15) exerts anticancer effects through the activities of lymphokine-activated killer (LAK) cells. However, its short half-life hinders its clinical application. Recombinant adeno-associated virus serotype?2 (rAAV2) is used for hIL15 gene transfer vectors, because of its low immunogenicity and long-term gene expression in human clinical trials. SV40?T/t antigens are related with many human epithelial cancers and are generally found in human breast cancer. In order to demonstrate the anticancer effects of hIL15 on SV40?T/t antigen-induced breast cancer, rAAV2-hIL15 was constructed and an SV40?T/t antigen-induced transgenic mouse breast cancer model was established. Our study showed that rAAV2-hIL15 could express the hIL15 protein with anticancer bioactivity. In addition, rAAV2-hIL15 could activate the cytotoxic activity of LAK cells in?vivo. Furthermore, the rAAV2-hIL15 successfully delayed cancer growth and eventually led to cancer cell death in SV40?T/t antigen-induced breast cancer transgenic mice. In summary, our study indicates that rAAV2-hIL15 may be applied for cancer immunotherapy of SV40?T/t antigen-induced breast cancer.     

Combination of cilostazol and clopidogrel attenuates Rat critical limb ischemia

ABSTRACT: Background and aimProcedural failure and untoward clinical outcomes after surgery remain problematic in critical limb ischemia (CLI) patients. This study tested a clopidogrel-cilostazol combination treatment compared with either treatment alone in attenuating CLI and improving CLI-region blood flow in rats. METHODS: Male Sprague--Dawley rats (n = 40) were equally divided into five groups: control, CLI induction only, CL I + cilostazol (12.0 mg/day/kg), CLI + clopidogrel (8.0 mg/kg/day) and CLI + combined cilostazol-clopidogrel. After treatment for 21 days, Laser Doppler imaging was performed. RESULTS: On day 21, the untreated CLI group had the lowest ratio of ischemic/normal blood flow (p < 0.001). Inflammation measured by VCAM-1 protein expression; oxidative stress; PAI-1, MMP-9 and TNF-alpha mRNA expressions; and immunofluorescence staining (IF) of CD68+ cells was lower with combined treatment than with the other treatments, and lower in the two single-treatment groups than the untreated CLI group (all p < 0.01). Anti-inflammatory mRNA expression of interleukin-10, and eNOS showed a reverse pattern among these groups. Apoptosis measured by Bax, caspase-3 and PARP; and muscle damage measured by cytosolic cytochrome-C, and serum and muscle micro-RNA-206 were all lowest with combination treatment, and the two single-treatment groups showed lower values than the untreated group (all p < 0.001). Angiogenesis measured by eNOS, IF staining of CD31+ and vWF + cells; and number of vessels in CLI region were highest with combination treatment and higher in the single-treatment groups than the untreated group (all p < 0.001). CONCLUSION: Combined cilostazol-clopidogrel therapy is superior to either agent alone in improving ischemia in rodent CLI.     

Continuing Exposure to Low-Dose Nonylphenol Aggravates Adenine-Induced Chronic Renal Dysfunction and Role of Rosuvastatin Therapy

ABSTRACT: BACKGROUND: Nonylphenol (NP), an environmental organic compound, has been demonstrated to enhance reactive-oxygen species (ROS) synthesis. Chronic exposure to low-dose adenine (AD) has been reported to induce chronic kidney disease (CKD). METHODS: In this study, we tested the hypothesis that chronic exposure to NP will aggravate AD-induced CKD through increasing generations of inflammation, ROS, and apoptosis that could be attenuated by rosuvastatin. Fifty male Wistar rats were equally divided into group 1 (control), group 2 (AD in fodder at a concentration of 0.25%), group 3 (NP: 2 mg/kg/day), group 4 (combined AD & NP), and group 5 (AD-NP + rosuvastatin: 20 mg/kg/day). Treatment was continued for 24 weeks for all animals before being sacrificed. RESULTS: By the end of 24 weeks, serum blood urea nitrogen (BUN) and creatinine levels were increased in group 4 than in groups 1-3, but significantly reduced in group 5 as compared with group 4 (all p<0.05). Histopathology scorings of renal-parenchymal and tubular damages were significantly higher in group 4 than in groups 1-3, but remarkably lower in group 5 compared with group 4 (all p<0.01). Both gene and protein levels of inflammation, oxidative stress, ROS, and cellular apoptosis were remarkably higher in group 4 compared with groups 1-3, but lowered in group 5 than in group 4 (all p<0.001). Conversely, both gene and protein levels of anti-oxidants, anti-inflammation and anti-apoptosis were markedly increased in group 5 compared with group 4 (all p<0.001). CONCLUSION: NP worsened AD-induced CKD that could be reversed by rosuvastatin therapy.