Mitochondrial DNA as a target for analyzing the biodistribution of cell therapy products.

Biodistribution tests are crucial for evaluating the safety of cell therapy (CT) products in order to prevent unwanted organ homing of these products in patients. Quantitative polymerase chain reaction (qPCR) using intronic Alu is a popular method for biodistribution testing owing to its ability to detect donor cells without modifying CT products and low detection limit. However, Alu-qPCR may generate inaccurate information owing to background signals caused by the mixing of human genomic DNA with that of experimental animals. The aim of this study was to develop a test method that is more specific and sensitive than Alu-qPCR, targeting the mitochondrial DNA (mtDNA) sequence that varies substantially between humans and experimental animals. We designed primers for 12S, 16S, and cytochrome B in mtDNA regions, assessed their specificity and sensitivity, and selected primers and probes for the 12S region. Human adipose-derived stem cells, used as CT products, were injected into the tail vein of athymic NCr-nu/nu mice and detected, 7 d after administration, in their lungs at an average concentration of 2.22 ± 0.69 pg/µg mouse DNA, whereas Alu was not detected. Therefore, mtDNA is more specific and sensitive than Alu and is a useful target for evaluating CT product biodistribution.

Early administration of high dose enoxaparin after traumatic brain injury.

BACKGROUND: Early enoxaparin 30 mg BID administration at 24 h post-injury has been demonstrated in patients with traumatic brain injury (TBI). However this dose can also yield subtherapeutic anti-Xa levels in 30-50% of trauma patients, suggesting that larger doses may be required for adequate prophylaxis against venous thromboembolism (VTE). The safety of enoxaparin 40 mg BID in trauma patients has previously been shown - however, these studies have largely excluded TBI patients. Therefore, we sought to demonstrate the safety of early enoxaparin 40 mg BID in a low-risk group of TBI patients. METHODS: A retrospective review of TBI patients at a Level 1 trauma center was performed. Patients with stable computed tomography (CT) of the head at 6 to 24 h post-injury who received enoxaparin 40 mg BID were included and serial GCS evaluations to identify possible clinical complications. To evaluate the safety of this dosing regimen, data was then compared to patients from our institution with similar TBI profiles who had received 5,000 units (U) of subcutaneous heparin (SQH) prophylaxis. RESULTS: 199 TBI patients were identified over a nine month period, 40/199 (19.7%) received DVT prophylaxis after traumatic injury. Of these 40, 19 (47.5%) received enoxaparin 40 mg BID and 21 (52.5%) received 5,000U of SQH. Low risk TBI patients who were either given enoxaparin (n = 7) or SQH (n = 4), demonstrated no clinical decline in mental status during their inpatient stay. CONCLUSION: Prior studies have demonstrated that enoxaparin 40 mg BID dosing is superior to traditional VTE prophylaxis in trauma patients. However, TBI patients are often excluded from this dosing due to concern for progression. Our study showed no clinical decline in mental status in a small cohort of low-risk TBI patients who received enoxaparin 40 mg BID.

Regulation of gene expression in the development of colitis-associated colon cancer in mice fed a high-fat diet.

Studies have shown that the higher prevalence of colorectal cancers among patients with inflammatory bowel disease. Thus, proinflammatory stimulus due to a high-fat diet may impose a higher risk on the development of colorectal cancer. In the present study, we applied a transcriptomic approach to characterize the molecular mechanism(s) by which high-fat feeding aggravates colitis-associated colorectal cancer (CAC). A high-fat diet was supplied in an azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced mouse model for 10 weeks and then the severity of CAC and global gene expression in colon were assessed. Although consumption of high-fat diet did not significantly aggravate CAC, it substantially changed gene expression profile in colon. In AOM/DSS treated mice (AD group) and AD mice fed a high-fat diet (AD + HF group), 34 and 54 DEGs were enriched in 'pathways in cancer', respectively. Notably, high-fat diet upregulated the expression of genes associated with spliceosome and ribosome biogenesis, and downregulated the expression of genes associated with lipid catabolism in mice treated with AOM/DSS. In addition, we identified that DEGs between the AD and AD + HF groups, were enriched in 'metabolic pathways', especially amino acid and nucleotide metabolism. Taken together, this study provides the molecular mechanism in understanding the high-fat diet-mediated CAC development.

Targeted suicide gene therapy for liver cancer based on ribozyme-mediated RNA replacement through post-transcriptional regulation.

Hepatocellular carcinoma (HCC) has high fatality rate and limited therapeutic options. Here, we propose a new anti-HCC approach with high cancer-selectivity and efficient anticancer effects, based on adenovirus-mediated Tetrahymena group I trans-splicing ribozymes specifically inducing targeted suicide gene activity through HCC-specific replacement of telomerase reverse transcriptase (TERT) RNA. To confer potent anti-HCC effects and minimize hepatotoxicity, we constructed post-transcriptionally enhanced ribozyme constructs coupled with splicing donor and acceptor site and woodchuck hepatitis virus post-transcriptional regulatory element under the control of microRNA-122a (miR-122a). Adenovirus encoding post-transcriptionally enhanced ribozyme improved trans-splicing reaction and decreased human TERT (hTERT) RNA level, efficiently and selectively retarding hTERT-positive liver cancers. Adenovirus encoding miR-122a-regulated ribozyme caused selective liver cancer cytotoxicity, the efficiency of which depended on ribozyme expression level relative to miR-122a level. Systemic administration of adenovirus encoding the post-transcriptionally enhanced and miR-regulated ribozyme caused efficient anti-cancer effects at a single dose of low titers and least hepatotoxicity in intrahepatic multifocal HCC mouse xenografts. Minimal liver toxicity, tissue distribution, and clearance pattern of the recombinant adenovirus were observed in normal animals administered either systemically or via the hepatic artery. Post-transcriptionally regulated RNA replacement strategy mediated by a cancer-specific ribozyme provides a clinically relevant, safe, and efficient strategy for HCC treatment.

Acute normovolemic hemodilution for a patient with secondary polycythemia undergoing aortic valve replacement due to severe aortic stenosis - A case report.

BACKGROUND: A high hematocrit level in patients with erythrocytosis is linked with increased blood viscosity and increased risk of thromboembolism. Therefore, it is necessary to adequately lower the hematocrit level before performing a high-risk surgery. CASE: A 67-year-old male was scheduled for aortic valve replacement due to severe aortic stenosis. The preoperative hematocrit level of this patient was very high due to secondary polycythemia by hypoxia. We decided to perform acute normovolemic hemodilution after anesthetic induction to reduce the risk of thromboembolism in the patient. The patient was discharged after a successful surgery and a post-operative period without any side effects. CONCLUSIONS: We estimate that patients with secondary polycythemia may benefit from acute normovolemic hemodilution to reduce their hematocrit levels while undergoing cardiac surgery using cardiopulmonary bypass. However, it is necessary to control the hematocrit level, since a significant decrease can cause side effects.

Electrically stimulable indium tin oxide plate for long-term in vitro cardiomyocyte culture.

BACKGROUND: We investigated whether electrical stimulation via indium tin oxide (ITO) could enhance the in vitro culture of neonatal rat ventricular myocytes (NRVMs), which are important in vitro models for studying the mechanisms underlying many aspects of cardiology. METHODS: Cardiomyocytes were obtained from 1-day-old neonatal rat heart ventricles. To evaluate function of NRVMs cultured on ITO with electrical stimulation, the cell viability, change of cell morphology, immunochemistry using cardiac-specific antibodies, and gene expression were tested. RESULTS: Defined sarcomeric structure, cell enlargement, and increased distribution of NRVMs appeared in the presence of electrical stimulation. These characteristics were absent in NRVMs cultured under standard culture conditions. In addition, the expression levels of cardiomyocyte-specific and ion channel markers were higher in NRVMs seeded on ITO-coated dishes than in the control group at 14 days after seeding. ITO-coated dishes could effectively provide electrical cues to support the in vitro culture of NRVMs. CONCLUSIONS: These results provide supporting evidence that electrical stimulation via ITO can be effectively used to maintain culture and enhance function of cardiomyocytes in vitro.

Effect of Cell Labeling on the Function of Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Cell labeling technologies are required to monitor the fate of transplanted cells in vivo and to select target cells for the observation of certain changes in vitro. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been transplanted for the treatment of heart injuries or used in vitro for preclinical cardiac safety assessments. Cardiomyocyte (CM) labeling has been used in these processes to facilitate target cell monitoring. However, the functional effect of the labeling agent on hiPSC-CMs has not been studied. Therefore, we investigated the effects of labeling agents on CM cellular functions. 3'-Dioctadecyloxacarbocyanine perchlorate (DiO), quantum dots (QDs), and a DNA plasmid expressing EGFP using Lipo2K were used to label hiPSC-CMs. We conclude that the hiPSC-CM labeling with DiO and QDs does not induce arrhythmogenic effects but rather improves the mRNA expression of cardiac ion channels and Ca2＋ influx by L-type Ca2＋ channels. Thus, DiO and QD labeling agents may be useful tools to monitor transplanted CMs, and further in vivo influences of the labeling agents should be investigated in the future.

Paroxetine Induces Apoptosis of Human Breast Cancer MCF-7 Cells through Ca2+-and p38 MAP Kinase-Dependent ROS Generation.

Depression is more common in women with breast cancer than the general population. Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are widely used for the treatment of patients with depression and a range of anxiety-related disorders. The association between the use of antidepressant medication and breast cancer is controversial. In this study, we investigated whether and how SSRIs induce the death of human breast cancer MCF-7 cells. Of the antidepressants tested in this study (amitriptyline, bupropion, fluoxetine, paroxetine, and tianeptine), paroxetine most reduced the viability of MCF-7 cells in a time-and dose-dependent manner. The exposure of MCF-7 cells to paroxetine resulted in mitochondrion-mediated apoptosis, which is assessed by increase in the number of cells with sub-G1 DNA content, caspase-8/9 activation, poly (ADP-ribose) polymerase cleavage, and Bax/Bcl-2 ratio and a reduction in the mitochondrial membrane potential. Paroxetine increased a generation of reactive oxygen species (ROS), intracellular Ca2+ levels, and p38 MAPK activation. The paroxetine-induced apoptotic events were reduced by ROS scavengers and p38 MAPK inhibitor, and the paroxetine's effect was dependent on extracellular Ca2+ level. Paroxetine also showed a synergistic effect on cell death induced by chemotherapeutic drugs in MCF-7 and MDA-MB-231 cells. Our results showed that paroxetine induced apoptosis of human breast cancer MCF-7 cells through extracellular Ca2+-and p38 MAPK-dependent ROS generation. These results suggest that paroxetine may serve as an anticancer adjuvant to current cancer therapies for breast cancer patients with or without depression.

Treatment of persistent postoperative hiccups with stellate ganglion block: Three case reports.

RATIONALE: Although persistent postoperative hiccups can cause various problems (such as sleep disorders, depression, fatigue) for the patient, there has been little research on this topic. The purpose of this study is to determine the effectiveness of treating persistent postoperative hiccups with a stellate ganglion block (SGB), an injection of local anesthetic in the sympathetic nerve tissue of the neck. PATIENT CONCERNS AND DIAGNOSES: Three patients each developed persistent hiccups within 3 days of abdominal surgery, lasting for 3 to 6 days. The patients were diagnosed as having persistent hiccups based on the hiccup duration. INTERVENTIONS AND OUTCOMES: The 3 patients were treated with an SGB. After the procedure, the frequency and intensity of hiccups decreased and then the hiccups stopped completely. CONCLUSION: An SGB is an effective method that can be considered in conjunction with other treatments for persistent hiccups. Clinicians should be mindful of the negative effects that persistent hiccups can exert on patients.

Role of Chemical Biology in Tuberculosis Drug Discovery and Diagnosis.

The use of chemical techniques to study biological systems (often referred to currently as chemical biology) has become a powerful tool for both drug discovery and the development of novel diagnostic strategies. In tuberculosis, such tools have been applied to identifying drug targets from hit compounds, matching high-throughput screening hits against large numbers of isolated protein targets and identifying classes of enzymes with important functions. Metabolites unique to mycobacteria have provided important starting points for the development of innovative tools. For example, the unique biology of trehalose has provided both novel diagnostic strategies as well as probes of in vivo biological processes that are difficult to study any other way. Other mycobacterial metabolites are potentially valuable starting points and have the potential to illuminate new aspects of mycobacterial pathogenesis.

Use of stellate ganglion block for treatment of recurrent syncope followed by chest pain.

Syncope is defined as a transient loss of consciousness and postural tone, characterized by rapid onset, short duration, and spontaneous recovery. Stellate ganglion block (SGB) is a nerve block method that is used for treatment of neuropathic pain in the head, neck and upper extremities, especially trigeminal neuralgia, postherpetic neuralgia and complex regional pain syndrome. SGB can modulate and stabilize the sympathetic nervous system, which prevents it from overexcitation and improves symptoms of syncope. The authors report a patient who was treated for pain and edema of both upper extremities with SGB, then showed improvement in recurrent syncope followed by chest pain and overall quality of life.

CHK2 is involved in the p53-independent radiosensitizing effects of valproic acid.

Radiotherapy is an effective treatment for the majority of types of localized solid cancer. However, the risk of side effects to the surrounding normal tissues limits radiotherapeutic approaches. Whilst the mechanism of action of valproic acid, an inhibitor of histone deacetylase, remains unknown, the inhibitor is a potential antineoplastic radiosensitizer. The present study demonstrated the in vitro radiosensitizing effects of valproic acid on the human breast cancer MCF7 cell line, and revealed that valproic acid increased the level of DNA breakage, apoptosis and senescence. In addition, western blot analyses revealed that valproic acid induced tumor suppressor protein (p)53 and p21 expression, and activated checkpoint kinase 2 (CHK2) in MCF7 cells and primary mouse embryonic fibroblasts. Notably, treatment with valproic acid also induced increases in the level of p21 protein levels and CHK2 activity in p53-null colon cancer HCT116 cells. Furthermore, the present study demonstrated that valproic acid-induced radiosensitization was largely dependent on the activity of CHK2. The results of the present study reveal that valproic acid may exhibit clinical utility with respect to increasing the anticancer efficacy of radiotherapy by affecting the level of p53.

Tempo-spatial Activation of Sequential Quadruple Stimuli for High Gene Expression of Polymeric Gene Nanocomplexes.

The clinical application of intracellular gene delivery via nanosized carriers is hindered by intracellular multistep barriers that limit high levels of gene expression. To solve these issues, four different intracellular or external stimuli that can efficiently activate a gene carrier, a gene, or a photosensitizer (pheophorbide A [PhA]) were assessed in this study. The designed nanosized polymeric gene complexes were composed of PhA-loaded thiol-degradable polycation (PhA@RPC) and cytomegalovirus (CMV) promoter-equipped pDNA. After cellular internalization of the resulting PhA@RPC/pDNA complexes, the complexes escaped endosomal sequestration, owing to the endosomal pH-induced endosomolytic activity of RPC in PhA@RPC. Subsequently, intracellular thiol-mediated polycation degradation triggered the release of PhA and pDNA from the complexes. Late exposure to light (for example, 12 h post-treatment) activated the released PhA and resulted in the production of reactive oxygen species (ROS). Intracellular ROS successively activated NF-κB, which then reactivated the CMV promoter in the pDNA. These sequential, stimuli-responsive chemical and biological reactions resulted in high gene expression. In particular, the time-point of light exposure was very significant to tune efficient gene expression as well as negligible cytotoxicity: early light treatment induced photochemical internalization but high cytotoxicity, whereas late light treatment influenced the reactivation of silent pDNA via PhA-generated ROS and activation of NF-κB. In conclusion, the quadruple triggers, such as pH, thiol, light, and ROS, successively influenced a gene carrier (RPC), a photosensitizer, and a genetic therapeutic, and the tempo-spatial activation of the designed quadruple stimuli-activatable nanosized gene complexes could be potential in gene delivery applications.

Inhaled nitric oxide for the brain dead donor with neurogenic pulmonary edema during anesthesia for organ donation: a case report.

Neurogenic pulmonary edema (NPE) in brain dead organ donors occurring after an acute central nervous system insult threatens organ preservation of potential organ donors and the outcome of organ donation. Hence the active and immediate management of NPE is critical. In this case, a 50-year-old male was admitted to the intensive care unit (ICU) for organ donation. He was hypoxic due to NPE induced by spontaneous intracerebral hemorrhage and intraventricular hemorrhage. Protective ventilatory management, intermittent recruitment maneuvers, and supportive treatment were maintained in the ICU and the operating room (OR). Despite this management, the hypoxemia worsened after the OR admission. So inhaled nitric oxide (NO) therapy was performed during the operation, and the hypoxic phenomena showed remarkable improvement. The organ retrieval was successfully completed. Therefore, NO inhalation can be helpful in the improvement of hypoxemia caused by NPE in brain dead organ donors during anesthesia for the organ donation.

Upregulation of human mammaglobin reduces migration and invasion of breast cancer cells.

Little is known about the biological role of human mammaglobin (hMAM) that is considered as a promising marker for breast cancer. Here, we investigated hMAM's role related to migration and invasion of human breast cancer cells (hBCC). Compared to normal cells, hBCC have high MAM mRNA expression levels. Of the hBCC tested, MAM mRNA expression levels were higher in noninvasive than in invasive cells. Overexpression of hMAM in breast cancer cells decreased migration and invasion, whereas knockdown of hMAM increased both. Taken together, these results suggest that metastasis of hBCC could be controlled by hMAM expression levels.

Needles as urethral foreign body in a child: successful removal using a new method with a laparoscopic needle holder.

Self-inserted foreign bodies in the lower urinary tract are rare among children. The treatment of foreign bodies in the urethra is determined by their type, size, location, shape, and mobility. We describe here a 13-year-old boy who had self-inserted a needle into his urethra out of curiosity. Initial attempts to remove the needle from the urethra by inserting a cystoscope failed due to the weak biting force of the forceps and because the needle was trapped in the urethral mucosa. We successfully used a laparoscopic needle holder to remove the needle through the perineum.

Cerebral oxygenation monitoring of patients during arthroscopic shoulder surgery in the sitting position.

BACKGROUND: Cerebral hypotension and desaturation can occur during shoulder surgery in the seated position. We evaluated the correlation of cerebral oxygen saturation (rSO(2)) using near infra-red spectroscopy (NIRS) and mean arterial pressures (MAP) (at the levels of the brain and heart). METHODS: Fifty patients, scheduled for the arthroscopic shoulder surgery in the seated position, were enrolled to monitor the rSO(2), bispectral Index (BIS), and MAPs at the levels of the brain and heart. The values of each parameter were collected at 5 min after intubation, immediately after placing the patient in the sitting position, 5 min after the patient was seated, immediately after the surgical incision, and every 30 min after incision. RESULTS: A correlation between the cerebral rSO(2) and the MAP at the level of brain were statistically significant. Cerebral rSO(2) and MAP after a change of posture from supine to sitting position were significantly decreased, compared to the baseline value. CONCLUSIONS: Monitoring cerebral rSO(2) and MAP at the level of brain can be helpful to detect the possibility of cerebral deoxygenation earlier.

Dual effects of fluoxetine on mouse early embryonic development.

Fluoxetine, a selective serotonin reuptake inhibitor, regulates a variety of physiological processes, such as cell proliferation and apoptosis, in mammalian cells. Little is known about the role of fluoxetine in early embryonic development. This study was undertaken to investigate the effect of fluoxetine during mouse early embryonic development. Late two-cell stage embryos (2-cells) were cultured in the presence of various concentrations of fluoxetine (1 to 50µM) for different durations. When late 2-cells were incubated with 5µM fluoxetine for 6h, the percentage that developed into blastocysts increased compared to the control value. However, late 2-cells exposed to fluoxetine (5µM) over 24h showed a reduction in blastocyst formation. The addition of fluoxetine (5µM) together with KN93 or KN62 (calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors) failed to increase blastocyst formation. Fluoxetine treatment inhibited TREK-1 and TREK-2, members of the two-pore domain K(+) channel family expressed in mouse embryos, activities, indicating that fluoxetine-induced membrane depolarization in late 2-cells might have resulted from TREK inhibition. In addition, long-term exposure to fluoxetine altered the TREK mRNA expression levels. Furthermore, injection of siRNA targeting TREKs significantly decreased blastocyst formation by ~30% compared to injection of scrambled siRNA. Long-term exposure of fluoxetine had no effect on blastocyst formation of TREK deficient embryos. These results indicate that low-dose and short-term exposures of late 2-cells to fluoxetine probably increase blastocyst formation through activation of CaMKII-dependent signal transduction pathways, whereas long-term exposure decreases mouse early embryonic development through inhibition of TREK channel gating.

Interfacial thermal conductance observed to be higher in semiconducting than metallic carbon nanotubes.

Thermal transport at carbon nanotube (CNT) interfaces was investigated by characterizing the interfacial thermal conductance between metallic or semiconducting CNTs and three different surfactants. We thereby resolved a difference between metallic and semiconducting CNTs. CNT portions separated by their electronic type were prepared in aqueous suspensions. After slightly heating the CNTs dispersed in the suspension, we obtained cooling curves by monitoring the transient changes in absorption, and from these cooling curves, we extracted the interfacial thermal conductance by modeling the thermal system. We found that the semiconducting CNTs unexpectedly exhibited a higher conductance of 11.5 MW/m(2)·K than that of metallic CNTs (9 MW/m(2)·K). Meanwhile, the type of surfactants hardly influenced the heat transport at the interface. The surfactant dependence is understood in terms of the coupling between the low-frequency vibrational modes of the CNTs and the surfactants. Explanations for the electronic-type dependency are considered based on the defect density in CNTs and the packing density of surfactants.