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In this study, we evaluated the effects of Cyclosporine A (CsA) on Lipopolysaccharide (LPS)-induced cytokine production in the genital tract of female rabbits. Twelve sexually mature and healthy female rabbits were randomly divided into four groups (n = 3 each). The rabbits in the LPS group were given an intrauterine infusion of Escherichia coli LPS (4 mg/kg body weight (BW)). Rabbits in the CsA group were given CsA (20 mg/kg BW). Rabbits in the LPS + CsA group were given LPS (4 mg/kg BW) and CsA (20 mg/kg BW). The control group received only LPS and CsA carrier. The gene expression and protein levels of pro- and anti-inflammatory cytokines were observed using qRT-PCR and immuno-histochemical (IHC) assay, respectively. Our study showed that IL-1β, IL-6, IL-8, TNF-α, IFN-γ, IL-4, IL-10, IL-13, and TGF-β were expressed in female genital organs. The LPS challenge increased the mRNA expression of IL-6 and TNF-α in the uterine body and IL-1β in the uterotubal junction compared to the control group. CsA increased the basal mRNA expression of anti-inflammatory cytokines (i.e., IL-4 in the uterine body, uterotubal junction, and oviductal ampulla; IL-10 in the cervix, oviductal isthmus, and ampulla; and TGF-β in the uterotubal junction and oviductal ampulla) and pro-inflammatory cytokines (i.e., IL-6 and IL-8 in the cervix; IL-1β in the oviductal isthmus; TNF-α in the oviductal ampulla; and IFN-γ in the uterine body compared to the control group). In addition, CsA inhibited the mRNA expression of pro-inflammatory cytokines, such as IL-6 in the uterine body, uterotubal junction, and oviductal isthmus; TNF-α in the uterine body; and IFN-γ in the uterotubal junction and oviductal isthmus induced by the LPS challenge. The IHC assay showed the LPS-induced increase in protein production of IL-6 in the uterine body and oviductal isthmus. CsA increased the protein production of IL-10 in the cervix, uterine body, oviductal ampulla, and isthmus. Moreover, CsA decreased the protein production of IL-6 in the uterine body and oviductal isthmus induced by LPS.

Histone deacetylases (HDACs) play a critical role in modulating cardiac function and ischemic injury. HDAC4 was found to be elevated and activated in response to injury. However, whether HDAC4 mediates cardiac function is currently unknown. In this study, we created myocyte-specific activated HDAC4 transgenic mice to examine the role of HDAC4 in mediating cardiac function during development and response to infarction. There are no differences in cardiac function and gross phenotype between wild-type and cardiomyocyte-specific HDAC4 transgenic mice at 1 mo of age. However, cardiac dysfunction and vascular growth deficiency were displayed in 6-mo-old HDAC4-transgenic mice compared with wild-type mice. Activation of HDAC4 increased heart and myocyte size, hypertrophic proteins, and interstitial fibrosis in 6-mo-old mice but not in 1-mo-old mice. To further define whether activated HDAC4 in the heart could impact myocardial function and remodeling, myocardial infarction was created in both wild-type and cardiomyocyte-specific HDAC4-transgenic mice. In myocardial infarction, the overexpression of activated HDAC4 exacerbated cardiac dysfunction and augmented cardiac remodeling and interstitial fibrosis, which was associated with the reduction of cardiokines in the heart. These results indicate the activation of HDAC4 as a crucial regulator for cardiac function in development and myocardial infarction. NEW & NOTEWORTHY We created myocyte-specific activated HDAC4-transgenic mice to examine the function of HDAC4 in mediating cardiac function. HDAC4 overexpression led to cardiac dysfunction, which was associated with increased hypertrophy and myocardial fibrosis. Furthermore, the overexpression of activated HDAC4 exacerbated cardiac dysfunction, augmented remodeling, and increased apoptosis in the infarcted heart. This is the first demonstration that transgenic overexpression of HDAC4 is crucial for modulation of cardiac function and remodeling.

Osteoarthritis (OA) is characterized by synovitis and synovial fibrosis. Synoviocytes are fibroblast-like resident cells of the synovium that are activated by transforming growth factor (TGF)-β to proliferate, migrate, and produce extracellular matrix. Synoviocytes secrete hyaluronan (HA) and proteoglycan-4 (PRG4). HA reduces synovial fibrosis in vivo, and the Prg4−/−mouse exhibits synovial hyperplasia. We investigated the antifibrotic effects of increased intracellular cAMP in TGF-β-stimulated human OA synoviocytes. TGF-β1 stimulated collagen I (COL1A1), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase (TIMP)-1, and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) expression, and procollagen I, α-SMA, HA, and PRG4 production, migration, and proliferation of OA synoviocytes were measured. Treatment of OA synoviocytes with forskolin (10 μM) increased intracellular cAMP levels and reduced TGF-β1-stimulated COL1A1, α-SMA, and TIMP-1 expression, with no change in PLOD2 expression. Forskolin also reduced TGF-β1-stimulated procollagen I and α-SMA content as well as synoviocyte migration and proliferation. Forskolin (10 μM) increased HA secretion and PRG4 expression and production. A cell-permeant cAMP analog reduced COL1A1 and α-SMA expression and enhanced HA and PRG4 secretion by OA synoviocytes. HA and PRG4 reduced α-SMA expression and content, and PRG4 reduced COL1A1 expression and procollagen I content in OA synoviocytes. Prg4−/−synovium exhibited increased α-SMA, COL1A1, and TIMP-1 expression compared with Prg4+/+synovium. Prg4−/−synoviocytes demonstrated strong α-SMA and collagen type I staining, whereas these were undetected in Prg4+/+synoviocytes and were reduced with PRG4 treatment. We conclude that increasing intracellular cAMP levels in synoviocytes mitigates synovial fibrosis through enhanced production of HA and PRG4, possibly representing a novel approach for treatment of OA synovial fibrosis.

Proton exchange membrane fuel cells (PEMFCs) have attracted tremendous attention as alternative energy sources because of their high energy density and practically zero greenhouse gas emission - water is their only direct by-product. Critical to the function of PEMFCs is fast proton and water transport in the proton exchange membrane (PEM); fast water transport facilitates optimized water management and optimal PEMFC operation. In order to design a PEM for outstanding PEMFC performance, we need to know how the membrane properties: chemistry and structure, affect the water transport. My PhD work focused on studying the water structure and transport in PEMs. I developed and applied in situ nonlinear spectroscopy to study fundamental physical chemical properties of water in PEMs. Specifically, I experimentally demonstrated the existence of two types of water subspecies in PEMs: 1) bulk water (bulkW) interacting only with other water molecules; 2) non-bulk water (nonbulkW) interacting with the membrane structure. Interestingly, the amount of these different water subspecies is tunable by simply changes the processing conditions of the polymer material that constitutes the PEM. Moreover, I further found that the different subspecies exhibit distinct diffusivities in the PEM and that overall water transport in PEMs can be explained as a linear combination of the diffusivity of each water subspecies, weighted by the respective fractional contributions. Based on these results, we suggest a general design target as maximizing the fractional contribution of nonbulkW, while maintaining large water retention in the membrane, to accelerate water transport while maintaining fast proton transport.

Objective. Congenital deficiency of the principal boundary lubricant in cartilage (i.e., lubricin, encoded by the gene PRG4) increases joint friction and causes progressive joint failure. This study was undertaken to determine whether restoring lubricin expression in a mouse model would prevent, delay, or reverse the disease process caused by congenital deficiency. Methods. Using genetically engineered lubricin-deficient mice, we restored gene function before conception or at ages 3 weeks, 2 months, or 6 months after birth. The effect of restoring gene function (i.e., expression of lubricin) on the tibiofemoral patellar joints of mice was evaluated histologically and by ex vivo biomechanical testing. Results. Restoring gene function in mice prior to conception prevented joint disease. In 3-week-old mice, restoring gene function improved, but did not normalize, histologic features of the articular cartilage and whole-joint friction. In addition, cyclic loading of the joints produced fewer activated caspase 3-containing chondrocytes when lubricin expression was restored, as compared to that in littermate mice whose gene function was not restored (nonrestored controls). Restoration of lubricin expression in 2-month-old or 6-month-old mice had no beneficial effect on histopathologic cartilage damage, extent of whole-joint friction, or activation of caspase 3 when compared to nonrestored controls. Conclusion. When boundary lubrication is congenitally deficient and cartilage becomes damaged, the window of opportunity for restoring lubrication and slowing disease progression is limited.

Objective To evaluate recombinant human proteoglycan 4 (rhPRG4) binding to CD44 receptor and its consequence on cytokine induced synoviocyte proliferation.

Deficiency of PRG4 (lubricin), the boundary lubricant in mammalian joints, contributes to increased joint friction accompanied by superficial and upper intermediate zone chondrocyte caspase-3 activation, as shown in lubricin-null (Prg4−/−) mice. Caspase-3 activity appears to be reversible upon the restitution of Prg4 either endogenously in vivo, in a gene trap mouse, or as an applied lubricant in vitro. In this study we show that intra-articular injection of human PRG4 in vivo in Prg4−/− mice prevented caspase-3 activation in superficial zone chondrocytes and was associated with a modest decrease in whole joint friction measured ex vivo using a joint pendulum method. Non-lubricated Prg4−/− mouse cartilage shows caspase cascade activation caused by mitochondrial dysregulation, and significantly higher levels of peroxynitrite (ONOO− and −OH) and superoxide (O−2) compared to Prg4+/+ and Prg4+/− cartilage. Enzymatic activity levels of caspase 8 across Prg4 mutant mice were not significantly different, indicating no extrinsic apoptosis pathway activation. Western blots showed caspase-3 and 9 activation in Prg4−/− tissue extracts, and the appearance of nitrosylated Cys163 in the active cleft of caspase-3 which inhibits its enzymatic activity. These findings are relevant to patients at risk for arthrosis, from camptodactyl-arthropathy-coxa vara-pericarditis (CACP) syndrome and transient lubricin insufficiency due to trauma and inflammation.

Recently, several biological assays have become available on the centrifugal microfluidic platform. Despite many innovative solutions developed for on-disc fluid handling for these assays, certain challenges, including liquid incubation and simplification of a multi-step assay on a plastic device, still need to be further addressed. Incubating fluids that require downstream processing, which we call “midstream incubation”, can often be difficult on the microfluidic disc platform due to surface tension changes induced by varying temperatures, thus causing operating instability. We describe here strategies for liquid reagent storage, release, incubation, and transfer, all of which utilize a single combination of actuation methods—wax valving and heat actuation by halogen lamp—on a centrifugal microfluidic device made using pristine materials. The strategies that we use to perform these steps, termed multifunctional wax valves, enable manipulation of a microlitre range fluid volume without the need for complex fabrication steps or hardware. This technology’s reliability and ease of use will hopefully allow for more powerful fluidics-based diagnostic tools to be created.

We have recently shown that in vivo inhibition of histone deacetylase (HDAC) stimulates endogenous myocardial regeneration in infarcted hearts (Zhang L et al. J Pharmacol Exp Ther 341: 285–293, 2012). Furthermore, our observation demonstrates that HDAC inhibition promotes cardiogenesis, which is associated with HDAC4 reduction. However, it remains unknown as to whether specific inhibition of HDAC4 modulates cardiac stem cells (CSCs) to facilitate myocardial repair and to preserve cardiac performance. c-kit+ CSCs were isolated from adult mouse hearts and were transfected with HDAC4 siRNA to knockdown HDAC4 of c-kit+ CSCs. The transfection of HDAC4 siRNA caused a marked reduction of HDAC4 mRNA and proteins in c-kit+ CSCs. Mouse myocardial infarction (MI) was created to assess the effect of HDAC4 inhibition in c-kit+ CSCs on myocardial regeneration in vivo when cells were introduced into MI hearts. Transplantation of HDAC4 siRNA-treated c-kit+ CSCs into MI hearts improved ventricular function, attenuated ventricular remodeling, and promoted CSC-derived regeneration and neovascularization. Furthermore, Ki67 and BrdU positively proliferative myocytes increased in MI hearts receiving HDAC4 siRNA-treated c-kit+ CSCs compared with MI hearts engrafted with control siRNA-treated c-kit+ CSCs. In addition, compared with MI hearts engrafted with control adenoviral GFP-infected c-kit+ CSCs, MI hearts receiving adenoviral HDAC4-infected c-kit+ CSCs exhibited attenuated cardiac functional recovery, CSC-derived regeneration, and neovascularization, which was accompanied with adverse ventricular remodeling and decrease in Ki67 and BrdU positively proliferative myocytes. HDAC4 inhibition facilitated c-kit+ CSCs into the differentiation into cardiac lineage commitments in vitro, while HDAC4 overexpression attenuated c-kit+ CSC-derived cardiogenesis. Our results indicate that HDAC4 inhibition promotes CSC-derived cardiac regeneration and improves the restoration of cardiac function.

Due to the high number of multiple embryo transfers into the uterus performed in assisted reproductive technology treatment (ART), the incidences of twin pregnancy and of vanishing twin syndrome (VTS) are correspondingly high. A number of studies have described the obstetric outcomes of the remaining fetus produced after the other twin had vanished compared with a singleton at the start following ART, but the results are mixed and contradictory. We performed a systematic review of the existing studies to explore the actual obstetric outcome of VTS to allow physicians to adequately advise their patients. A detailed search strategy was used to conduct electronic literature searches (spanning 1978–2015) on Medline, EMBASE, the Cochrane library and Web of Science. As randomized trials are not feasible in this aspect, we included observational (cohort and case–control) studies which compared the obstetric outcomes of the VTS group and singleton at the start control group after ART. The outcomes were evaluated by two aspects, the duration of pregnancy (gestational age, preterm delivery rate, extremely preterm delivery rate) and the birth weight of the fetus [mean birth weight, low birth weight rate, very low birth weight rate and small for gestational age (SGA)]. 1271 publications were identified by the initial search. 499 studies were excluded following duplication checks. 760 were excluded after reviewing the abstracts. Of the remaining 12 articles, 7 were excluded after a detailed full-text review. Two case–control studies and three cohort studies were included in the final analysis. The pooled mean gestational age difference (95% confidence intervals) was −0.27 (−0.60, 0.06) and failed to demonstrate a difference between the two groups. A similar result was found in the preterm delivery rate, with a pooled risk ratio of 1.33 (0.91, 1.94). The prevalence of extremely preterm delivery rate was higher in the VTS group, with a pooled risk ratio of 3.5 (1.72, 7.12). The mean birth weight was lower in the VTS group, with a mean difference of −0.3 (−0.59, −0.01). No difference was found in low birth weight rate, very low birth weight rate and rate of small for gestational age, with risk ratio of 1.85 (0.88, 3.86), 4.86 (0.91, 25.91) and 1.29 (0.52, 3.18), correspondingly. There is a slight adverse effect of VTS on the remaining fetus for birth weight and extremely preterm delivery rate, but sensitivity analysis shows these effects to be statistically unstable. It is too early to draw conclusions for adverse obstetric outcomes for VTS patients. It could reduce much of the anxiety of couples who experience early embryonic loss of one of their twins. More research with rigorously designed and standardized methodologies are required that include larger, better clinically defined populations. Studies that show no correlation should be published in the future to avoid any possible impact of publication bias. After that, patients can receive the most accurate information.

Based on the conclusions from the experimental investigation in Part I, empirical equations for the accumulated shear strain, accumulated direction ratio and elastic modulus of frozen soil are derived. The basic issues observed in the test results can also be observed in these empirical equations. Subsequently, an elaborate constitutive model for frozen soil subjected to long-term low-level repeated loading is produced by combining the empirical equations and classical elasto-plastic theory. This model accounts for the dependency of the accumulated behaviour on the initial stress state, repeated stress amplitude and frozen soil strength. In addition, the evolution behaviour of the elastic modulus with accumulated strain is also considered. The model is verified for frozen soil with the aid of the triaxial test results represented in Part I. This study is the first attempt to model the elasto-plastic behaviour under long-term low-level repeated loading for frozen soil.

We have recently shown that the inhibition of histone deacetylases (HDAC) protects the heart against ischemia and reperfusion (I/R) injury. The mechanism by which HDAC inhibition induces cardioprotection remains unknown. We sought to investigate whether the genetic disruption of gp-91, a subunit of NADPH-oxidase, would mitigate cardioprotection of HDAC inhibition. Wild-type and gp-91(-)(/-) mice were treated with a potent inhibitor of HDACs, trichostatin A (TSA, 0.1 mg/kg, i.p.). Twenty-four hours later, the perfused hearts were subjected to 30 min of ischemia and 30 min of reperfusion. HDAC inhibition in wild-type mice produced marked improvements in ventricular functional recovery and the reduction of infarct size. TSA-induced cardioprotection was eliminated with genetic deletion of gp91. Notably, Western blot and immunostaining displayed a significant increase in gp-91 in myocardium following HDAC inhibition, which resulted in a mildly subsequent increase in the production of reactive oxygen species (ROS). The pre-treatment of H9c2 cardiomyoblasts with TSA (50 nmol/l) decreased cell necrosis and increased viability in response to simulated ischemia (SI), which was abrogated by the transfection of cells with gp-91 siRNA, but not by scrambled siRNA. Furthermore, treatment of PLB-985 gp91(+/+) cells with TSA increased the resistance to SI, which also diminished with genetic disruption of gp91 in gp91(phox)-deficient PLB-985 cells. TSA treatment inhibited the increased active caspase-3 in H9c2 cardiomyoblasts and PLB-985 gp91(+/+) cells exposed to SI, which were prevented by knockdown of gp-91 by siRNA. These results suggest that a cascade consisting of gp-91 and HDAC inhibition plays an essential role in orchestrating the cardioprotective effect.

We have recently demonstrated that the inhibition of histone deacetylases (HDAC) protects the heart against ischemia-reperfusion (I/R) injury. The mechanism by which HDAC inhibition confers myocardial protection remains unknown. The purpose of this study is to investigate whether the disruption of NF-kappaB p50 would eliminate the protective effects of HDAC inhibition. Wild-type and NF-kappaB p50-deficient mice were treated with trichostatin A (TSA; 0.1 mg/kg ip), a potent inhibitor of HDACs. Twenty-four hours later, the hearts were perfused in Langendorff model and subjected to 30 min of ischemia and 30 min of reperfusion. Inhibition of HDACs by TSA in wild-type mice produced marked improvements in left ventricular end-diastolic pressure, left ventricular rate pressure product, and the reduction of infarct size compared with non-TSA-treated group. TSA-induced cardioprotection in wild-type animals was absent with genetic deletion of NF-kappaB p50 subunit. Notably, Western blot displayed a significant increase in nuclear NF-kappaB p50 and the immunoprecipitation demonstrated a remarkable acetylation of NF-kappaB p50 at lysine residues following HDAC inhibition. EMSA exhibited a subsequent increase in NF-kappaB DNA binding activity. Luciferase assay demonstrated an activation of NF-kappaB by HDAC inhibition. The pretreatment of H9c2 cardiomyoblasts with TSA (50 nmol/l) decreased cell necrosis and increased in cell viability in simulated ischemia. The resistance of H9c2 cardiomyoblasts to simulated ischemia by HDAC inhibition was eliminated by genetic knockdown of NF-kappaB p50 with transfection of NF-kappaB p50 short interfering RNA but not scrambled short interfering RNA. These results suggest that NF-kappaB p50 acetylation and activation play a pivotal role in HDAC inhibition-induced cardioprotection.

Background Diabetic men generally have reduced efficacy with PDE-5 inhibitors (PDE5i) for the treatment of erectile dysfunction (ED). Objective To determine whether chronic vardenafil exposure alters cavernous protein expression predicting improved erectile function in diabetes. Design Forty-two adult male Sprague Dawley rats with streptozotocin-induced (50mg/kg IP) diabetes for 4 wk, were exposed to either vehicle or vardenafil for 6 wk. Assessments compared the impact of vardenafil given at 1h and 20h to erectile function and cellular alterations and downstream translation of cavernous protein profiles were aimed. Intervention Vehicle or vardenafil 0.5mg/kg/day by oral gavage for 6 wk. Measurements Erectile function, penile tissue morphology, protein expression and surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI) protein profiling were determined. Results and Limitations A significant increase of intracavernous pressure was seen in both treatment arms compared to diabetic rats not receiving vardenafil. Immunohistochemical staining showed improved endothelial and smooth muscle cell staining with chronic vardenafil use. Western blot analysis demonstrated increased endothelial cell eNOS and smooth muscle α-actin protein content. SELDI protein profiling showed enhanced proteins expression at molecular weights of 14.7, 20, 41.9, 66.2, and 83.9kDa in the chronically treated vardenafil group. Conclusions Vardenafil was effective in treating diabetic-induced ED with the greatest effect achieved through chronic dosing, with no additive effect measured with the final acute dose. Changes noted in the histology and protein expression indicate that vardenafil may have a protective effect in this disease state. This finding may serve as a basis for further work evaluating the utility of chronic vardenafil dosing in diabetic men.