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Insects comprise more than a million species and many authors have attempted to explain this success by evolutionary innovations. A much overlooked evolutionary novelty of insects is the serosa, an extraembryonic epithelium around the yolk and embryo. We have shown previously that this epithelium provides innate immune protection to eggs of the beetle Tribolium castaneum. It remained elusive, however, whether this immune competence evolved in the Tribolium lineage or is ancestral to all insects. Here, we expand our studies to two hemimetabolous insects, the bug Oncopeltus fasciatus and the swarming grasshopper Locusta migratoria . For Oncopeltus , RNA sequencing reveals an extensive response upon infection, including the massive upregulation of antimicrobial peptides (AMPs). We demonstrate antimicrobial activity of these peptides using in vitro bacterial growth assays and describe two novel AMP families called Serosins and Ovicins. For both insects, quantitative polymerase chain reaction shows immune competence of the eggs when the serosa is present, and in situ hybridizations demonstrate that immune gene expression is localized in the serosa. This first evidence from hemimetabolous insect eggs suggests that immune competence is an ancestral property of the serosa. The evolutionary origin of the serosa with its immune function might have facilitated the spectacular radiation of the insects. This article is part of the theme issue ‘Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom’.

This study tested the hypothesis that preactivated and disaggregated shape-changed platelet (PreD-SCP) therapy significantly protected rat kidney from ischemia-reperfusion (IR) injury. Adult-male Sprague-Dawley rats (n = 24) were equally categorized into Groups 1 (sham-operated control [SC]), 2 (SC + PreD-SCP), 3 (IR only), and 4 (IR + PreD-SCP). By 72 hr after IR procedure, the circulatory levels of creatinine, blood urine nitrogen and inflammatory biomarkers (interleukin [IL]-6/tumor necrosis factor [TNF]-α), and ratio of urine protein to urine creatinine were significantly higher in Group 3 than in other groups and significantly higher in Group 4 than in Groups 1 and 2, but they showed no different between Groups 1 and 2 (all p < .001). The microscopic findings showed that the expressions of kidney injury score, cellular inflammation (MMP-9/CD14//F4/80), and fibrotic area were identical to the circulatory inflammation, whereas the integrity of podocyte components (ZO-1/synaptopodin/podocin) exhibited an opposite to circulatory inflammation among the four groups (all p < .0001). The protein expressions of inflammatory (TNF-α/IL-1s/NF-κB/iNOS/TRAF6/MyD88/TLR-4), apoptotic/cell death (mitochondrial Bax/cleaved caspase-3/p-53), oxidized protein, mitogen-activated protein kinase family (p-38/p-JNK/p-c-JUN), and mitochondrial-damaged biomarkers displayed a similar pattern, whereas the antiapoptotic (Bcl-2/Bcl-XL) and integrity of mitochondrial biomarkers followed an opposite trend to circulatory inflammation among the four groups (all p < .001). PreD-SCP therapy effectively protected the kidney against IR injury.

Background This study tested the hypothesis that combined melatonin (Mel) and adipose-derived mesenchymal stem cells (ADMSCs) treatment was superior to either one alone on protecting the testis against acute testicular torsion-induced ischemia-reperfusion (TTIR) injury. Methods and results Male adult SD rats (n = 30) were equally categorized into group 1 (sham-operated control), group 2 [TTIR/by torsion of right/left testis (i.e., ischemia) with rotated 720° counterclockwise for 2 h, then detorsion (i.e., reperfusion) to the original position for 72 h], group 3 (TTIR + Mel/intraperitoneal administration/50 mg/kg at 30 min after ischemia, followed by 20 mg at 3 h and days 1/2/3 after TTIR), group 4 (TTIR + ADMSCs/1.2 × 106 cells/by tail-vein administration at 30 min after ischemia, followed by days 1/2 TTIR), and group 5 (TTIR + Mel + ADMSCs/tail-vein administration). The result showed that the protein expressions of oxidative-stress (NOX-1/NOX-2/oxidized-protein), apoptotic/mitochondrial-damaged (mitochondrial-Bax/cleaved-caspase3/cleaved-PARP/cytosolic-cytochrome C), and fibrotic (TGF-ß/Smad3) biomarkers as well as testicular damage scores were lowest in group 1, highest in group 2, and significantly higher in groups 3/4 than in group 5, but they showed no difference between groups 3/4, whereas the protein expressions of androgen receptor (AR) and vimentin showed an opposite pattern of oxidative stress (all p < 0.0001). The cellular levels of inflammation (MMP-9/MPO/CD68) exhibited an identical pattern, whereas the numbers of Sertoli cells, α-tubulin, AR and vimentin as well as thickness of seminiferous tubule exhibited an opposite pattern of oxidative stress among the groups (all p < 0.0001). Conclusion Mel-ADMSCs effectively protected the testis against TTIR injury.

Dorsoventral pattering relies on Toll and BMP signalling in all insects studied so far, with variations in the relative contributions of both pathways.Drosophilaand the beetleTriboliumshare extensive dependence on Toll, while representatives of more distantly related lineages like the waspNasoniaand bugOncopeltusrely more strongly on BMP signalling. Here, we show that in the cricketGryllus bimaculatus, an evolutionarily distant outgroup, Toll has, like inDrosophila, a direct patterning role for the ventral half of the embryo. In addition, Toll polarises BMP signalling, although this does not involve the conserved BMP inhibitor Sog/Chordin. Finally, Toll activation relies on ovarian patterning mechanisms with striking similarity toDrosophila. Our data suggest two surprising hypotheses: (1) that Toll's patterning function inGryllusandDrosophilais the result of convergent evolution or (2) aDrosophila-likesystem arose early in insect evolution and was extensively altered in multiple independent lineages.

Background: This study assessed the expression of Jagged2 in human bladder cancer (BC) tested the hypothesis that melatonin (Mel) inhibited the tumorigenesis of BC cells mainly through downregulating the Notch/Jagged2 and PI3K/AKT/mTOR/MMPs(2&9) signaling pathways. Methods and Results: Tissue array from BC patients showed that the gene and protein expressions of JAG2/Jagged2 were significantly upregulated from T1 to T3 (primary tumor size) and from stage I to III (all p

Background: The Hemiptera (aphids, cicadas, and true bugs) are a key insect order, with high diversity for feeding ecology and excellent experimental tractability for molecular genetics. Building upon recent sequencing of hemipteran pests such as phloem-feeding aphids and blood-feeding bed bugs, we present the genome sequence and comparative analyses centered on the milkweed bug Oncopeltus fasciatus, a seed feeder of the family Lygaeidae. Results: The 926-Mb Oncopeltus genome is well represented by the current assembly and official gene set. We use our genomic and RNA-seq data not only to characterize the protein-coding gene repertoire and perform isoform-specific RNAi, but also to elucidate patterns of molecular evolution and physiology. We find ongoing, lineage-specific expansion and diversification of repressive C2H2 zinc finger proteins. The discovery of intron gain and turnover specific to the Hemiptera also prompted the evaluation of lineage and genome size as predictors of gene structure evolution. Furthermore, we identify enzymatic gains and losses that correlate with feeding biology, particularly for reductions associated with derived, fluid nutrition feeding. Conclusions: With the milkweed bug, we now have a critical mass of sequenced species for a hemimetabolous insect order and close outgroup to the Holometabola, substantially improving the diversity of insect genomics. We thereby define commonalities among the Hemiptera and delve into how hemipteran genomes reflect distinct feeding ecologies. Given Oncopeltus’s strength as an experimental model, these new sequence resources bolster the foundation for molecular research and highlight technical considerations for the analysis of medium-sized invertebrate genomes., La lista completa de autores puede verse en el archivo asociado., Facultad de Ciencias Exactas, Centro Regional de Estudios Genómicos

Background: This study tested the hypothesis that Entresto could safely and effectively preserve heart and kidney function in rats with cardiorenal syndrome (CRS) induced by 5/6 nephrectomy and intra-peritoneal doxorubicin administration (accumulated dosage up to 7.5 mg/kg) together with daily high-protein-diet (HPD).Methods and Results: Adult male Sprague-Dawley rats (n = 24) were equally categorized into Group 1 (sham-operated control + HPD), Group 2 (CRS + HPD) and Group 3 [CRS + HPD + Entresto (100 mg/kg/day orally) since Day 14 after CRS induction] and euthanized by Day 63 after CRS induction. By Day 63, circulatory BUN and creatinine levels and ratios of urine protein to creatinine were significantly higher in Group 2 than those in Groups 1 and 3, and significantly higher in Group 3 than in Group 1, whereas left-ventricular ejection fraction and kidney weight showed an opposite pattern among all groups (all p

We investigated the cardioprotective effect of melatonin (Mel) and exendin-4 (Ex4) treatment in a rat model of cardiorenal syndrome (CRS). Male-adult SD rats (n=48) were randomly and equally divided into sham-control (SC), dilated cardiomyopathy (DCM) (doxorubicin 7 mg/kg i.p. every five days/ 4 doses), CRS (defined as DCM + CKD) only, CRS-Mel (20 mg/kg/day), CRS-Ex4 (10 μg/kg/day) and CRS-Mel-Ex4. In-vitro results showed protein expressions of oxidative-stress (NOX-1/NOX-2/oxidized protein), DNA/mitochondrial-damaged (γ-H2AX/cytosolic cytochrome-C) and apoptotic (cleaved caspase-3/PARP) biomarkers, and senescence (β-galactosidase cells) were upregulated, whereas mitochondrial ATP level was decreased in doxorubicin/ p-Cresol-treated H9C2 cells that were revised by Mel and Ex4 treatments (all p

In this study, we tested the hypothesis that a combined adipose-derived mesenchymal stem cell (ADMSC) and ADMSC-derived exosome therapy protected rat kidney from acute ischemia-reperfusion (IR) injury (i.e., ligation of both renal arteries for 1h and reperfusion for 72h prior to euthanization).Adult-male SD rats (n=40) were equally categorized into group 1 (sham control), group 2 (IR), group 3 [IR+exosome (100μg)], group 4 [IR+ADMSC (1.2×10(6) cells)], and group 5 (IR-exosome-ADMSC). All therapies were performed at 3h after IR procedure from venous administration. By 72h, the creatinine level and kidney injury score were the lowest in group 1 and the highest in group 2, significantly higher in group 3 than in groups 4 and 5, and significantly higher in group 4 than in group 5 (all P0.0001). The protein expression of inflammatory (TNF-α/NF-κB/IL-1β/MIF/PAI-1/Cox-2), oxidative-stress (NOX-1/NOX-2/oxidized protein), apoptotic (Bax/caspase-3/PARP), and fibrotic (Smad3/TGF-β) biomarkers showed an identical pattern, whereas the anti-apoptotic (Smad1/5, BMP-2) and angiogenesis (CD31/vWF/angiopoietin) biomarkers and mitochondrial cytochrome-C showed an opposite pattern of creatinine level among the five groups (all P0.001). The microscopic findings of glomerular-damage (WT-1), renal tubular-damage (KIM-1), DNA-damage (γ-H2AX), inflammation (MPO/MIF/CD68) exhibited an identical pattern, whereas the podocyte components (podocin/p-cadherin/synaptopodin) displayed a reversed pattern of creatinine level (all P0.0001).Combined exosome-ADMSC therapy was superior to either one for protecting kidney from acute IR injury.

One of the key morphogenetic processes used during development is the controlled intercalation of cells between their neighbors. This process has been co-opted into a range of developmental events, and it also underlies an event that occurs in each major group of bilaterians: elongation of the embryo along the anterior-posterior axis [1]. In Drosophila, a novel component of this process was recently discovered by Paré et al., who showed that three Toll genes function together to drive cell intercalation during germband extension [2]. This finding raises the question of whether this role of Toll genes is an evolutionary novelty of flies or a general mechanism of embryonic morphogenesis. Here we show that the Toll gene function in axis elongation is, in fact, widely conserved among arthropods. First, we functionally demonstrate that two Toll genes are required for cell intercalation in the beetle Tribolium castaneum. We then show that these genes belong to a previously undescribed Toll subfamily and that members of this subfamily exhibit striped expression (as seen in Tribolium and previously reported in Drosophila [3-5]) in embryos of six other arthropod species spanning the entire phylum. Last, we show that two of these Toll genes are required for normal morphogenesis during anterior-posterior embryo elongation in the spider Parasteatoda tepidariorum, a member of the most basally branching arthropod lineage. From our findings, we hypothesize that Toll genes had a morphogenetic function in embryo elongation in the last common ancestor of all arthropods, which existed over 550 million years ago.

We tested the hypothesis that melatonin (Mel) enhances exogenous mitochondria (Mito) treatment against rodent hepatic ischemia-reperfusion (IR) injury. In vitro study utilized three groups of hepatocytes (i.e. nontreatment, menadione, and menadione-melatonin treatment, 4.0 × 10(5) each), while in vivo study used adult male Sprague Dawley rats (n = 40) equally divided into sham-control (SC), IR (60-min left-lobe ischemia + 72-hr reperfusion), IR-Mel (melatonin at 30 min/6/8 hr after reperfusion), IR-Mito (mitochondria 15,000 μg/rat 30 min after reperfusion), and IR-Mel-Mito. Following menadione treatment in vitro, oxidative stress (NOX-1/NOX-2/oxidized protein), apoptotic (cleaved caspase-3/PARP), DNA damage (γ-H2AX/CD90/XRCC1), mitochondria damage (cytosolic cytochrome c) biomarkers, and mitochondrial permeability transition were found to be lower, whereas mitochondrial cytochrome c were found to be higher in hepatocytes with melatonin treatment compared to those without (all P < 0.001). In vivo study demonstrated highest liver injury score and serum AST in IR group, but lowest in SC group and higher in IR-Mito group than that in groups IR-Mel and IR-Mel-Mito, and higher in IR-Mel group than that in IR-Mel-Mito group after 72-hr reperfusion (all P < 0.003). Protein expressions of inflammatory (TNF-α/NF-κB/IL-1β/MMP-9), oxidative stress (NOX-1/NOX-2/oxidized protein), apoptotic (caspase-3/PARP/Bax), and mitochondria damage (cytosolic cytochrome c) biomarkers displayed an identical pattern, whereas mitochondria integrity marker (mitochondrial cytochrome c) showed an opposite pattern compared to that of liver injury score (all P < 0.001) among five groups. Microscopically, expressions of apoptotic nuclei, inflammatory (MPO(+) /CD68(+) /CD14(+) cells), and DNA damage (γ-H2AX(+) cells) biomarkers exhibited an identical pattern compared to that of liver injury score (all P < 0.001) among five groups. Melatonin-supported mitochondria treatment offered an additional benefit of alleviating hepatic IR injury.

This study tested the hypothesis that early administration of empagliflozin (Empa), an inhibitor of glucose recycling in renal tubules, could preserve heart function in cardiorenal syndrome (CRS) in rat. Chronic kidney disease (CKD) was caused by 5/6 subtotal nephrectomy and dilated cardiomyopathy (DCM) by doxorubicin (DOX) treatment. In vitro results showed that protein expressions of cleaved-caspase3 and autophagy activity at 24 h/48 h in NRK-52P cells were significantly upregulated by para-Creso treatment; these were significantly downregulated by Empa treatment. Flow cytometric analysis showed that annexin-V (i.e., early/late apoptosis) in NRK-52P cells expressed an identical pattern to cleaved-caspase3 between the two groups (all p

Background We tested the hypothesis that extracorporeal shockwave treatment (ESWT) can abolish inflammation and restore urothelial barrier integrity in acute interstitial cystitis by upregulating the fatty acid receptor GPR120. Methods A total of 30 female Sprague-Dawley rats were categorized into five groups: (1) sham-operated rats (SC); (2) rats treated with ESWT (SC + ESWT); (3) rats with bladder irritation using 150 mg/kg cyclophosphamide through intraperitoneal injection; (4) cyclophosphamide rats treated with ESWT (cyclophosphamide+ESWT); (5) cyclophosphamide rats treated with GPR120 agonist (cyclophosphamide+GW9508). Results On Day 3, urine and bladder specimens were collected for biochemical, histopathological, immunological, and immunoblotting analysis. Following stimulation with cyclophosphamide, the inhibition of the elevated levels of TAK1/NF-κB and phospho-TAK1/NF-κB by ESWT and GPR120 agonists in RT4 cells was associated with a suppression of NF-κB translocation from the cytosol to the nucleus. Accordingly, this anti-inflammatory effect was abolished by GPR120 antagonist and knockdown of GPR120. Histologically, bladder inflammation in cyclophosphamide-treated rats was suppressed by GW9508 or ESWT. Masson’s trichrome and Sirius red staining revealed that cyclophosphamide treatment enhanced synthesis of extracellular matrix in rats that was reversed by GW9508 or ESWT. Upregulated pro-inflammatory mediators and cytokines in the cyclophosphamide-treated rats were also suppressed in the GW9508- or ESWT-treated rats. The significantly increased inflammatory cell infiltration as well as the impaired urothelial integrity of the bladder after cyclophosphamide treatment were reversed by treatment with GW9508 or ESWT. Conclusions These findings suggest that GPR120, the sensing receptor for ESWT, may be useful in the treatment of interstitial cystitis by inhibiting inflammatory response in bladder cells.

We tested whether combined melatonin (Mel) and exendin-4 (Ex4) treatment can better preserve glomerular structural integrity after ischemia-reperfusion (IR) injury compared with either alone. Adult male Sprague Dawley rats (n = 50) were equally divided into sham control (SC), IR, IR-Ex4 (10 μg/kg subcutaneously 30 min after reperfusion and daily for 5 days), IR-Mel (20 mg/kg intraperitoneally at 30 min postreperfusion and 50 mg/kg at 6 and 18 hr), and IR-Ex4-Mel were euthanized at day 14. Serum creatinine level and urine protein-to-creatinine ratio at days 3 and 14 were highest in IR group and lowest in SC, significantly higher in IR-Ex4 and IR-Mel groups than in IR-Ex4-Mel group (all P < 0.001) without significant difference between IR-Ex4 and IR-Mel groups. Changes in podocyte injury score (PIS) and kidney injury score were highest in IR group and lowest in SC, significantly higher in IR-Ex4 and IR-Mel groups than in IR-Ex4-Mel, and significantly higher in IR-Mel group than in IR-Ex4 group (all P < 0.001). Immunohistochemical microscopic findings of the expressions of FSP-1 and WT-1 (two glomerular damage indicators) and KIM-1 and snail (two renal tubular-damaged indicators) showed an identical pattern, whereas the expressions of ZO-1, p-cadherin, podocin, dystroglycan, fibronectin, and synaptopodin (six indices of glomerular integrity) demonstrated an opposite pattern compared to that of PIS among five groups (all P < 0.001). Protein expressions of inflammatory (TNF-α/NF-κB/MMP-9) and oxidative stress (NOX-1, NOX-2, oxidized protein) biomarkers exhibited an identical pattern to that of PIS among five groups (all P < 0.001). Combined melatonin-exednin-4 therapy further protected glomerulus from IR injury.

Aim: Sitagliptin, an oral glucose-lowering agent, has been found to produce cardiovascular protection possibly via anti-inflammatory and anti-atherosclerotic activities of glucagon-like peptide-1 receptor (GLP-1). The aim of this study was to investigate whether sitagliptin protected the kidney function from acute ischemia-reperfusion (IR) injury in rats. Methods: Adult male SD rats were categorized into 4 groups: sham control, IR injury, IR+sitagliptin (300 mg/kg) and IR+sitagliptin (600 mg/kg). Acute renal IR injury of both kidneys was induced by clamping the renal pedicles for 1 h. The drug was orally administered at 1, 24 and 48 h after acute IR. Blood samples and 24-h urine were collected before and at 72 h after acute IR. Then the rats were sacrificed, and the kidneys were harvested for biochemical and immunohistochemical studies. Results: Acute IR procedure markedly increased serum levels of creatinine and BUN and the ratio of urine protein to creatinine. The kidney injury score, inflammatory biomarkers (MMP-9, TNF-α and NF-κB) levels and CD68+ cells in IR kidneys were considerably increased. The expression of oxidized protein, reactive oxygen species (NOX-1, NOX-2) and apoptosis proteins (Bax, caspase-3, PARP) in IR kidneys was also significantly upregulated. All these pathological changes were suppressed by sitagliptin in a dose-dependent manner. Furthermore, the serum GLP-1 level, and the expression of GLP-1 receptor, anti-oxidant biomarkers (HO-1 and NQO-1 cells, as well as SOD-1, NQO-1 and HO-1 proteins), and angiogenesis markers (SDF-1α+ and CXCR4+ cells) in IR kidneys were significantly increased, and further upregulated by sitagliptin. Conclusion: Sitagliptin dose-dependently protects rat kidneys from acute IR injury via upregulation of serum GLP-1 and GLP-1 receptor expression in kidneys.

The potent immunosuppressive effect of systemic tacrolimus is limited by the high incidence of severe adverse effects, including nephrotoxicity and hypertension. Intravesical application of tacrolimus is hindered by its poor aqueous solubility, justifying the search for novel delivery platforms such as liposomes. We evaluated the pharmacokinetics of tacrolimus encapsulated in liposomes (lipo-tacrolimus), which is being developed as a potential orphan drug indication for hemorrhagic cystitis.A single dose of lipo-tacrolimus was instilled in the bladder with the rat under anesthesia. Also, tacrolimus was instilled intravesically or injected intraperitoneally in other rat groups. The tacrolimus dose was constant in all formulations at 200 μg/ml. At different times blood, urine and bladder samples were collected and stored at -80C until analysis. Tacrolimus levels in samples were analyzed using microparticle enzyme immunoassay II.The AUC of lipo-tacrolimus in serum at 0 to 24 hours was significantly lower than that of tacrolimus instillation or injection. Noncompartmental pharmacokinetic data analysis revealed maximum concentration of lipo-tacrolimus and tacrolimus in serum and urine at 1 and at 2 hours, respectively. Urine AUC(0-24) after intravesical administration was significantly higher than in the intraperitoneal group (p0.05). Bladder tacrolimus AUC(0-24) did not differ significantly between the groups.Single dose pharmacokinetics revealed that bladder instillation of liposome encapsulated tacrolimus significantly decreased systemic exposure to instilled tacrolimus as well as vehicle related toxicity. Intravesical liposomal tacrolimus may be a promising approach as an orphan drug indication for hemorrhagic cystitis.

The origin of germline cells was a crucial step in animal evolution. Therefore, in both developmental biology and evolutionary biology, the mechanisms of germline specification have been extensively studied over the past two centuries. However, in many animals, the process of germline specification remains unclear. Here, we show that in the cephalochordate amphioxus Branchiostoma floridae, the germ cell-specific molecular markers Vasa and Nanos become localized to the vegetal pole cytoplasm during oogenesis and are inherited asymmetrically by a single blastomere during cleavage. After gastrulation, this founder cell gives rise to a cluster of progeny that display typical characters of primordial germ cells (PGCs). Blastomeres separated at the two-cell stage grow into twin embryos, but one of the twins fails to develop this Vasa-positive cell population, suggesting that the vegetal pole cytoplasm is required for the formation of putative PGCs in amphioxus embryos. Contrary to the hypothesis that cephalochordates may form their PGCs by epigenesis, our data strongly support a preformation mode of germ cell specification in amphioxus. In addition to the early localization of their maternal transcripts in the putative PGCs, amphioxus Vasa and Nanos are also expressed zygotically in the tail bud, which is the posterior growth zone of amphioxus. Thus, in addition to PGC specification, amphioxus Vasa and Nanos may also function in highly proliferating somatic stem cells.

There still lacking effective treatment for bladder cancer. This study investigated whether melatonin (Mel) can suppress the growth and invasion of bladder cancer cells. Male C57B/L6 mice were categorized into control group (ie, subcutaneous injection of HT1197 bladder cancer cell line at the back] and treatment group [subcutaneous HT1197 cells + intraperitoneal Mel (100 mg/kg/d) from day 8 to day 21 after tumor cell injection]. In vitro Mel suppressed cell growth of four bladder cancer cell lines (ie, T24, RT4, HT1197, HT1376), cell migration in HT1197/HT1376, mitochondrial membrane potential (MMP) in T24 and colony formation in RT4 cells as well as arrested the cell cycle at G0 phase and inhibited the mitotic phase of T24 cells (all P < 0.0001). Protein expression of ZNF746 in RT4/T24 cells and protein expression phosphorylated (p)-AKT/MMP-2/MMP-9 in HT1197/HT1376 cells were reduced following Mel treatment (all P < 0.001). Transfection of T24 cells with plasmid-based shRNA (ie, ZNF746-silencing) downregulated the protein expression of MMP-9, cell growth, and invasion and attachment to endothelial cells but upregulated the colony formation (all P < 0.001). Mel suppressed oxidative stress and MMP but upregulated mitochondria mass in ZNF746-silenced T24 cells, whereas these parameters exhibited a similar patter to Mel treatment in ZNF746-silenced T24 cells (all P < 0.0001). In vivo study demonstrated that Mel treatment significantly suppressed cellular expressions of MMP-9/MMP-2, protein expressions of ZNF746/p-AKT, and tumor size (all P < 0.001). Mel treatment suppressed the growth, migration, and invasion of bladder carcinoma cells through downregulating ZNF746-regulated MMP-9/MMP-2 signaling.

The Fox genes are united by encoding a fork head domain, a deoxyribonucleic acid (DNA)-binding domain of the winged-helix type that marks these genes as encoding transcription factors. Vertebrate Fox genes are classified into 23 subclasses named from FoxA to FoxS. We have surveyed the genome of the amphioxus Branchiostoma floridae, identifying 32 distinct Fox genes representing 21 of these 23 subclasses. The missing subclasses, FoxR and FoxS, are specific to vertebrates, and in addition, B. floridae has one further group, FoxAB, that is not found in vertebrates. Hence, we conclude B. floridae has maintained a high level of Fox gene diversity. Expressed sequence tag and complementary DNA sequence data support the expression of 23 genes. Several linkages between B. floridae Fox genes were noted, including some that have evolved relatively recently via tandem duplication in the amphioxus lineage and others that are more ancient.

Androgen and the androgen receptor (AR) have been shown to play critical roles in male fertility. Our previous data demonstrated that mice lacking AR (AR(-/y)) revealed incomplete germ cell development and lowered serum testosterone levels, which resulted in azoospermia and infertility. However, the consequences of AR loss in Leydig cells remain largely unknown. Using a Cre-LoxP conditional knockout strategy, we generated a tissue-specific knockout mouse (L-AR(-/y)) with the AR gene deleted by the anti-Müllerian hormone receptor-2 (Amhr2) promoter driven Cre expressed in Leydig cells. Phenotype analyses show that the outside appearance of L-AR(-/y) mice was indistinguishable from wild type mice (AR(+/y)), but with atrophied testes and epididymis. L-AR(-/y) mice were infertile, with spermatogenic arrest predominately at the round spermatid stage and no sperm could be detected in the epididymis. L-AR(-/y) mice also have lower serum testosterone concentrations and higher serum leuteinizing hormone and follicle-stimulating hormone concentrations than AR(+/y) mice. Further mechanistic studies demonstrated that hypotestosteronemia in L-AR(-/y) mice is not caused by reducing numbers of Leydig cells, but instead by the alterations of several key steroidogenic enzymes, including 17beta-HSD3, 3beta-HSD6, and P450c17. Together, L-AR(-/y) mice provide in vivo evidence that functional AR in Leydig cells is essential to maintain normal spermatogenesis, testosterone production, and required for normal male fertility.

Androgens and the androgen receptor (AR) play important roles in male fertility, although the detailed mechanisms, particularly how androgen/AR influences spermatogenesis in particular cell types, remain unclear. Using a Cre-Lox conditional knockout strategy, we generated a tissue-specific knockout mouse with the AR gene deleted only in Sertoli cells (S-AR -/y ). Phenotype analyses show the S-AR -/y mice were indistinguishable from WT AR mice (B6 AR +/y ) with the exception of testes, which were significantly atrophied. S-AR -/y mice were infertile, with spermatogenic arrest predominately at the diplotene premeiotic stage and almost no sperm detected in the epididymides. S-AR -/y mice also have lower serum testosterone concentrations and higher serum leuteinizing hormone concentrations than B6 AR +/y mice. Further mechanistic studies demonstrated that S-AR -/y mice have defects in the expression of anti-Müllerian hormone, androgen-binding protein, cyclin A1, and sperm-1, which play important roles in the control of spermatogenesis and/or steroidogenesis. Together, our Sertoli cell-specific AR knockout mice provide in vivo evidence of the need for functional AR in Sertoli cells to maintain normal spermatogenesis and testosterone production, and ensure normal male fertility.

Apolipoprotein E (apoE) is synthesized in many tissues, and the liver is the primary site from which apoE redistributes cholesterol and other lipids to peripheral tissues. Here we demonstrate that the TR4 orphan nuclear receptor (TR4) can induce apoE expression in HepG2 cells. This TR4-mediated regulation of apoE gene expression was further confirmed in vivo using TR4 knockout mice. Both serum apoE protein and liver apoE mRNA levels were significantly reduced in TR4 knockout mice. Gel shift and luciferase reporter gene assays further demonstrated that TR4 can induce apoE gene expression via a TR4 response element located in the hepatic control region that is 15 kb downstream of the apoE gene. Furthermore our in vivo data from TR4 knockout mice prove that TR4 can also regulate apolipoprotein C-I and C-II gene expression via the TR4 response element within the hepatic control region. Together our data show that loss of TR4 down-regulates expression of the apoE/C-I/C-II gene cluster in liver cells, demonstrating important roles of TR4 in the modulation of lipoprotein metabolism.

This study tested whether combined therapy with melatonin and apoptotic adipose-derived mesenchymal stem cells (A-ADMSCs) offered additional benefit in ameliorating sepsis-induced acute kidney injury. Adult male Sprague-Dawley rats (n = 65) were randomized equally into five groups: Sham controls (SC), sepsis induced by cecal-ligation and puncture (CLP), CLP-melatonin, CLP-A-ADMSC, and CLP-melatonin-A-ADMSC. Circulating TNF-α level at post-CLP 6 hr was highest in CLP and lowest in SC groups, higher in CLP-melatonin than in CLP-A-ADMSC and CLP-melatonin-A-ADMSC groups (all P < 0.001). Immune reactivity as reflected in the number of splenic helper-, cytoxic-, and regulatory-T cells at post-CLP 72 hr exhibited the same pattern as that of circulating TNF-α among all groups (P < 0.001). The histological scoring of kidney injury and the number of F4/80+ and CD14+ cells in kidney were highest in CLP and lowest in SC groups, higher in CLP-melatonin than in CLP-A-ADMSC and CLP-melatonin-A-ADMSC groups, and higher in CLP-A-ADMSC than in CLP-melatonin-A-ADMSC groups (all P < 0.001). Changes in protein expressions of inflammatory (RANTES, TNF-1α, NF-κB, MMP-9, MIP-1, IL-1β), apoptotic (cleaved caspase 3 and PARP, mitochondrial Bax), fibrotic (Smad3, TGF-β) markers, reactive-oxygen-species (NOX-1, NOX-2), and oxidative stress displayed a pattern identical to that of kidney injury score among the five groups (all P < 0.001). Expressions of antioxidants (GR+, GPx+, HO-1, NQO-1+) were lowest in SC group and highest in CLP-melatonin-A-ADMSC group, lower in CLP than in CLP-melatonin and CLP-A-ADMSC groups, and lower in CLP-melatonin- than in CLP-A-ADMSC-tretaed animals (all P < 0.001). In conclusion, combined treatment with melatonin and A-ADMSC was superior to A-ADMSC alone in protecting the kidneys from sepsis-induced injury.

Although chronic exposure to environmental contaminants is hazardous to health, the association between chronic kidney disease (CKD) and nonylphenol (NP), a common environmental compound, remains unclear. This study tested the hypothesis that chronic NP exposure aggravated adenine (AD)-induced CKD that could be mitigated with rosuvastatin treatment. Fifty Wistar rats were randomly (n=10/each group) categorized into normal controls (N(C)), NP only (2.0mg/kg/day), AD only (0.25% AD in fodder), combined NP-AD, and NP-AD with rosuvastatin (20.0mg/kg/day) (NP-AD-R(OSU)). All animals received treatment for 24 weeks prior to being sacrificed. Results showed that ratio of urine protein to creatinine were increased in NP-AD group than in groups N(C), NP, and AD, but reduced in NP-AD-R(OSU) group compared with NP-AD group (all p

Background Successful reproductive efforts require the establishment of a situation favorable for reproduction that requires integration of both behavior and internal physiological events. TR4 nuclear receptor is known to be involved in male fertility via controlling spermatogenesis, yet its roles in regulating other biological events related to reproduction have not been completely revealed. Methods Male TR4 knockout (TR4-/-) and wild type mice were used for the sexual behavior and penile dysfunction studies. Mice were sacrificed for histological examination and corresponding genes profiles were analyzed by quantitative RT-PCR. Reporter gene assays were performed. Results We describe an unexpected finding of priapism in TR4-/- mice. As a transcriptional factor, we demonstrated that TR4 transcriptionally modulates a key enzyme regulating penis erection and neuronal nitric oxide synthese NOS (nNOS). Thereby, elimination of TR4 results in nNOS reduction in both mRNA and protein levels, consequently may lead to erectile dysfunction. In addition, male TR4-/- mice display defects in sexual and social behavior, with increased fear or anxiety, as well as reduced mounting, intromission, and ejaculation. Reduction of ER alpha, ER beta, and oxytocin in the hypothalamus may contribute to defects in sexual behavior and stress response. Conclusions Together, these results provide in vivo evidence of important TR4 roles in penile physiology, as well as in male sexual behavior. In conjunction with previous finding, TR4 represents a key factor that controls male fertility via regulating behavior and internal physiological events.

Background Reactive oxygen species are important mediators exerting toxic effects on various organs during ischemia-reperfusion (IR) injury. We hypothesized that adipose-derived mesenchymal stem cells (ADMSCs) protect the kidney against oxidative stress and inflammatory stimuli in rat during renal IR injury. Methods Adult male Sprague-Dawley (SD) rats (n = 24) were equally randomized into group 1 (sham control), group 2 (IR plus culture medium only), and group 3 (IR plus immediate intra-renal administration of 1.0 × 106 autologous ADMSCs, followed by intravenous ADMSCs at 6 h and 24 h after IR). The duration of ischemia was 1 h, followed by 72 hours of reperfusion before the animals were sacrificed. Results Serum creatinine and blood urea nitrogen levels and the degree of histological abnormalities were markedly lower in group 3 than in group 2 (all p < 0.03). The mRNA expressions of inflammatory, oxidative stress, and apoptotic biomarkers were lower, whereas the anti-inflammatory, anti-oxidative, and anti-apoptotic biomarkers were higher in group 3 than in group 2 (all p < 0.03). Immunofluorescent staining showed a higher number of CD31+, von Willebrand Factor+, and heme oxygenase (HO)-1+ cells in group 3 than in group 2 (all p < 0.05). Western blot showed notably higher NAD(P)H quinone oxidoreductase 1 and HO-1 activities, two indicators of anti-oxidative capacity, in group 3 than those in group 2 (all p < 0.04). Immunohistochemical staining showed higher glutathione peroxidase and glutathione reductase activities in group 3 than in group 2 (all p < 0.02) Conclusion ADMSC therapy minimized kidney damage after IR injury through suppressing oxidative stress and inflammatory response.

Background This study aimed at investigating the cellular and molecular impacts of bone marrow-derived mononuclear cells (BMCs) on regeneration of fibrotic liver in rats. Materials and Methods Thirty male adult Fisher rats were randomized into three groups: group 1 (normal controls, n = 10); group 2 (carbon tetrachloride-induced liver fibrosis, n = 10), and group 3 (liver fibrosis with portal venous infusion of autologous BMCs, 1 × 106, n = 10). After 7-d culturing, BMCs were characterized by flow cytometry. Groups 2 and 3 received BMC aspiration through bilateral femurs 5 d before hepatectomy. All animals received 70% hepatectomy, whereas only group 3 received a bolus of intra-portal BMC infusion 48 h after hepatectomy. Liver-to-body weight ratio, degree of fibrosis (Masson trichrome staining), oxidative stress, peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α), collagen I, tumor necrosis factor-α (TNF-α), and transforming growth factor- β (TGF-β) expressions were analyzed 14 d after hepatectomy. Immunohistochemical staining for albumin, α-smooth muscle actin, and CD31 was performed for tracing cellular differentiation. Results Cellular phenotypes typical of hepatocyte, hepatic stellate cell (HSC), and endothelial cell were identified in the engrafted BMCs. Liver-to-body weight ratio was enhanced with PGC-1α significantly preserved, whereas oxidative index, collagen I, α-SMA, TNF-α, and TGF-β expressions were all decreased in group 3 compared with group 2 (all P Conclusions This study demonstrated a positive impact of intra-portal BMC infusion in enhancing regeneration and reducing fibrosis of the regenerating fibrotic liver in rats through suppressing HSC activation and inflammatory cytokine expressions, preserving mitochondrial function and reducing oxidative stress.

We previously reported a very high cumulative incidence of urothelial carcinoma in Taiwanese kidney transplant recipients. Rapamycin, the inhibitor of mTOR Complex 1, provides alternative immunosuppressive therapy after kidney transplantation with less neoplastic potential. We examined the in vivo and in vitro effects of rapamycin on urothelial carcinoma.The rat model of urothelial carcinoma was induced by 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in Fischer F344 rats. The anti-tumor effect of rapamycin was assessed grossly, microscopically, and by Western blot analysis. The mechanism of rapamycin's attenuation of urothelial carcinoma was also evaluated by T24 cells.Rapamycin significantly reduced urinary bladder tumor growth in the rat model of 0.05% BBN-induced urothelial carcinoma (P0.001). The blood trough levels of rapamycin were correlated with the occurrence of urothelial carcinoma. In vitro, rapamycin also inhibited the cell proliferation, migration, and invasion, as well as the protein expression of vascular endothelial growth factor-A of T24 urothelial carcinoma cells, whereas rapamycin did not induce significant apoptosis in T24 cells. Rapamycin decreased the expression of phospho-mTOR, phospho-S6K, cyclin D1, and VEGF-A. Rapamycin also activated AKT in T24 cells in the rat model of urothelial carcinoma. The rapamycin-associated activation of AKT was inhibited by rictor siRNA, but not raptor siRNA.This study provides in vitro and in vivo evidence that rapamycin may inhibit the development of urothelial carcinoma. The present findings also suggest rictor-dependent AKT activation as a consequence of mTORC1 inhibition.

We investigated whether early combined cilostazol and bone marrow-derived endothelial progenitor cell (BMDEPC) treatment offers synergistic benefit in ameliorating monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) in rats. Male Sprague-Dawley rats (n = 10/group) were randomized to receive saline injection only (group 1), MCT (70 mg/kg) (group 2), and MCT plus cilostazol (20 mg/kg/day) (group 3), MCT plus BMDEPCs (2.0 x 10(6) cells) (group 4), and MCT plus combined cilostazol/BMDEPCs (group 5). Intravenous BMDEPCs and oral cilostazol were given on day 3 after MCT administration. By day 42, connexin43 protein expression in right ventricle (RV) was reduced in group 2 compared with other groups and also was decreased in groups 3 and 4 compared with groups 1 and 5 (all p < 0.05). In addition, mRNA expressions of matrix metalloproteinase-9, tumor necrosis factor-alpha, and caspase-3 were higher, whereas Bcl-2 and endothelial nitric-oxide synthase were lower in lung and RV in group 2 compared with the other groups (all p < 0.05). The number of alveolar sacs and lung arterioles was lower in group 2 than in other groups and lower in groups 3 and 4 than in group 5 (all p < 0.05). RV systolic pressure (RVSP) and weight were increased in group 2 compared with the other groups (all p < 0.0001). Moreover, RVSP and RV-to-left ventricle plus septum weight ratio were higher in groups 3 and 4 than in groups 1 and 5 (p < 0.001) but showed no difference between groups 1 and 5. In conclusion, early combined autologous BMDEPC/cilostazol treatment is superior to BMDEPC or cilostazol only for preventing MCT-induced PAH.

Purpose New-onset diabetes mellitus (PTDM), a major metabolic complication after renal transplantation, examined for incidence and risk factors. Methods The records of 358 renal transplant recipients with functioning grafts, from 1986 to 2006, were categorized into two groups according to the usage of tacrolimus (FK): FK-based (n = 120 patients) and non–FK-based (n = 238). Using Kaplan-Meier survival analysis and a Cox regression model, this study analyzed the cumulative incidence of PTDM and risk factors, including gender, age, and presence of hepatitis. Results Cumulative incidences of PTDM after 1, 3, and 5 years posttransplantation in the FK-based group were 11%, 18%, and 22%, respectively. In the non–FK-based group, the cumulative incidences were 5%, 9%, and 12% (P = .01). Taking into account the risk factors, the cumulative incidence of PTDM was significant among patients 51 years or older (odds ratio, 3.965; P = .005), but not with regard to gender or presence of hepatitis B and/or C. Overall cumulative incidence of PTDM in our series was 15% (54/358), including 44% (24/54) of cases that occurred within 1 year after renal transplantation. Conclusion FK is more diabetogenic than cyclosporine or sirolimus. Older age (≧51 years) is a significant risk factor, in contrast to hepatitis and gender. About half of these cases of PTDM occurred within 1 year after transplantation. These results suggest that aggressive monitoring of blood sugar is necessary for early detection of PTDM.

Androgens and the androgen receptor (AR) play important roles in the testes. Previously we have shown that male total AR knockout (T-AR −/y ) mice revealed incomplete germ cell development and lowered serum testosterone levels, which resulted in azoospermia and infertility. However, the consequences of AR loss in particular types of testicular cells remain unclear. Using a Cre-loxP conditional knockout strategy, we generated a tissue-selective knockout mouse with the AR gene deleted in testis peritubular myoid cells (PM-AR −/y ). Phenotype analyses showed that PM-AR −/y mice were indistinguishable from WT AR (AR +/y ) mice with the exception of smaller testes size. PM-AR −/y mice have serum testosterone concentrations comparable with AR +/y mice. PM-AR −/y mice have oligozoospermia in the epididymis; however, fertility was normal. Although normal germ cell distribution ratio was found, total germ cell number decreased in PM-AR −/y mice. Further mechanistic studies demonstrated that PM-AR −/y mice have defects in the expression of Sertoli cells' functional marker genes such as tranferrin, epidermal fatty acid-binding protein, androgen-binding protein, and other junction genes including occludin, testin, nectin, zyxin, vinculin, lamininγ3, gelsolin, connection43, and N-cadherin. Furthermore, there were defects in peritubular myoid cell contractility-related genes such as endothelin-1, endothelin receptor A and B, adrenomedullin, adrenomedullin receptor, and vasopressin receptor 1a. Together, our PM-AR −/y mice provide in vivo evidence for the requirement of functional AR in peritubular myoid cells to maintain normal Sertoli cells function and peritubular myoid cell contractility, thus ensuring normal spermatogenesis and sperm output.

BACKGROUND: Testosterone provokes Sertoli cell maturation and represses AMH production. In adult patients with Sertoli-cells-only syndrome (SCOS) and androgen insensitivity syndrome (AIS), high level of AMH expression is detected in Sertoli cells due to defect of androgen/AR signaling. OBJECTIVE: We postulated that up-regulation of SOX9 due to impairment of androgen/AR signaling in Sertoli cells might explain why high level of anti-Mullerian hormone (AMH) expression occur in these testiculopathic patients. METHODS: Biological research of testicular specimens from men with azoospermia or mouse. The serum hormone levels were studied in 23 men with obstructive azoospermia, 33 men with SCOS azoospermia and 21 volunteers with normal seminograms during a period of 4 years. Immunohistochemical staining and reverse-transcription PCR were used to examine the relationships among AR, SOX9 and AMH expression in adult human and mouse testes. The ability of AR to repress the expression of SOX9 and AMH was evaluated in vitro in TM4 Sertoli cells and C3H10T1/2 cells. RESULTS: SCOS specimens showed up-regulation of SOX9 and AMH proteins but down-regulation of AR proteins in Sertoli cells. The mRNA levels of AR were significantly lower and the SOX9, AMH mRNA levels higher in all SCOS patients compared to controls (P< 0.05). The testosterone levels in the SCOS patients were within the normal range, but most were below the median of the controls. Furthermore, our in vitro cell line experiments demonstrated that androgen/AR signaling suppressed the gene and protein levels of AMH via repression of SOX9. CONCLUSIONS: Our data show that the functional androgen/AR signaling to repress SOX9 and AMH expression is essential for Sertoli cell maturation. Impairment of androgen/AR signaling promotes SOX9-mediated AMH production, accounts for impairments of Sertoli cells in SCOS azoospermic patients.

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 Conclusion NP worsened AD-induced CKD that could be reversed by rosuvastatin therapy.

Background This study tested the hypothesis that exendin-4 and sitagliptin can effectively protect kidney from acute ischemia-reperfusion (IR) injury. Methods Adult SD-rats (n = 48) equally divided into group 1 (sham control), group 2 (IR injury), group 3 [IR + sitagliptin 600 mg/kg at post-IR 1, 24, 48 hr)], and group 4 [IR + exendin-4 10 μm/kg at 1 hr after procedure] were sacrificed after 24 and 72 hrs (n = 6 at each time from each group) following clamping of bilateral renal pedicles for 60 minutes (groups 2–4). Results Serum creatinine level and urine protein to creatinine ratio were highest in group 2 and lowest in group 1 (all p Conclusion Exendin-4 and sitagliptin provided significant protection for the kidneys against acute IR injury.

Introduction This study tested the hypothesis that cyclosporine (CsA)-supported syngeneic adipose-derived mesenchymal stem cell (ADMSC) therapy offered superior attenuation of acute ischemia–reperfusion (IR) kidney injury to either therapy alone. Methods Adult Sprague–Dawley rats (n = 40) were equally divided into group 1 (sham controls), group 2 (IR injury), group 3 (IR + CsA (20 mg/kg at 1 and 24 hours after procedure)), group 4 (syngeneic ADMSC (1.2×106) at 1, 6 and 24 hours after procedure), and group 5 (IR + CsA-ADMSC). Results By 72 hours after the IR procedure, the creatinine level and the ratio of urine protein to creatinine were highest in group 2 and lowest in group 1, and significantly higher in groups 3 and 4 than in group 5 (all P 0.05). The inflammatory biomarkers at mRNA (matrix metalloproteinase-9, RANTES, TNF-α), protein (TNF-α, NF-κB, intercellular adhesion molecule-1, platelet-derived growth factor), and cellular (CD68+) levels of IR kidney showed a similar pattern compared with that of creatinine in all groups (all P