Customized small-sized clinostat using 3D printing and gas-permeable polydimethylsiloxane culture dish.

Over the past few decades, research on life in space has increased. Owing to the expensive nature of and the challenges associated with conducting experiments in real space, clinostats, which continuously randomize the gravity vector by using motors, have been used to generate simulated microgravity (SMG) on Earth. Herein, by using a 3D printing method, we develop a customized small-sized clinostat (CS clinostat) that is easy to manufacture, inexpensive, and robust. Moreover, we develop and fabricate a gas-permeable polydimethylsiloxane culture dish that fits inside the CS clinostat. To validate SMG generation, ovarian cancer cells (OV- 90, TOV-21G, and Caov-3) were applied to demonstrate a significant reduction in caveolin-1 expression, a biomarker of SMG, indicating SMG generation. The proposed CS clinostat system has good accessibility for SMG research, which makes it useful as a tool for biologists, who are unfamiliar with conventional clinostat equipment, to conduct preliminary studies in the space environment.

CFTR regulates brown adipocyte thermogenesis via the cAMP/PKA signaling pathway.

BACKGROUND: Cystic fibrosis (CF) is characterized by reduced growth and lower body weight, which are multifactorial. CF mouse models lack key disease characteristics that predispose to a negative energy balance, such as pulmonary infections or exocrine pancreatic insufficiency, and yet they still exhibit a growth defect and an abnormally increased energy expenditure. Whether adipocyte thermogenesis contributes to the elevated resting energy expenditure in CF mice is unknown. METHODS: We examined the expression of CFTR in thermogenic brown adipose tissue (BAT) and investigated a functional role for CFTR using BAT-specific CFTR null mice (CFTRBATKO). RESULTS: The CFTR protein is expressed in mouse BAT at levels comparable to those in the lungs. BAT-specific inactivation of CFTR in mice increases whole-body energy expenditure associated with sympathetic stimulation by cold exposure. Weight gain on a high-fat diet is attenuated in these mice. However, CFTR-deficient brown adipocytes themselves have impaired, rather than enhanced, thermogenic responses. These cells feature decreased lipolysis and blunted activation of the cAMP/PKA signaling pathway in response to adrenergic stimulation. This suggests that compensatory heat production in other tissues likely accounts for the increased systemic energy expenditure seen in CFTRBATKO mice. CONCLUSIONS: Our data reveal a new role for CFTR in the regulation of adipocyte thermogenesis.

The interferon-inducible protein viperin controls cancer metabolic reprogramming to enhance cancer progression.

Metabolic reprogramming is an important cancer hallmark. However, the mechanisms driving metabolic phenotypes of cancer cells are unclear. Here, we show that the interferon-inducible (IFN-inducible) protein viperin drove metabolic alteration in cancer cells. Viperin expression was observed in various types of cancer and was inversely correlated with the survival rates of patients with gastric, lung, breast, renal, pancreatic, or brain cancer. By generating viperin knockdown or stably expressing cancer cells, we showed that viperin, but not a mutant lacking its iron-sulfur cluster-binding motif, increased lipogenesis and glycolysis via inhibition of fatty acid ß-oxidation in cancer cells. In the tumor microenvironment, deficiency of fatty acids and oxygen as well as production of IFNs upregulated viperin expression via the PI3K/AKT/mTOR/HIF-1α and JAK/STAT pathways. Moreover, viperin was primarily expressed in cancer stem-like cells (CSCs) and functioned to promote metabolic reprogramming and enhance CSC properties, thereby facilitating tumor growth in xenograft mouse models. Collectively, our data indicate that viperin-mediated metabolic alteration drives the metabolic phenotype and progression of cancer.

DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the ß-Catenin-P-Glycoprotein Signaling Pathway.

Dickkopf-3 (DKK3), a tumor suppressor, is frequently downregulated in various cancers. However, the role of DKK3 in ovarian cancer has not been evaluated. This study aimed to assess aberrant DKK3 expression and its role in epithelial ovarian carcinoma. DKK3 expression was assessed using immunohistochemistry with tissue blocks from 82 patients with invasive carcinoma, and 15 normal, 19 benign, and 10 borderline tumors as controls. Survival data were analyzed using Kaplan-Meier and Cox regression analysis. Paclitaxel-resistant cells were established using TOV-21G and OV-90 cell lines. Protein expression was assessed using Western blotting and immunofluorescence analysis. Cell viability was assessed using the MT assay and 3D-spheroid assay. Cell migration was determined using a migration assay. DKK3 was significantly downregulated in invasive carcinoma compared to that in normal, benign, and borderline tumors. DKK3 loss occurred in 56.1% invasive carcinomas and was significantly associated with disease-free survival and chemoresistance in serous adenocarcinoma. DKK3 was lost in paclitaxel-resistant cells, while ß-catenin and P-glycoprotein were upregulated. Exogenous secreted DKK3, incorporated by cells, enhanced anti-tumoral effect and paclitaxel susceptibility in paclitaxel-resistant cells, and reduced the levels of active ß-catenin and its downstream P-glycoprotein, suggesting that DKK3 can be used as a therapeutic for targeting paclitaxel-resistant cancer.

CD4+/CD8+ Ratio and Growth Differentiation Factor 8 Levels in Peripheral Blood of Large Canine Males Are Useful Parameters to Build an Age Prediction Model.

PURPOSE: To build an age prediction model, we measured CD4+ and CD8+ cells, and humoral components in canine peripheral blood. MATERIALS AND METHODS: Large Belgian Malinois (BGM) and German Shepherd Dog (GSD) breeds (n=27), aged from 1 to 12 years, were used for this study. Peripheral bloods were obtained by venepuncture, then plasma and peripheral blood mononuclear cells (PBMCs) were separated immediately. Six myokines, including interleukin (IL)-6, IL-8, IL-15, leukemia inhibitory factor (LIF), growth differentiation factor 8 (GDF8), and GDF11 were measured from plasma and CD4+/CD8+ T-lymphocytes ratio were measured from PBMC. These parameters were then tested with age prediction models to find the best fit model. RESULTS: We found that the T-lymphocyte ratio (CD4+/CD8+) was significantly correlated with age (r=0.46, p=0.016). Among the six myokines, only GDF8 showed a significant correlation with age (r=0.52, p=0.005). Interestingly, these two markers showed better correlations in male dogs than females, and BGM breed than GSD. Using these two age biomarkers, we could obtain the best fit in a quadratic linear mixed model (r=0.77, p=3×10-6). CONCLUSIONS: Age prediction is a challenging task because of complication with biological age. Our quadratic linear mixed model using CD4+/CD8+ ratio and GDF8 level showed a meaningful age prediction.

Defective brown adipose tissue thermogenesis and impaired glucose metabolism in mice lacking Letmd1.

Manipulation of energy-dissipating adipocytes has the potential to produce metabolic benefits. To this end, it is valuable to understand the mechanisms controlling the generation and function of thermogenic fat. Here, we identify Letm1 domain containing 1 (Letmd1) as a regulator of brown fat formation and function. The expression of Letmd1 is induced in brown fat by cold exposure and by ß-adrenergic activation. Letmd1-deficient mice exhibit severe cold intolerance concomitant with abnormal brown fat morphology, reduced thermogenic gene expression, and low mitochondrial content. The null mice exhibit impaired ß3-adrenoreceptor-dependent thermogenesis and are prone to diet-induced obesity and defective glucose disposal. Letmd1 was previously described as a mitochondrial protein, and we find that it also localizes to the nucleus and interacts with the transcriptional coregulator and chromatin remodeler Brg1/Smarca4, thus providing a way to impact thermogenic gene expression. Our study uncovers a role for Letmd1 as a key regulatory component of adaptive thermogenesis.

Targeting ODC1 inhibits tumor growth through reduction of lipid metabolism in human hepatocellular carcinoma.

Ornithine decarboxylase 1 (ODC1), a metabolic enzyme critically involved in the polyamine biosynthesis, is commonly upregulated in hepatocellular carcinoma (HCC). Despite its altered expression in human HCC tissues, the molecular mechanism by which ODC1 alters the course of HCC progression and functions in HCC cell survival is unknown. Here we identified that silencing of ODC1 expression with small interfering (si) RNA causes inhibition of HCC cell growth through blockade of cell cycle progression and induction of apoptosis. Next, to obtain insights into the molecular changes in response to ODC1 knockdown, global changes in gene expression were examined using RNA sequencing. It revealed that 119 genes show same directional regulation (76 up- and 43 down-regulated) in both Huh1 and Huh7 cells and were considered as a common ODC1 knockdown signature. Particularly, we found through a network analysis that KLF2, which is known to inhibit PPARγ expression and adipogenesis, was commonly up-regulated. Subsequent Western blotting affirmed that the downregulation of ODC1 was accompanied by a decrease in the levels of PPARγ as well as of PARP-1, cyclin E1 and pro-caspase 9 delaying cell cycle progression and accelerating apoptotic signaling. Following the down-regulation of PPARγ expression, ODC1 silencing resulted in a strong inhibition in the expression of important regulators of glucose transport and lipid biogenesis, and caused a marked decrease in lipid droplet accumulation. In addition, ODC1 silencing significantly inhibited the growth of human HCC xenografts in nude mice. These findings indicate that the function of ODC1 is correlated with HCC lipogenesis and suggest that targeting ODC1 could be an attractive option for molecular therapy of HCC.

Whole-transcriptome analysis of mouse adipose tissue in response to short-term caloric restriction.

Caloric restriction (CR) has been shown to extend the lifespan of many species by improving cellular function and organismal health. Additionally, fat reduction by CR may play an important role in lengthening lifespan and preventing severe age-related diseases. Interestingly, CR induced the greatest transcriptome change in the epididymal fat of mice in our study. In this transcriptome analysis, we identified and categorized 446 genes that correlated with CR level. We observed down-regulation of several signaling pathways, including insulin/insulin-like growth factor 1 (insulin/IGF-1), epidermal growth factor (EGF), transforming growth factor beta (TGF-ß), and canonical wingless-type mouse mammary tumor virus integration site (Wnt). Many genes related to structural features, including extracellular matrix structure, cell adhesion, and the cytoskeleton, were down-regulated, with a strong correlation to the degree of CR. Furthermore, genes related to the cell cycle and adipogenesis were down-regulated. These biological processes are well-identified targets of insulin/IGF-1, EGF, TGF-ß, and Wnt signaling. In contrast, genes involved in specific metabolic processes, including the tricarboxylic acid cycle and the electron transport chain were up-regulated. We performed in silico analysis of the promoter sequences of CR-responsive genes and identified two associated transcription factors, Paired-like homeodomain 2 (Pitx2) and Paired box gene 6 (Pax6). Our results suggest that strict regulation of signaling pathways is critical for creating the optimal energy homeostasis to extend lifespan.

Mitochondrial Efficiency-Dependent Viability of Saccharomyces cerevisiae Mutants Carrying Individual Electron Transport Chain Component Deletions.

Mitochondria play a crucial role in eukaryotic cells; the mitochondrial electron transport chain (ETC) generates adenosine triphosphate (ATP), which serves as an energy source for numerous critical cellular activities. However, the ETC also generates deleterious reactive oxygen species (ROS) as a natural byproduct of oxidative phosphorylation. ROS are considered the major cause of aging because they damage proteins, lipids, and DNA by oxidation. We analyzed the chronological life span, growth phenotype, mitochondrial membrane potential (MMP), and intracellular ATP and mitochondrial superoxide levels of 33 single ETC component-deleted strains during the chronological aging process. Among the ETC mutant strains, 14 (sdh1Δ, sdh2Δ, sdh4Δ, cor1Δ, cyt1Δ, qcr7Δ, qcr8Δ, rip1Δ, cox6Δ, cox7Δ, cox9Δ, atp4Δ, atp7Δ, and atp17Δ) showed a significantly shorter life span. The deleted genes encode important elements of the ETC components succinate dehydrogenase (complex II) and cytochrome c oxidase (complex IV), and some of the deletions lead to structural instability of the membrane-F1F0-ATP synthase due to mutations in the stator stalk (complex V). These short-lived strains generated higher superoxide levels and produced lower ATP levels without alteration of MMP. In summary, ETC mutations decreased the life span of yeast due to impaired mitochondrial efficiency.

Tumor-specific delivery of siRNA using supramolecular assembly of hyaluronic acid nanoparticles and 2b RNA-binding protein/siRNA complexes.

Anticancer therapeutics delivering exogenous siRNA have been explored to suppress the tumor-associated genes, but several limitations of siRNA delivery such as tumor-targeted delivery, controlled siRNA release at the sites of interest, or instabilities of siRNA in physiological fluids should be preferentially addressed for its clinical applications. As an attempt to meet these criteria, we designed a supramolecular assembly, which was composed of cholesterol-bearing hyaluronic acid (HA-Chol) conjugates and 2b RNA-binding protein (2b)/siRNA complexes. In contrast to the traditional siRNA polyplexes using electrostatic interactions, HA-Chol nanoparticles, as a results of self-assembly of HA-Chol conjugates, provide the hydrophobic core that acts as the container for 2b protein/siRNA complexes, where a high affinity of 2b protein for siRNA could neutralize the negative-charged siRNA. Here, we investigated the potential of HA-Chol/2b/siRNA complexes as the siRNA carriers that provide encapsulation, protection, and targeted delivery of siRNA. The HA-Chol nanoparticles could selectively deliver 2b protein/siRNA complexes to the tumor cells with up-regulated CD44 receptors and suppress the expression of target gene. The pH-associated binding properties of siRNA for 2b proteins allowed the controlled release of siRNA in the endocytic compartments, and ultimately the released siRNA could obtain the RNAi acitivities in the cells, whereas the encapsulated 2b proteins still stayed within the HA-Chol nanoparticles. Our delivery systems demonstrate the promising potential of the efficient siRNA carriers in the anticancer therapeutic applications.

Enhancement of mitochondrial function correlates with the extension of lifespan by caloric restriction and caloric restriction mimetics in yeast.

Caloric restriction mimetics (CRMs) have been developed to mimic the effects of caloric restriction (CR). However, research reports for the effects of CRMs are often times inconsistent across different research groups. Therefore, in this study, we compared seven identified CRMs which extend the lifespans of various organisms including caffeine, curcumin, dapsone, metformin, rapamycin, resveratrol, and spermidine to CR for mitochondrial function in a single model, Saccharomyces cerevisiae. In this organism, rapamycin extended chronological lifespan (CLS), but other CRMs failed to extend CLS. Rapamycin enhanced mitochondrial function like CR did, but other CRMs did not. Both CR and rapamycin worked on mitochondrial function, but they worked at different windows of time during the chronological aging process.

Self-assembled amphiphilic DNA-cholesterol/DNA-peptide hybrid duplexes with liposome-like structure for doxorubicin delivery.

DNA nanoparticles have been proposed for drug encapsulation and intracellular delivery, but it has remained a challenge to rationally design DNA nanoparticles for delivery of drug to human cancer cells, not to normal cells. In this study, we synthesized an amphiphilic DNA hybrid duplex by using Watson-Crick base pairing and DNA bioconjugation with cholesterol or tLyp-1 tumor-homing peptide. The resulting amphiphilic DNA hybrid duplexes can self-assemble in an aqueous solution into liposome-like nanoparticles (c-DNA-p nanoparticles) with the exposure of tLyp-1 peptides to their outside. As a nanocarrier for doxorubicin, c-DNA-p nanoparticles can efficiently intercalate doxorubicin and also show the pH-dependent complexing/dissociation behaviors with doxorubicin, resulting in release of doxorubicin into cytosol after cell uptake. Moreover, tLyp-1 peptides with cell penetrating properties and specific binding ability for Neuropilin-1 receptors enable doxorubicin-loaded c-DNA-p nanoparticles to be delivered into the target cells through the NRP-1-dependent internalization pathway. Here, we demonstrated the targeted delivery of doxorubicin to MDA-MB231 breast cancer cells, compared to HFF normal cells. These results provide an alternative approach to specifically delivering doxorubicin into targeted cells for cancer therapy as well as controlling drug release under the acidic conditions such as endosomes or lysosomes.

Efficient siRNA delivery into tumor cells by p19-YSA fusion protein.

For the efficient cytoplasmic delivery of siRNA in a receptor-specific fashion, we designed a p19-YSA fusion protein composed of p19 RNA binding protein and ephrin mimetic peptide (YSA peptide). The resulting recombinant protein had the high affinity for EphA2 receptor overexpressed on cancer cells as well as the complexing ability with siRNA, thus leading to tumor-targeted delivery of siRNA. The buried structure of siRNA within p19-YSA/siRNA complexes allowed the bound siRNAs to be protected from the external RNases, resulting in the enhanced stability of siRNA in serum conditions. The p19-YSA carriers could complex with siRNA in a size-dependent and sequence-independent manner and showed the pH-dependent complexing/dissocation behaviors with siRNA. In contrast to electrostatic interaction-mediated siRNA delivery systems such as cationic polymers/siRNA or cationic polypeptides/siRNA complexes, the bound siRNA within p19-YSA/siRNA complexes showed enhanced stability against large polyanions found outside cells, due to the nanomolar levels of affinity. Here, we demonstrated the superior efficiency of p19-YSA/siRNA complexes in RFP gene silencing, compared to untreated cells. These results provide an alternative approach to enhance the stability of siRNA as well as to achieve the targeted siRNA delivery.

Systemic delivery of siRNA by chimeric capsid protein: tumor targeting and RNAi activity in vivo.

Recently, we reported that a chimeric capsid protein assembled into a macromolecular container-like structure with capsid shell and the resulting siRNA/capsid nanocarrier complexes efficiently suppressed RFP gene expression in the cell culture system. To extend RNAi to the in vivo applications, we here demonstrated that the siRNA/capsid nanocarrier complexes could have tumor-specific targeting ability in vivo as well as the increased stability of siRNA during body circulation. When systemically administered, our siRNA/capsid nanocarrier complexes delivered siRNA to tumor tissues and efficiently suppressed RFP gene expression in tumor-bearing mice. The enhanced longevity of siRNA in vivo could be explained by shielding effect derived from the capsid shell, where the encapsulated siRNAs are protected from nucleases in plasma. The multivalent RGD peptides on shell surface, as a result of self-assembling of capsid protein subunits, showed efficient delivery of siRNA to the tumor tissues in vivo, due to the RGD-mediated binding to integrin receptors overexpressed on tumor cells. Moreover, the prolonged in vivo circulation time of our siRNA/capsid nanocarrier complexes increased the potential to serve as siRNA carriers for optimal in vivo RNAi. These results provide an alternative approach to systemically deliver siRNA to the tumor sites as well as to enhance the stability of siRNA in vivo. Therefore, our results revealed the promising potential of our capsid nanocarrier system as a therapeutic siRNA carrier for cancer treatment.

N-myc downstream-regulated gene 1 is involved in the regulation of cystogenesis in transgenic mice overexpressing human PKD2 gene.

Autosomal dominant polycystic kidney disease (ADPKD) is an inheritable and progressive kidney disease featured by the formation of fluid-filled cysts. In a previous study, transgenic mice overexpressing human PKD2 gene were produced as an ADPKD animal model. To select genes controlled by PKD2, 2DE was performed using kidney tissues of 12- and 18-month-old transgenic mice. The protein localization was detected by immunohistochemistry, and 3D culture was utilized to observe in vitro cystogenesis. As a result, N-myc downstream-regulated gene 1 (NDRG1) was chosen as a candidate regulator gene of cystogenesis. NDRG1 is an intracellular protein involved in cellular proliferation and differentiation. This gene was expressed much higher in the kidney of hPKD2 TG mice. Also, the high level of NDRG1 protein was detected in the cyst lining epithelial cells. The hypothesis that PKD2 gene regulates NDRG1 expression was supported, and NDRG1 knockdown resulted in attenuation of cyst growth in vitro. Furthermore, NDRG1 knockdown suppressed cellular growth in mouse inner medullary collecting duct-3 cells. We found that early growth response 1, a transcription factor that binds to the NDRG1 promoter, was mediated in the NDRG1 expression regulation by PKD2. In this study, we found the novel gene that was involved in cystogenesis, which will provide the new insight in ADPKD.

Chimeric capsid protein as a nanocarrier for siRNA delivery: stability and cellular uptake of encapsulated siRNA.

For the efficient cytoplasmic delivery of siRNA, we designed a chimeric capsid protein composed of a capsid shell, integrin targeting peptide, and p19 RNA binding protein. This recombinant protein assembled into a macromolecular container-like structure with capsid shell and provided a nanocarrier for siRNA delivery. Our capsid nanocarriers had dual affinity both for siRNA within the interior and integin receptors on the exterior, and the capsid shell structure allowed the encapsulated siRNAs to be protected from the external nucleases, leading to the enhanced stability of siRNA in serum conditions. The capsid nanocarriers could complex with siRNA in a size-dependent and sequence-independent manner and showed the pH-dependent complexing/dissocation behaviors with siRNA. Moreover, RGD peptides on the exterior surface of the capsid shell enabled the capsid nanocarriers to deliver siRNA into the cytosol of the target cells. Here, we demonstrated the superior efficiency of our siRNA/capsid nanocarrier complexes in RFP gene silencing, compared to untreated cells. These results provide an alternative approach to enhancing the stability of siRNA as well as to achieving targeted siRNA delivery.

A monitoring method for Atg4 activation in living cells using peptide-conjugated polymeric nanoparticles.

To date, several principal methods are presently used to monitor the autophagic process, but they have some potential experimental pitfalls or limitations that make them not applicable to living cells. In order to improve on the currently developed detection methods for autophagy, we report here fluorescent peptide-conjugated polymeric nanoparticles loaded with a lysosome staining dye in their core. The fluorescent peptide is designed to be specifically cleaved by the Atg4 cysteine protease, which plays a crucial role in autophagy activation. In this study, we demonstrate that peptide-conjugated polymeric nanoparticles can be used to visualize Atg4 activity in both cell-free and cell culture systems. The fluorescence imaging of cells incubated with nanoparticles demonstrates that Atg4 activity is activated in the autophagy-induced conditions, but suppressed in the autophagy-inhibited conditions. These results indicate that Atg4 activity is correlated with autophagic flux through its own regulatory pathway. Therefore, our strategy provides an alternative detection method that can clearly distinguish between an "autophagy active" and "autophagy inactive" state in cultured cells. As our nanoparticles are highly cell-permeable and biocompatible, this detection system has general applicability to living cells and can be extended to cell-based screening to evaluate newly developed compounds.

Murine immune responses to a Plasmodium vivax-derived chimeric recombinant protein expressed in Brassica napus.

BACKGROUND: To develop a plant-based vaccine against Plasmodium vivax, two P. vivax candidate proteins were chosen. First, the merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen that is currently considered a strong vaccine candidate. Second, the circumsporozoite protein (CSP), a component of sporozoites that contains a B-cell epitope. METHODS: A synthetic chimeric recombinant 516 bp gene encoding containing PvMSP-1, a Pro-Gly linker motif, and PvCSP was synthesized; the gene, named MLC, encoded a total of 172 amino acids. The recombinant gene was modified with regard to codon usage to optimize gene expression in Brassica napus. The Ti plasmid inducible gene transfer system was used for MLC chimeric recombinant gene expression in B. napus. Gene expression was confirmed by polymerase chain reaction (PCR), beta-glucuronidase reporter gene (GUS) assay, and Western blot. RESULTS: The MLC chimeric recombinant protein expressed in B. napus had a molecular weight of approximately 25 kDa. It exhibited a clinical sensitivity of 84.21% (n=38) and a clinical specificity of 100% (n=24) as assessed by enzyme-linked immunosorbent assay (ELISA). Oral immunization of BALB/c mice with MLC chimeric recombinant protein successfully induced antigen-specific IgG1 production. Additionally, the Th1-related cytokines IL-12 (p40), TNF, and IFN-γ were significantly increased in the spleens of the BALB/c mice. CONCLUSIONS: The chimeric MLC recombinant protein produced in B. napus has potential as both as an antigen for diagnosis and as a valuable vaccine candidate for oral immunization against vivax malaria.

Laparoscopic ureteroureteral anastomosis for distal ureteral injuries during gynecologic laparoscopic surgery.

STUDY OBJECTIVE: To estimate the feasibility and surgical outcomes of laparoscopic ureteroureteral for treatment of distal ureteral injuries. DESIGN: Retrospective clinical study (Canadian Task Force classification II-2). SETTING: University teaching hospital. PATIENTS: Four women with ureteral transection or ureterovaginal fistula. INTERVENTION: Laparoscopic ureteroureteral . MEASUREMENTS AND MAIN RESULTS: Median age of patients was 44 (range, 33-63) years, and median operating time was 110 (range, 85-150) minutes. There were no conversions to laparotomy. No intraoperative or postoperative complications occurred. Follow-up ranged from 20 to 46 months. All patients have been asymptomatic, and follow-up intravenous pyelograms and ultrasound examinations have been normal. CONCLUSION: Laparoscopic ureteroureteral anastomosis is an alternative surgical option in women with distal ureteral injuries during gynecologic laparoscopic surgery.

Ionizing radiation-induced micronucleus formation is mediated by reactive oxygen species that are produced in a manner dependent on mitochondria, Nox1, and JNK.

Ionizing radiation (IR) is known to induce genotoxic damage to DNA, chromosomes, and the nucleus. However, the damage that IR causes to the nucleus has received much less attention. Given that reactive oxygen species (ROS) are involved in IR-induced DNA breaks and chromosomal aberrations, this study examined the role of ROS in IR-induced damage to the nucleus. Human Jurkat T cells were irradiated with gamma-rays at a dose of 2.5 Gy, which resulted in a dramatic increase in both the cellular ROS levels and the number of micronuclei. This latter event was attenuated when the IR-induced ROS were eliminated through the exogenous application of an antioxidant enzyme catalase. The ability of IR to induce the accumulation of ROS and micronucleus formation was also reduced either when the cells were irradiated in the presence of rotenone, a mitochondrial respiratory chain inhibitor, or when the cellular Nox1 levels were reduced by RNA interference. These results suggest that IR stimulates both the mitochondria and Nox1 to produce ROS, and that these ROS are involved in the IR-induced formation of micronuclei. IR also activated c-Jun N-terminal kinase (JNK), which was reversed by catalase, rotenone, or Nox1 RNA interference. SP600125, a JNK-specific inhibitor, suppressed the IR-induced accumulation of ROS. This inhibitor consistently attenuated the IR-induced formation of micronuclei. Therefore, ROS and JNK appear to act in a positive mutual manner in IR-induced signaling processes. Overall, IR appears to induce the formation of micronuclei by inducing ROS through mitochondria, Nox1, and JNK.