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Sestrin (SESN) is known as a cysteine sulfinic acid reductase. Recently, nonredox functions of SESN in metabolic regulation and antitumor property have been recognized. While mechanisms underlying the expression of SESN are not fully understood. Here we report that insulin markedly increased SESN2 level in HepG2 cells through mTOR activation. To determine whether insulin affects SESN2 degradation, we assessed SESN2 turnover by applying the protein synthesis inhibitor, cycloheximide (CHX), and found that following insulin treatment SESN2 protein levels were reduced significantly slower than non-insulin-treated cells. Furthermore, the proteasomal inhibitor, MG132, dramatically increased SESN2 protein and its ubiquitination level while in the presence of MG132 insulin did not further increase SESN2 content, suggesting that insulin increases SESN2 content mainly via inhibiting its proteasomal degradation. We then explored the potential feedback role of SESN2 in insulin signaling by SESN2 siRNA knockdown in HepG2 cells. Following SESN2 knockdown insulin-stimulated PKB phosphorylation was enhanced and accompanied by reduced PTEN content. Taken together, our study suggests that insulin upregulates SESN2 content via the PI3K/mTOR signaling pathway and this effect is attributed to decreased SESN2 degradation. Furthermore, SESN2 via modulating PTEN plays a negative feedback role in insulin signaling.

Dopamine, as a universal material for surface treatment, can effectively improve the surface performance of aramid fibers. However, directly processing the surface of aramid fibers using dopamine currently incurs a high cost. To seek dopamine substitutes, one must first explore the adhesion mechanism responsible for binding the dopamine to the surface of the fiber. In this study, we construct an all-atomic molecular dynamics model of an aramid fiber before and after surface modification using dopamine. A force field based on condensed-phase optimized molecular potentials for atomistic simulation studies (COMPASS) is used. Using it, we analyze the surface adhesion mechanism of polydopamines aggregated by 21 kinds of molecular structures typically found on the surface of aramid fibers. The results show that a clear and smooth interface is formed between the polydopamine nanofilm layer and the surface of the aramid fiber. The high atomic density of the polydopamine in the small interface region is found to be conducive to noncovalent bonds of polydopamines with the surface of the aramid fiber. In addition, we investigate the works of adhesion of the 21 molecular structures typically found on the surface of aramid fibers. The results suggest that the work of adhesion of 5,6-indolequinone is the highest, followed by annular eumelanin molecules with annular planar structure. Straight-chain shaped dimers proved to be the molecules with the highest adhesion ability of the dihydroxyindole chain oligomers. Therefore, there is reason to suppose that more molecular structures (as above) can be formed by processing the surface of aramid fibers using dopamine by controlling the processing conditions. These molecular structures help improve the adhesion ability of the dopamine on the surface of the aramid fiber. Additionally, if these polydopamine molecules with high adhesion ability can be synthesized on a large scale, then new surface-processing materials are possible.

Background: Βeta-2-microglobulin (β2-M) has been demonstrated as a growth factor and signaling molecule in breast cancer and leukemia. The purpose of the study is to characterize β2-M expression in molecular subtypes of breast cancer, thereby investigating the mechanism of β2-M action in breast cancer., Methods: β2-M and B-Cell Lymphoma/Leukemia 2 (Bcl-2) transcript expression levels in breast cancer tissue and the corresponding normal tissue were quantified using real-time PCR. The protein expression levels of β2-M, estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), tumor protein 53 (p53) and Ki67 were determined by immunohistochemical (IHC) staining. Following silencing of the β2-M by siRNA, the levels of Bcl-2, ER, PR and HER-2 transcripts and the protein expression levels in human breast cancer cells were measured by real-time PCR and western blotting, respectively., Results: The expression of β2-M transcripts demonstrated no significant differences between the four breast cancer molecular subtypes and no significant correlations with age, clinical stage or lymph node metastasis. β2-M transcript expression demonstrated a positive correlation when compared to Bcl-2 transcript expression (P<0.05). The β2-M protein expression was significantly higher in breast cancer when compared with benign breast tumors (P<0.01), and have no significant correlation with age, clinical stage or lymph node metastasis. There was a significant difference demonstrated in β2-M protein expression in the four breast cancer molecular subtypes (P<0.05), and between the ER+ and ER- groups (P<0.01); however, no significant difference was demonstrated between the HER-2+ and HER-2- groups. β2-M protein expression had a negative correlation with ER protein expression (P<0.01), a positive correlation with p53 protein expression (P<0.01), and no correlation with Ki67 protein expression. β2-M silencing significantly inhibited Bcl-2 mRNA expression, but did not inhibit ER, PR and HER-2 mRNA expression in MCF-7 cells (ER+, PR+ and HER-2-). In addition, Bcl-2 and HER-2 mRNA expression were significantly up-regulated in MDA-MB-231 cells (ER-, PR- and HER-2-), which is consistent with the silencing effect seen at the protein level., Conclusions: β2-M expression demonstrated a significant difference in the four breast cancer molecular subtypes, and may be related to apoptosis regulation in breast cancer.

AB The nuclear transcription factor hypoxia-inducible factor 1[alpha] (HIF-1α) is a key regulator of gene expression under hypoxic and inflammatory conditions. The germline-encoded pattern recognition receptor toll-like receptor 4 (TLR4) recognizes molecular motifs shared by large groups of microorganisms as well as by endogenous ligands released from stressed and/or injured tissues. We have previously demonstrated that local inhibition of HIF-1α ameliorates lung injury induced by trauma/hemorrhagic shock (T/HS) in rats. In the current study, we directly determined the role of TLR4 in HIF-1α activation during T/HS-induced acute lung injury in mice. C3H/HeJ mice that harbor a TLR4 mutation and wild-type (WT) mice were infused T/HS or trauma/sham shock (T/SS) lymph from Sprague-Dawley rats. Evans blue dye lung permeability, lung water content, myeloperoxidase levels, and lung histological analysis confirmed that TLR4-deficient mice are resistant to lung injury after T/HS lymph infusion. Lungs from WT and TLR4mut mice after T/SS lymph infusion expressed negligible levels of HIF-1α. The induction of HIF-1α in lung homogenates from TLR4mut mice after T/HS or T/HS lymph infusion was markedly reduced as compared with their WT counterparts but remained elevated as compared with TLR4mut mice after T/SS lymph infusion. Endothelial cells from TLR4mut mice and silence of TLR4 in cells from WT mice showed a remarkable reduction of HIF-1α on T/HS lymph stimulation. Blocking of nuclear factor-κB activity by SN50 or Bay 11-7085 in WT cells diminished T/HS lymph-induced HIF-1α accumulation when compared with T/SS lymph incubation. Thus, our data suggest that TLR4 activation by T/HS is necessary for T/HS-induced lung injury and an augmented pulmonary HIF-1α response, which will provide more insights into the pathogenesis of shock-induced acute lung injury and identify potential therapeutic targets.

Objective: To research the change of concentration of the amino acid neurotransmitters in the striatum focal cerebral ischemia in rat and the effect of Acorus tatarinowii Schott, one of inducing resuscitation drugs, for 4 of amino acid neurotransmitters., Methods: Twenty four rats were divided into four groups (n = 6): control group, cerebral ischemia group, sham operation group and Acorns tatarinowii Schott treated group. Rats were established into models of cerebral ischemia by occluding bilateral thread cork method. Formation sampling were performed in a striatum area using microdialysis and the detection of biological sample including aspartic acid, glutamic acid, glycine and gamma-aminobutyric acid by high performance liquid chromatography (HPLC) electrochemical detector system., Results: Compared with the control, the all contents of 4 kinds of the amino acids were significantly increased during cerebral ischemia (P < 0.01). Compared with the cerebral ischemia group, the contents of aspartic acid, glutamic acid that were excitatory amino acids were remarkably decreased in the striatum for Acorus tatarinowii Schott treated group (P < 0.01), It was no significant influence on gamma-aminobutyric acid and glycine that belonged to inhibitory amino acid in a nascent condition but with a elevating in the later period of microdialysis., Conclusion: Acorus tatarinowii Schott can enter the cerebral parenchyma through blood brain barrier and cut down glutamic acid,aspartic acid increased during cerebral ischemia. As a result, the neurotoxicity attributed to the excitatory amino acid has been released in excessive amounts declined so as to avoid the secondary impairment of neurons caused by excitatory amino acids pernicious effects after ischemia. It may be one of the protective mechanism of drugs for inducing resuscitation resembling EAA receptor antagonists to ischemi brain.

To investigate the role of collagen structure in corneal biomechanics, measurement of localized corneal elasticity with minimal destruction to the tissue is necessary. We adopted the recently developed acoustic radiation force elastic microscopy (ARFEM) technique to measure localize biomechanical properties of the human cornea. In ARFEM, a low-frequency, high-intensity acoustic force is used to displace a femtosecond laser-generated microbubble, while high-frequency, low-intensity ultrasound is used to monitor the position of the microbubble within the cornea. Two ex vivo human corneas from a single donor were dehydrated to physiologic thickness, embedded in gelatin and then evaluated using the ARFEM technique. In the direction perpendicular to the corneal surface, ARFEM measurements provided elasticity values of E = 1.39 ± 0.28 kPa for the central anterior cornea and E = 0.71 ± 0.21 kPa for the central posterior cornea in pilot studies. The increased value of corneal elasticity in the anterior cornea correlates with the higher density of interweaving lamellae in this region., (Copyright © 2014. Published by Elsevier Inc.)

Objective: To explore the effect of retinoid X receptor (RXR) agonist bexarotene on atherosclerosis and the potential mechanism in streptozotocin (STZ) induced diabetic apolipoprotein E knockout (apoE(-/-)) mice., Methods: Eight C57BL/6 mice served as control, 46 apoE(-/-) mice were randomized into 4 groups: apoE(-/-) group (n = 10), STZ+apoE(-/-) group (n = 12), STZ+apoE(-/-)+Bex 10 (10 mg×kg⁻¹×d⁻¹)group (n = 12), STZ+ apoE(-/-)+Bex 30 (30 mg×kg⁻¹×d⁻¹) group (n = 12). Diabetic apoE(-/-) animal model was established by intraperitoneal injection of STZ. Blood glucose was determined by glucose oxidase method. Patch area in thoracic aorta was measured by HE staining. Western blotting was used to determine the RXR and gp91(phox) protein level in thoracic aorta. Reactive oxygen species (ROS) level in blood and thoracic aorta homogenates was detected by Fenton and Griess method., Results: (1) Patch areas of thoracic aorta were larger in apoE(-/-) group than in C57BL/6 group [(38.40 ± 8.95)µm² vs. (0.10 ± 0.01) µm², P < 0.01], further increased in STZ+apoE(-/-) group [(94.06 ± 8.04)µm², P < 0.05 vs. apoE(-/-) group] and significantly reduced in STZ+apoE(-/-)+Bex 10 group [(78.72 ± 4.62)µm², P < 0.05 vs. STZ+apoE(-/-) group] and further educed in STZ+apoE(-/-)+Bex 30 group [(46.13 ± 7.56)µm², P < 0.05 vs. STZ+apoE(-/-)+Bex 10 group]. (2) Blood glucose level, TG, TC, LDL-C, thoracic aorta gp91(phox) protein level and ROS level in blood and thoracic aorta homogenates were significantly higher in STZ+apoE(-/-) group than in apoE(-/-) group (all P < 0.05). Blood glucose level and TG, TC, LDL-C levels were similar between STZ+apoE(-/-)+Bex10 and STZ+apoE(-/-) group. Thoracic aorta gp91(phox) protein level and ROS level in blood and thoracic aorta homogenates were lower in STZ+apoE(-/-)+Bex 10 group than in STZ+apoE(-/-) group (P < 0.05). Blood glucose level, TG, TC, LDL-C levels, gp91(phox) expression in thoracic aorta, ROS level in blood and thoracic in STZ+apoE(-/-)+Bex 30 group were lower than in STZ+apoE(-/-) group (all P < 0.05)., Conclusion: Bexarotene treatment could attenuate arteriosclerosis progression in STZ induced diabetic apoE(-/-) mice, the underlying mechanism might be related to suppressing oxidative stress and decreasing blood glucose level and improving lipids metabolism.

Intellectual disability affects about 3% of individuals globally, with∼50% idiopathic. We designed an exonic-resolution array targeting all known submicroscopic chromosomal intellectual disability syndrome loci, causative genes for intellectual disability, and potential candidate genes, all genes encoding glutamate receptors and epigenetic regulators. Using this platform, we performed chromosomal microarray analysis on 165 intellectual disability trios (affected child and both normal parents). We identified and independently validated 36 de novo copy-number changes in 32 trios. In all, 67% of the validated events were intragenic, involving only exon 1 (which includes the promoter sequence according to our design), exon 1 and adjacent exons, or one or more exons excluding exon 1. Seventeen of the 36 copy-number variants involve genes known to cause intellectual disability. Eleven of these, including seven intragenic variants, are clearly pathogenic (involving STXBP1, SHANK3 (3 patients), IL1RAPL1, UBE2A, NRXN1, MEF2C, CHD7, 15q24 and 9p24 microdeletion), two are likely pathogenic (PI4KA, DCX), two are unlikely to be pathogenic (GRIK2, FREM2), and two are unclear (ARID1B, 15q22 microdeletion). Twelve individuals with genomic imbalances identified by our array were tested with a clinical microarray, and six had a normal result. We identified de novo copy-number variants within genes not previously implicated in intellectual disability and uncovered pathogenic variation of known intellectual disability genes below the detection limit of standard clinical diagnostic chromosomal microarray analysis.

Background: The Fibroblast Growth Factor (FGF) pathway is driving various aspects of cellular responses in both normal and malignant cells. One interesting characteristic of this pathway is the biphasic nature of the cellular response to some FGF ligands like FGF2. Specifically, it has been shown that phenotypic behaviors controlled by FGF signaling, like migration and growth, reach maximal levels in response to intermediate concentrations, while high levels of FGF2 elicit weak responses. The mechanisms leading to the observed biphasic response remains unexplained., Results: A combination of experiments and computational modeling was used to understand the mechanism behind the observed biphasic signaling responses. FGF signaling involves a tertiary surface interaction that we captured with a computational model based on Ordinary Differential Equations (ODEs). It accounts for FGF2 binding to FGF receptors (FGFRs) and heparan sulfate glycosaminoglycans (HSGAGs), followed by receptor-phosphorylation, activation of the FRS2 adapter protein and the Ras-Raf signaling cascade. Quantitative protein assays were used to measure the dynamics of phosphorylated ERK (pERK) in response to a wide range of FGF2 ligand concentrations on a fine-grained time scale for the squamous cell lung cancer cell line H1703. We developed a novel approach combining Particle Swarm Optimization (PSO) and feature-based constraints in the objective function to calibrate the computational model to the experimental data. The model is validated using a series of extracellular and intracellular perturbation experiments. We demonstrate that in silico model predictions are in accordance with the observed in vitro results., Conclusions: Using a combined approach of computational modeling and experiments we found that competition between binding of the ligand FGF2 to HSGAG and FGF receptor leads to the biphasic response. At low to intermediate concentrations of FGF2 there are sufficient free FGF receptors available for the FGF2-HSGAG complex to enable the formation of the trimeric signaling unit. At high ligand concentrations the ligand binding sites of the receptor become saturated and the trimeric signaling unit cannot be formed. This insight into the pathway is an important consideration for the pharmacological inhibition of this pathway.

Objective: To explore new hallmarks affecting the prognosis of patients with laryngeal squamous cell carcinoma (LSCC) via investigating the expression of CyclinE and p27 in LSCC tissues., Method: The expression of CyclinE and p27 was detected via Elivision immunohistochemical staining in 160 LSCC tissues and 20 normal laryngeal tissues (NLT). The relationship between CyclinE/ p27 and LSCC/ NLT was analyzed via Log-rank analysis. The relationship of CyclinE and p27 protein was statistically analyzed by spearman correlation analysis. The relationship between CyclinE/p27 and clinical-pathology-factors of patients with LSCC, such as age, gender, tumor site, diameter, differentiation, lymph node metastasis and PTNM stage were analyzed by Chi-square test. The relationship between clinical-pathology-factors, CyclinE, p27 and overall survival time of patients with LSCC was analyzed via Cox multiplicity and Kaplan-Meier survival analysis. A significant difference was recognized by P<0.05., Result: In LSCC the positive rates of CyclinE and p27 protein was 62.50% and 41.25% respectively (P<0.05). In NLT the positive rates of CyclinE and p27 protein was 35% and 70% respectively (P<0.05). The expression of CyclinE or p27 protein was closely correlated with lymph node metastasis, PTNM stage of patients with LSCC (P<0.05). The expression of CyclinE and p27 had no significant correlations with patients' gender, age and tumor site, diameter differentiation (P>0.05 for all). A negative correlation was found between the expression of CyclinE and p27 protein, r= -0.767(P<0.05). Kaplan-Meier survival analysis showed that the overall survival rate of patients with LSCC was 36.9% (P<0.05). The 5-year survival rate in positive group of CyclinE was 8%, in negative group was 80% (P<0.05). On the contrary, the 5-year survival rate of patients with LSCC in positive group of p27 protein was 77.27%, the rate was 5.32% in negative group (P<0.05). Cox multivariate regression analysis revealed that lymph node metastasis, PTNM stage, CyclinE and p27 were independent risk factors of prognosis for patients with LSCC., Conclusion: It is the molecular basis underlying the development and invasion/ metastasis of LSCC that activation of CyclinE gene accompanying inactivation of p27 gene. It is very important of co-detecting CyclinE and p27 protein to predict the prognosis of patients with LSCC.

We tested the hypothesis that endometriosis can be induced in baboons more successfully by intra-pelvic injection of the pellet of menstrual endometrium when compared to its supernatant. Menstrual endometrium, separated into pellet (n = 5) and supernatant fractions ( n = 8), or phosphate buffered saline (1 ml, n = 7, controls) was injected laparoscopically into the pelvis. During laparoscopy 25 days later, the number (ρ = 0.027) and surface area (ρ <0.0001) of endometriosis-like lesions were significantly higher in the pellet group, than in the supernatant group, or in the control group. Histological typical endometriosis was present only in the endometrial pellet group (1/15), whereas stromal endometriosis was observed in both the pellet group (6/15), and the supernatant group (6/20). Peritoneal endometrial like glands were observed in both the endometrial pellet group (3/15), and in the supernatant group (1/20). In conclusion, we confirmed our hypothesis that endometriosis can be induced in baboons more successfully by intrapelvic injection of the pellet of menstrual endometrium when compared to its supernatant.

A comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC/TOFMS) method has been developed for the formation and identification of unresolved complex mixtures (UCMs) in lacustrine biodegraded oils that with the same source rock, similar maturity, and increasing degradation rank from Nanxiang Basin, China. Normal alkanes, light hydrocarbons, isoprenoids, steranes, and terpanes are degraded gradually from oil B330 to oil G574. The compounds in biodegraded oil (oil G574) have fewer types, the polarity difference of compounds in different types is minor, and the relative content of individual compounds is similar. All the features make the compounds in biodegraded oil coelute in GC analysis and form the raised "baseline hump" named UCMs. By injecting standard materials and analyzing mass spectrums of target compounds, it is shown that cyclic alkanes with one to five rings are the major components of UCMs. Furthermore, UCMs were divided into six classes. Classes I and II, composed of alkyl-cyclohexanes, alkyl-naphthanes, and their isomers, are originated from the enrichment of hydrocarbons resistant to degradation in normal oils. Classes III ~ VI, composed of sesquiterpenoids, tricyclic terpanes, low molecular steranes, diasteranes, norhopanes, and their isomers, are probably from some newly formed compounds during the microbial transformation of oil.

Coxsackievirus B3 (CVB3) infection is considered as the most common cause of viral myocarditis with no available vaccine. Considering that CVB3 mainly invades through the gastrointestinal mucosa, the development of CVB3-specific mucosal vaccine, which is the most efficient way to induce mucosal immune responses, gains more and more attention. In this study, we used absent in melanoma 2 (AIM2) as a mucosal adjuvant to enhance the immunogenicity and immunoprotection of CVB3-specific chitosan-pVP1 vaccine. Mice were intranasally co-immunized with 50 μg chitosan-pAIM2 and equal amount of chitosan-pVP1 vaccine 4 times at 2 week-intervals, and then challenged with CVB3 2 weeks after the last immunization. Compared with chitosan-pVP1 vaccine immunization alone, chitosan-pAIM2 co-immunization enhanced resistance to CVB3-induced myocarditis evidenced by significantly enhanced ejection fractions from 55.40 ± 9.35 to 80.31 ± 11.35, improved myocarditis scores from 1.50 ± 0.45 to 0.30 ± 0.15, reduced viral load from 3.33 ± 0.50 to 0.50 ± 0.65, and increased survival rate from 40.0% to 75.5%. This increased immunoprotection might be attributed to the augmented level of CVB3-specific fecal SIgA with high affinity and neutralizing ability. In addition, co-immunization with chitosan-pAIM2 remarkably facilitated dendritic cells (DCs) recruitment to mesenteric lymph nodes (MLN), and promoted the expression of IgA-inducing factors (BAFF, APRIL, iNOS, RALDH1, IL-6, TGF-β), which might account for its mucosal adjuvant effect. This strategy may represent a promising prophylactic vaccine against CVB3-induced myocarditis.

An inflammatory response induced by high glucose is a cause of endothelial dysfunction in diabetes and is an important contributing link to atherosclerosis. Diabetes is an independent risk factor of atherosclerosis and activation of retinoid X receptor (RXR) has been shown to exert anti-atherogenic effects. In the present study, we examined the effects of the RXR ligands 9-cis-retinoic acid (9-cis-RA) and SR11237 on high glucose-induced inflammation in human umbilical endothelial vein endothelial cells (HUVECs) and explored the potential mechanism. Our results showed that the inflammation induced by high-glucose in HUVECs was mainly mediated by the activation of nuclear factor-B (NF- κB). High glucose-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were in comparison, significantly decreased by treatment with RXR. The effect of RXR agonists was mainly due to the inhibition of NF-κB activation. Using pharmacological inhibitors and siRNA, we confirmed that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was an upstream activator of NF-κB. Furthermore, RXR agonists significantly inhibited high glucose-induced activation of NADPH oxidase and significantly decreased the production of reactive oxygen species (ROS). To explore whether the rapid inhibitory effects of RXR agonists were in fact mediated by RXR, we examined the effect of RXR downregulation by RXR siRNA. Our results showed that RXR siRNA largely abrogated the effects of RXR agonists, suggesting the requirement of RXR expression. Therefore, we have shown that RXR is involved in the regulation of NADPH oxidase- NF-κB signal pathway, as the RXR ligands antagonized the inflammatory response in HUVECs induced by high glucose.

A sensitive, specific and simple LC-MS/MS method was developed for the identification and quantification of bivalirudin in human plasma using diazepam as an internal standard (IS). The API-4000 LC-MS/MS was operated under multiple-reaction monitoring mode using electrospray ionization. The sample preparation consisted of an easy protein precipitation sample pretreatment with methanol. Chromatographic separation was achieved on a Zorbax Eclipse plus C18 100 × 2.1 mm column with a mobile phase of water-methanol-0.1% formic acid. The analytes were detected with a triple quadrupole Quantum Access with positive ionization. Ions monitored in the multiple-reaction monitoring mode were m/z 1091 → 650 for bivalirudin (at 2.70 min) and m/z 285 → 193 for diazepam (at 3.85 min). The developed method was validated in human plasma with a lower limit of quantitation of 20 µg/L for bivalirudin. A linear response function was established for the range of concentrations 20-10,000 µg/L (r > 0.998) for bivalirudin. The intra- and inter-day precision values for bivalirudin met the acceptance criteria as per US Food and Drug Administration guidelines. Bivalirudin was stable in the battery of stability studies, viz. bench-top, freeze-thaw cycles and long-term stability. The developed assay method was applied to an intravenous administration study in humans., (Copyright © 2013 John Wiley & Sons, Ltd.)

Background: Most normal cells in the presence of oxygen utilize glucose for mitochondrial oxidative phosphorylation. In contrast, many cancer cells rapidly convert glucose to lactate in the cytosol, a process termed aerobic glycolysis. This glycolytic phenotype is enabled by lactate dehydrogenase (LDH), which catalyzes the inter-conversion of pyruvate and lactate. The purpose of this study was to identify and characterize potent and selective inhibitors of LDHA., Methods: High throughput screening and lead optimization were used to generate inhibitors of LDHA enzymatic activity. Effects of these inhibitors on metabolism were evaluated using cell-based lactate production, oxygen consumption, and 13C NMR spectroscopy assays. Changes in comprehensive metabolic profile, cell proliferation, and apoptosis were assessed upon compound treatment., Results: 3-((3-carbamoyl-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyquinolin-4-yl) amino) benzoic acid was identified as an NADH-competitive LDHA inhibitor. Lead optimization yielded molecules with LDHA inhibitory potencies as low as 2 nM and 10 to 80-fold selectivity over LDHB. Molecules in this family rapidly and profoundly inhibited lactate production rates in multiple cancer cell lines including hepatocellular and breast carcinomas. Consistent with selective inhibition of LDHA, the most sensitive breast cancer cell lines to lactate inhibition in hypoxic conditions were cells with low expression of LDHB. Our inhibitors increased rates of oxygen consumption in hepatocellular carcinoma cells at doses up to 3 microM, while higher concentrations directly inhibited mitochondrial function. Analysis of more than 500 metabolites upon LDHA inhibition in Snu398 cells revealed that intracellular concentrations of glycolysis and citric acid cycle intermediates were increased, consistent with enhanced Krebs cycle activity and blockage of cytosolic glycolysis. Treatment with these compounds also potentiated PKM2 activity and promoted apoptosis in Snu398 cells., Conclusions: Rapid chemical inhibition of LDHA by these quinoline 3-sulfonamids led to profound metabolic alterations and impaired cell survival in carcinoma cells making it a compelling strategy for treating solid tumors that rely on aerobic glycolysis for survival.

S-adenosylhomocysteine hydrolase (SAHH) is an enzyme, which catalyzes the hydrolysis of S-adenosylhomocysteine (SAH) which is formed after the donation of the methyl group of S-adenosylmethionine (SAM) to a methyl acceptor in methylation reaction. As a potent regulator of methylation, SAHH plays a critical role in methylation reaction in the cells. Here we cloned the SAHH gene from unicellular green alga Dunaliella salina (dsSAHH) and investigated its effects on flagellar regeneration of D. salina, and found that dsSAHH was upregulated both at the protein and the transcription levels during pH shock-triggered flagellar regeneration of D. salina. The flagellar regeneration was accelerated when dsSAHH was overexpressed, but it was inhibited by SAHH inhibitor 3-deazaadenosine (DZA). Moreover, a receptor for activated C kinase 1 from D. salina (dsRACK1), which was identified to interact with dsSAHH, was increased when dsSAHH was overexpressed in D. salina as shown by real-time PCR. The findings of this study suggest that dsSAHH may participate in the regulation of flagellar regeneration of D. salina.

Langerhans cell histiocytosis (LCH) is characterized by uncontrolled proliferation of Langerhans cells accompanying eosinophils. It often attacks children under 10 years of age. LCH in identical twins is very rare and its prognosis is different. Here we report identical-twin sisters with LCH. Computed tomography (CT) revealed osteolytic change in each twin's skull, and the elder exhibited poor eyesight. There were massive histiocyte-like cells surrounded by eosinophils in pathologic specimen of the abnormal lesions, which is typical pathologic finding in LCH. These pathologic cells were positive for S-100 and the cell surface protein CD1 antigen (CD1α), the known markers of LCH. After treating them with surgery, no symptoms were seen in the younger until now. While the older was found another soft mass (about 2.0 cm in diameter) in the left temporal area 18 months later. The same treatment was given to the older after admission, and she is healthy to date. To explore the relationship between hallmarks and the prognosis of identical-twin patients with LCH, we retrieved the 16 literatures (16 identical-twin pairs, 31 patients) listed in PubMed during the past 60 years. The data revealed all those patients who have disseminated to the bone marrow, spleen and liver with symptoms of fever and hepatosplenomegaly exhibited worse prognosis (9 out of the 31 patients). The other identical-twin subjects without infiltration of those organs recovered well. In conclusion, this study reveals the adverse hallmarks of prognosis in identical-twin patients with LCH by reviewing relevant literatures.

Objective: To determine the effect of inhibiting aromatase activity on endometrial lesion growth and aromatase expression in a baboon model of induced endometriosis., Design: Prospective study., Setting: Primate research institute., Animal(s): Sixteen olive baboons., Intervention(s): Sixteen olive baboons with induced endometriosis were examined with laparoscopy 10 months after disease inoculation. Animals in group 1 (n = 10) were treated with 1.25 mg/d of the aromatase inhibitor (AI) letrozole, and animals in group 2 (n = 6) were given a placebo for a total of 6 months., Main Outcome Measure(s): Total number of endometriotic lesions, morphology, and volume of lesions, as well as semiquantitative reverse transcription-polymerase chain reaction and quantitative polymerase chain reaction for levels of aromatase cytochrome messenger RNA were measured. Ovarian volumes were evaluated before treatment initiation and every 2 months during the study., Result(s): Treatment of group 1 animals with an AI significantly decreased lesion volume from baseline measurements, whereas the placebo-treated animals showed an increase in lesion volume. Aromatase messenger RNA levels in lesions in the AI-treated animals were significantly lower compared with the placebo-treated animals. Ovarian volumes were significantly increased at 6 months of AI treatment compared with pretreatment volumes., Conclusion(s): These findings suggest that suppression of aromatase cytochrome P450 may inhibit the in vivo growth of endometriotic lesions in baboons., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

In this study we test the hypothesis that nonlinear optical (NLO) multiphoton photoactivation of riboflavin using a focused femtosecond (FS) laser light can be used to induce cross-linking (CXL) and mechanically stiffen collagen as a potential clinical therapy for the treatment of keratoconus and corneal ectasia. Riboflavin-soaked, compressed collagen hydrogels are cross-linked using a FS laser tuned to 760 nm and set to either 100 mW (NLO CXL I) or 150 mW (NLO CXL II) of laser power. FS pulses are focused into the hydrogel using a 0.75 NA objective lens, and the hydrogel is three-dimensionally scanned. Measurement of hydrogel stiffness by indentation testing show that the calculated elastic modulus (E) values are significantly increased over twofold following NLO CXL I and II compared with baseline values (P<0.05). Additionally, no significant differences are detected between NLO CXL and single photon, UVA CXL (P>0.05). This data suggests that NLO CXL has a comparable effect to conventional UVA CXL in mechanically stiffening collagen and may provide a safe and effective approach to localize CXL at different regions and depths within the cornea.

Objectives: The objective was to evaluate the pharmacokinetic and pharmacodynamic properties of a single intravenous fixed dose compared with a weight-adjusted dose of linezolid., Methods: A Phase I, comparative clinical trial was conducted involving 20 healthy male Chinese volunteers, assigned into low weight (LW) (50 kg < weight ≤ 55 kg) and high weight (HW) (≥ 80 kg) groups. All subjects were administrated single dose of linezolid (600 mg/30 min) and, after 72 h washout period, another single-dose (10 mg/kg/30 min). Plasma linezolid concentrations were measured by liquid chromatography-tandem mass spectrometry. A Monte Carlo simulation was used to evaluate the probability of pharmacodynamic target attainment (PTA)., Results: With 600 mg dose, plasma concentrations in LW group were much higher than that in HW group. A persistent serum inhibitory activity was observed in LW group; the inhibitory activity was lower in HW group. The PTA in HW group was lower than in LW group. For 10 mg/kg dose, both HW and LW groups had similar plasma concentrations. The HW and LW groups had similar serum inhibitory effects. The PTA in HW and LW groups also showed no difference., Conclusions: Our findings suggest that a weight-adjusted, 10 mg/kg regimen of linezolid may be more appropriate than fixed dosing for patients of different body weight.

Genital Alphapapillomavirus (αPV) infections are one of the most common sexually transmitted human infections worldwide. Women infected with the highly oncogenic genital human papillomavirus (HPV) types 16 and 18 are at high risk for development of cervical cancer. Related oncogenic αPVs exist in rhesus and cynomolgus macaques. Here the authors identified 3 novel genital αPV types (PhPV1, PhPV2, PhPV3) by PCR in cervical samples from 6 of 15 (40%) wild-caught female Kenyan olive baboons (Papio hamadryas anubis). Eleven baboons had koilocytes in the cervix and vagina. Three baboons had dysplastic proliferative changes consistent with cervical squamous intraepithelial neoplasia (CIN). In 2 baboons with PCR-confirmed PhPV1, 1 had moderate (CIN2, n = 1) and 1 had low-grade (CIN1, n = 1) dysplasia. In 2 baboons with PCR-confirmed PhPV2, 1 had low-grade (CIN1, n = 1) dysplasia and the other had only koilocytes. Two baboons with PCR-confirmed PhPV3 had koilocytes only. PhPV1 and PhPV2 were closely related to oncogenic macaque and human αPVs. These findings suggest that αPV-infected baboons may be useful animal models for the pathogenesis, treatment, and prophylaxis of genital αPV neoplasia. Additionally, this discovery suggests that genital αPVs with oncogenic potential may infect a wider spectrum of non-human primate species than previously thought.

Background: To investigate on expressions and clinical significances of CD133 protein and vasculogenic mimicry (VM) in primary non-small cell lung cancer (NSCLC)., Methods: The specimens of NSCLC from 305 Chinese patients with follow-up were analyzed for CD133 protein expression and VM by immunohistochemical and histochemical staining., Results: In NSCLC, positive rates of 48.9% and 35.7% were obtained for CD133 and VM, respectively. The VM and expression of CD133 were significantly higher in carcinoma than in normal. There were a positive relationship between the VM and expression of CD133 and the tumor grade, lymph node metastasis and clinical stage (all P<0.05). The overall mean survival time of the patients with CD133 and VM positive expression was lower than that of patients with negative expression. Microvessel density (MVD) was positive corresponded with the grade, lymph node metastasis and clinical stage (all P<0.05). The overall mean survival time of the patients with MVD≥22's group was shorter than that of patients with MVD<22's group. Pathological-tumor-node-metastasis (pTNM) stage, positive expression of CD133 and VM, postoperative therapy and MVD were independent prognostic factors of NSCLC (P<0.05). Immunohistochemistry revealed an important intratumoral heterogeneity in all four CD133 expression profiles., Conclusions: VM, MVD and expression of CD133 are related to differentiation, lymph node metastasis, clinical stage, and prognosis. It is suggested that CD133, VM and MVD should be considered as a potential marker for the prognosis.

Colistin is the last resort for treatment of multidrug-resistant Acinetobacter baumannii. Unfortunately, resistance to colistin has been reported all over the world. The highest resistance rate was reported in Asia, followed by Europe. The heteroresistance rate of A. baumannii to colistin is generally higher than the resistance rate. The mechanism of resistance might be loss of lipopolysaccharide or/and the PmrAB two-component system. Pharmacokinetic/pharmacodynamic studies revealed that colistin monotherapy is unable to prevent resistance, and combination therapy might be the best antimicrobial strategy against colistin-resistant A. baumannii. Colistin/rifampicin and colistin/carbapenem are the most studied combinations that showed promising results in vitro, in vivo and in the clinic. New peptides showing good activity against colistin-resistant A. baumannii are also being investigated.

Background: Uterus transplantation (UTx) may provide the first available treatment for women affected by uterine infertility. The present study aimed to further develop a surgical technique for autologous UTx in a non-human primate species and to assess long-term function., Methods: Female baboons (n= 16) underwent autologous transplantation of the uterus with the Fallopian tubes and ovaries, performed with a previously published surgical technique (n= 6, Group 1) or using a modified technique (n= 10; Group 2). The uterine arteries were dissected to the proximal end of the anterior branch (Group 1) or the entire (Group 2) internal iliac artery, and the ovarian veins were dissected to the crossing over the ureter (Group 1) or further cranially to include greater lengths and patches of the cava/renal vein (Group 2). Back-table preparation created common venous and arterial ends with arterial anastomosis either end-to-side to the left external iliac artery (Group 1) or end-to-end to the left internal iliac artery (Group 2)., Results: Overall short-time survival of the animals was 88% (66% in Group 1 and 100% in Group 2). Of all the operated animals, 75% (66% in Group 1 and 80% in Group 2) resumed ovarian cyclicity. Regular menstruation after UTx was demonstrated only in Group 2 (60%). Menstruating animals (n= 6) were each exposed to timed mating for ≥5 menstrual cycles, but pregnancy did not occur. Adhesions and tubal blockage were seen in post-mortem analysis., Conclusions: The modified UTx model of Group 2 is a safe procedure and shows resumed long-term uterine function in a majority of the animals, although pregnancy could not be demonstrated.

Background and Objective: In recent years, many studies have revealed the prognosis in patients with non-small cell lung cancer (NSCLC). In general, some clinic-pathological parameters have been related with prognosis. The aim of this study is to detect the relationship among lymphatic vessel density (LVD), microvessel density (MVD), expression of carcinoembryonic antigen (CEA) mRNA, metastasis suppressor genes (KAI1 and Kiss-1), and the prognosis of NSCLC patients., Methods: Blood samples were collected from 57 cases of NSCLC. The transcription of CEA mRNA was detected via nested reverse transcriptase-polymerase chain reaction and micro-fluid chip. Immunohistochemistry was performed to detect the expression of LVD, MVD, KAI1 and Kiss-1 in the patients. All follow-up data were collected and analyzed., Results: The overall five-year survival rate was 18%, and the median survival was 34 months. TNM stage, lymph node metastasis, and expression of MVD, LVD, CEA mRNA and Kiss-1 were factors to survival, as determined via single survival analysis. Multivariate analysis demonstrated that TNM stage, lymph node metastasis, and expression of CEA mRNA were independent prognostic factors for NSCLC patients., Conclusions: The expression of MVD, LVD, Kiss-1 and CEA mRNA is related to the prognosis of NSCLC.

Linezolid is an important therapeutic option for infections from multi-drug resistant Gram-positive pathogens. However, prolonged linezolid treatment (>14 days) is considered to increase the risk of hematological adverse events. We aimed to evaluate the hematological safety profile of an i.v. single dose of linezolid in healthy volunteers of different body weight. We conducted a phase I clinical trial involving 20 healthy male Chinese volunteers that received an i.v. single dose of linezolid (600 mg). The study participants were assigned to two groups: low-weight (LW) group: 50 kg

Esophageal squamous cell carcinoma (ESCC) is a predominant type of esophageal cancer, which is a malignant tumor originating from the esophageal mucosa or gland and is aggressive with poor prognosis. Identification of new gene expression patterns would be helpful for providing new targets for the early detection and treatment of ESCC patients. In the present study, we employed cDNA array technology to compare gene expression profiles between ESCC tissues and adjacent normal epithelial tissues from ESCC patients. There was at least a 4-fold change in the expression levels of 72 genes that were significantly increased and 107 genes that were decreased in ESCC compared with normal esophageal epithelium. Among them, genes known to be involved in ESCC were found, including matrix metalloproteinases, transcription factors SOX-4 and SOX-17, the Wingless-type MMTV integration site family member 2, and cell cycle regulators. Moreover, we have newly identified the two genes that are down-regulated in ESCC: monoamine oxidase A, an enzyme that catalyzes monoamines oxidation and 15-hydroxyprostaglandin dehydrogenase [NAD+], a prostaglandin-synthesizing enzyme that physiologically antagonizes COX-2. Likewise, we found the three genes that are up-regulated in ESCC: CD7, a cell surface glycoprotein member of the immunoglobulin superfamily, LIM-domain kinase 1, a small subfamily with an unique combination of two N-terminal LIM motifs and a C-terminal protein kinase domain, and TTK protein kinase, a previously unidentified member of the kinase family. These newly identified genes may be involved in the progression of the tumor and/or represent properties specific to ESCC.

Objectives: To investigate the safety, pharmacokinetics and food effect of iptakalim in healthy adult Han Chinese volunteers., Methods: Study 1 was a randomized open-label, Latin square designed, single-dose, three-period, self-control crossover study. Six men and six women received 5, 10 and 20 mg of iptakalim orally. Study 2 was a randomized, open-label, single-dose, two-period, self-control crossover study. Ten men were included and each subject received 5 mg iptakalim orally, fasting and nonfasting., Key Findings: No adverse effects were reported and no clinically meaningful changes in vital signs were found. Cmax, AUC(0-t) and AUC(0-∞) were proportional over the dose levels of 5, 10 and 20 mg. Tmax, t½ and CL/F were similarly independent of dose level. In the 5 mg and 20 mg group, the Cmax, AUC(0-t) and AUC(0-∞) in women were significantly higher than in men, although they showed no difference after correction by mg/kg doses in the 5 mg group. At the 5-mg dose level, no significant difference in pharmacokinetics was found in nonfasting and fasting subjects., Conclusions: Single-dose pharmacokinetics of iptakalim showed dose proportionality over the dose levels of 5-20 mg. The pharmacokinetics showed gender differences in the 5 and 20 mg groups. Food had almost no impact on the pharmacokinetics at the 5 mg level., (© 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.)

Up to 90% of individuals affected by Sotos syndrome have a pathogenic alteration of NSD1 (encodes nuclear receptor-binding Su-var, enhancer of zeste, and trithorax domain protein 1), a histone methyltransferase that functions as both a transcriptional activator and a repressor. Genomic copy number variations may also cause a Sotos-like phenotype. We evaluated a three-generation family segregating a Sotos-like disorder characterized by typical facial features, overgrowth, learning disabilities, and advanced bone age. Affected individuals did not have a detectable NSD1 mutation, but rather were found to have a 1.9 Mb microduplication of 19p13.2 with breakpoints in two highly homologous Alu elements. Because the duplication included the DNA methyltransferase gene (DNMT1), we assessed DNA methylation of peripheral blood and buccal cell DNA and detected no alterations. We also examined peripheral blood gene expression and found evidence for increased expression of genes within the duplicated region. We conclude that microduplication of 19p13.2 is a novel genomic disorder characterized by variable neurocognitive disability, overgrowth, and facial dysmorphism similar to Sotos syndrome. Failed compensation of gene duplication at the transcriptional level, as seen in peripheral blood, supports gene dosage as the cause of this disorder., (© 2011 John Wiley & Sons A/S.)

Background: Invasive fungal infections such as candidiasis and mold infections cause significant morbidity and mortality in seriously ill patients. Micafungin is an echinocandin antifungal agent with potent activity against most species of Candida and Aspergillus. We did this meta-analysis to clarify whether micafungin offers superior efficacy and safety compared with other antifungal agent for treating infections associated with invasive candidiasis., Methods: We did a meta-analysis of randomized controlled trials to examine whether micafungin has superior efficacy and safety compared with other antifungal agents recommended by the treatment guidelines for fungal infection. Seven trials involving 2913 patients were included in this analysis. Odds ratios (OR) and 95% confidence intervals (CI) were calculated., Results: Micafungin was associated with significantly better treatment success compared with the comparator antifungal agents (modified intention to treat, 2851 patients; random-effects model, OR 1.20, 95%CI 1.00 - 1.45, P = 0.0487). In addition, micafungin was more effective than the comparators for antifungal prophylaxis of neutropenic patients undergoing hematopoietic stem cell transplantation (OR 1.47, 95%CI 1.08 - 2.00, P = 0.01). Although there was no significant difference between the compared regimens in terms of the incidence of adverse drug effects (OR 0.94, 95%CI 0.77 - 1.11), fewer patients treated with micafungin withdrew from the studies because of adverse events (OR 0.64, 95%CI 0.44 - 0.94)., Conclusions: Micafungin has a good safety and tolerability profile, with an efficacy at least comparable to the other antifungal agents. Micafungin offers advantages over other agents for antifungal prophylaxis. Micafungin offers an appropriate alternative for antifungal prophylaxis rather than the treatment of invasive candida infections.

Tetraloop-receptor interactions are prevalent structural units in RNAs, and include the GAAA/11-nt and GNRA-minor groove interactions. In this study, we have compiled a set of 78 nonredundant loop-helix interactions from X-ray crystal structures, and examined them for the extent of their sequence and structural variation. Of the 78 interactions in the set, only four were classical GAAA/11-nt motifs, while over half (48) were GNRA-minor groove interactions. The GNRA-minor groove interactions were not a homogeneous set, but were divided into five subclasses. The most predominant subclass is characterized by two triple base pair interactions in the minor groove, flanked by two ribose zipper contacts. This geometry may be considered the "standard" GNRA-minor groove interaction, while the other four subclasses are alternative ways to form interfaces between a minor groove and tetraloop. The remaining 26 structures in the set of 78 have loops interacting with mostly idiosyncratic receptors. Among the entire set, a number of sequence-structure correlations can be identified, which may be used as initial hypotheses in predicting three-dimensional structures from primary sequences. Conversely, other sequence patterns are not predictive; for example, GAAA loop sequences and GG/CC receptors bind to each other with three distinct geometries. Finally, we observe an example of structural evolution in group II introns, in which loop-receptor motifs are substituted for each other while maintaining the larger three-dimensional geometry. Overall, the study gives a more complete view of RNA loop-helix interactions that exist in nature.

Purpose: To characterize and quantify the collagen fiber (lamellar) organization of human corneas in three dimensions by using nonlinear optical high-resolution macroscopy (NLO-HRMac) and to correlate these findings with mechanical data obtained by indentation testing of corneal flaps., Methods: Twelve corneas from 10 donors were studied. Vibratome sections, 200 μm thick, from five donor eyes were cut along the vertical meridian from limbus to limbus (arc length, 12 mm). Backscattered second harmonic-generated (SHG) NLO signals from these sections were collected as a series of overlapping 3-D images, which were concatenated to form a single 3-D mosaic (pixel resolution: 0.44 μm lateral, 2 μm axial). Collagen fiber intertwining was quantified by determining branching point density as a function of stromal depth. Mechanical testing was performed on corneal flaps from seven additional eyes. Corneas were cut into three layers (anterior, middle, and posterior) using a femtosecond surgical laser system and underwent indentation testing to determine the elastic modulus for each layer., Results: The 3-D reconstructions revealed complex collagen fiber branching patterns in the anterior cornea, with fibers extending from the anterior limiting lamina (ALL, Bowman's layer), intertwining with deeper fibers and reinserting back to the ALL, forming bow spring-like structures. Measured branching-point density was four times higher in the anterior third of the cornea than in the posterior third and decreased logarithmically with increasing distance from the ALL. Indentation testing showed an eightfold increase in elastic modulus in the anterior stroma., Conclusions: The axial gradient in lamellar intertwining appears to be associated with an axial gradient in the effective elastic modulus of the cornea, suggesting that collagen fiber intertwining and formation of bow spring-like structures provide structural support similar to cross-beams in bridges and large-scale structures. Future studies are necessary to determine the role of radial and axial structural-mechanical heterogeneity in controlling corneal shape and in the development of keratoconus, astigmatism, and other refractive errors.

Background: The baboon (Papio hamadryas anubis) can be transcervically instrumented, facilitating studies of intrauterine contraception and reproductive tract infection. We sought to determine if the baboon could become infected with a single cervical inoculation of Chlamydia trachomatis., Methods: Ten female baboons were randomized and inoculated cervically with C. trachomatis serovar E (or buffer alone). Animals underwent weekly clinical and laparoscopic evaluations for four weeks and at post-inoculation week 8, to monitor upper tract infection. Cervical culture and nucleic acid amplification testing (NAAT) were completed weekly throughout the study. Animals were euthanized at week 16 and the reproductive tracts were examined histologically., Results: All inoculated animals developed cervical infection. The average duration of positive NAAT results was 6.8 weeks (range 2-16). Two of eight (25%) animals tested positive from fallopian tube samples. Infected animals showed histological findings consistent with chlamydial infection, such as germinal centers. Five of ten animals seroconverted to C. trachomatis., Conclusions: Baboons cervically inoculated once with C. trachomatis develop infection similar to humans, with a low incidence of upper tract infection. This novel model of Chlamydia infection closely resembles human disease and opens new avenues for studying the pathogenesis of sexually transmitted infections and contraceptive safety.

Purpose: Corneal collagen cross-linking (CXL) by the use of riboflavin and ultraviolet-A light (UVA) is a promising and novel treatment for keratoconus and other ectatic disorders. Since CXL results in enhanced corneal stiffness, this study tested the hypothesis that CXL-induced stiffening would be proportional to the collagen autofluorescence intensity measured with nonlinear optical (NLO) microscopy., Methods: Rabbit eyes (n = 50) were separated into five groups including: (1) epithelium intact; (2) epithelium removed; (3) epithelium removed and soaked in riboflavin, (4) epithelium removed and soaked in riboflavin, with 15 minutes of UVA exposure; and (5) epithelium removed and soaked in riboflavin, with 30 minutes of UVA exposure. Corneal stiffness was quantified by measuring the force required to displace the cornea 500 μm. Corneas were then fixed in paraformaldehyde and sectioned, and the collagen autofluorescence over the 400- to 450-nm spectrum was recorded., Results: There was no significant difference in corneal stiffness among the three control groups. Corneal stiffness was significantly and dose dependently increased after UVA (P < 0.0005). Autofluorescence was detected only within the anterior stroma of the UVA-treated groups, with no significant difference in the depth of autofluorescence between different UVA exposure levels. The signal intensity was also significantly increased with longer UVA exposure (P < 0.001). Comparing corneal stiffness with autofluorescence intensity revealed a significant correlation between these values (R(2) = 0.654; P < 0.0001)., Conclusions: The results of this study indicate a significant correlation between corneal stiffening and the intensity of collagen autofluorescence after CXL. This finding suggests that the efficacy of CXL in patients could be monitored by assessing collagen autofluorescence.

Objectives: To evaluate the efficacy and safety of macrolides in cystic fibrosis (CF)., Methods: Randomized controlled trials (RCTs) of macrolides for the treatment of CF published in PubMed, the Cochrane Library and Embase were searched. Application of inclusion and exclusion criteria, data extraction, and assessment of methodological quality were independently performed in duplicate. The primary efficacy outcome was the impact on the deterioration of lung function (changes in FEV(1) and FVC). Safety outcomes included adverse events and mortality., Results: Eight RCTs (seven with azithromycin and one with clarithromycin) were found in the systematic review and six RCTs with azithromycin (654 patients) were included in the meta-analysis. Azithromycin treatment showed a significant increase in FEV(1)% (3.22%, 95% CI = 1.38-5.06, P = 0.0006, I(2) = 0%) and FVC% (3.23%, 95% CI = 1.62-4.85, P < 0.0001, I(2) = 0%) compared with placebo. In individuals with baseline Pseudomonas aeruginosa colonization, both FEV(1)% (4.80%, 95% CI = 1.66-7.94, P = 0.003, I(2) = 42%) and FVC% (4.74%, 95% CI = 1.92-7.57, P = 0.001, I(2) = 0%) increased significantly. The incidence rates of the main side effects (cough, headache, abdominal pain, vomiting, nausea and diarrhoea) were not significantly different between the azithromycin-treated group and the placebo group. The RCT of clarithromycin, involving 18 patients, showed its effects on clinical improvement; however, the small sample size made comparisons with azithromycin difficult., Conclusions: Long-term use of azithromycin can improve lung function, especially for P. aeruginosa-colonized CF patients. There was no evidence of increased adverse events with azithromycin. More data are needed to verify the best azithromycin regimen and to evaluate other macrolides in CF patients.

Background: Clinical laboratories are adopting array genomic hybridization as a standard clinical test. A number of whole genome array genomic hybridization platforms are available, but little is known about their comparative performance in a clinical context., Methods: We studied 30 children with idiopathic MR and both unaffected parents of each child using Affymetrix 500 K GeneChip SNP arrays, Agilent Human Genome 244 K oligonucleotide arrays and NimbleGen 385 K Whole-Genome oligonucleotide arrays. We also determined whether CNVs called on these platforms were detected by Illumina Hap550 beadchips or SMRT 32 K BAC whole genome tiling arrays and tested 15 of the 30 trios on Affymetrix 6.0 SNP arrays., Results: The Affymetrix 500 K, Agilent and NimbleGen platforms identified 3061 autosomal and 117 X chromosomal CNVs in the 30 trios. 147 of these CNVs appeared to be de novo, but only 34 (22%) were found on more than one platform. Performing genotype-phenotype correlations, we identified 7 most likely pathogenic and 2 possibly pathogenic CNVs for MR. All 9 of these putatively pathogenic CNVs were detected by the Affymetrix 500 K, Agilent, NimbleGen and the Illumina arrays, and 5 were found by the SMRT BAC array. Both putatively pathogenic CNVs identified in the 15 trios tested with the Affymetrix 6.0 were identified by this platform., Conclusions: Our findings demonstrate that different results are obtained with different platforms and illustrate the trade-off that exists between sensitivity and specificity. The large number of apparently false positive CNV calls on each of the platforms supports the need for validating clinically important findings with a different technology.

Objective: The paucity of data on the fetal effects of prenatal exposure to chemotherapy prompted us to study transplacental transport of chemotherapeutic agents., Methods: Fluorouracil-epirubicin-cyclophosphamide (FEC) and doxorubicin-bleomycin-vinblastine-dacarbazine (ABVD) were administered to pregnant baboons. At predefined time points over the first 25 h after drug administration, fetal and maternal blood samples, amniotic fluid (AF), urine, fetal and maternal tissues, and cerebrospinal fluid (CSF) were collected. High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) were used for bioanalysis of doxorubicin, epirubicin, vinblastine, and cyclophosphamide., Results: In nine baboons, at a median gestational age of 139 days (range, 93-169), FEC 100% (n = 2), FEC 200% (n=1), ABVD 100% (n = 5), and ABVD 200% (n = 1) were administered. The obtained ratios of fetal/maternal drug concentration in the different simultaneously collected samples were used as a measure for transplacental transfer. Fetal plasma concentrations of doxorubicin and epirubicin averaged 7.5 ± 3.2% (n = 6) and 4.0 ± 1.6% (n = 8) of maternal concentrations, respectively. Fetal tissues contained 6.3 ± 7.9% and 8.7 ± 8.1% of maternal tissue concentrations for doxorubicin and epirubicin, respectively. Vinblastine concentrations in fetal plasma averaged 18.5 ± 15.5% (n=9) of maternal concentrations. Anthracyclines and vinblastine were neither detectable in maternal nor in fetal brain/CSF. 4-Hydroxy-cyclophosphamide concentrations in fetal plasma and CSF averaged 25.1 ± 6.3% (n = 3) and 63.0% (n = 1) of the maternal concentrations, respectively., Conclusion: This study shows limited fetal exposure after maternal administration of doxorubicin, epirubicin, vinblastine, and 4-hydroxy-cyclophosphamide., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Objective: To determine the impact of physiologic changes of pregnancy on pharmacokinetics of chemotherapeutic agents., Design: A preclinical and a clinical case-control trial., Setting: Institute of Primate Research Nairobi and collaborating hospitals in Belgium, the Netherlands and Czech Republic., Population: Pregnant and nonpregnant women and baboons receiving chemotherapy., Methods: Chemotherapy pharmacokinetics was compared between the pregnant and nonpregnant state. Standard-dosed chemotherapy regimens were administered in pregnant and nonpregnant baboons/women, followed by serial blood samplings. Drug plasma levels were determined using high performance liquid chromatography and atomic absorption spectrometry., Main Outcome Measures: Area under the curve (AUC), maximal plasma concentration, terminal elimination half-life, clearance and distribution volume of each drug in pregnant and nonpregnant state., Results: Intraindividual comparative pharmacokinetic data were obtained for doxorubicin and paclitaxel/platinum in three and two baboons, respectively. In the clinical trial, two patients were exposed to doxorubicin and one patient was exposed to paclitaxel/platinum during and after pregnancy. Furthermore, a pooled analysis was performed based on 16 cycles of pregnant and 11 cycles of nonpregnant women. Numbers of pregnant/nonpregnant patients were 5/2, 7/5, 4/4 and 2/2 for paclitaxel, doxorubicin, epirubicin and platinum, respectively. For all drugs tested in the preclinical and clinical study, a decreased AUC and maximal plasma concentration and an increased distribution volume and clearance were observed in pregnancy., Conclusions: Although numbers were too small for statistical significance, pregnancy-associated physiologic alterations appear to lead to a decrease in plasma exposure of chemotherapeutic drugs. The importance of long-term follow-up of women treated with chemotherapy during pregnancy is underscored.

Objectives: We aimed to evaluate the cardiac safety of macrolides either used alone or co-administered with other agents., Methods: Pubmed and Embase databases were searched for the cardiac safety of macrolides in the treatment of infected patients. Identified studies were evaluated by two independent reviewers. Case reports, case series, controlled trials and randomly controlled trials were included., Results: A total of 48 reports (18 clinical studies and 30 case reports) were included in the present study. Among these reports, 25 were about macrolides used alone, and 23 about combination therapy. Based on the available data, we found that erythromycin carries the greatest risk of QT prolongation and Tdp from all macrolides in clinical practice (21/48), followed by clarithromycin (12/48) and azithromycin (6/48). Old age, high dosage, rapid administration and cardiac related diseases are additional risk factors., Conclusions: Macrolides may induce cardiotoxicity themselves when used alone. When co-administered, they may also increase the risk of other drugs that potentially prolong the QTc interval or induce Tdp. Therefore, early and correct adjustment of the dosage, close daily ECG monitoring and the avoidance of co-administration of other known QT-prolonging agents should be used in order to prevent the development of adverse effects.

Background and Objectives: The progression of glaucoma can be reduced or delayed by reducing intraocular pressure (IOP). The properties of femtosecond laser surgery, such as markedly reduced collateral tissue damage, coupled with the ability to achieve isolated subsurface surgical effects in the sclera, make this technology a promising candidate in glaucoma management. In this pilot study we demonstrate the in vivo creation of partial thickness subsurface drainage channels with the femtosecond laser in the sclera of rabbit eyes in order to increase aqueous humor (AH) outflow., Study Design/materials and Methods: A femtosecond laser beam tuned to a 1.7 microm wavelength was scanned along a rectangular raster pattern to create the partial thickness subsurface drainage channels in the sclera of one eye of each of the four rabbits included in this pilot study. IOP was measured before and 20 minutes after the laser treatment to evaluate the acute effect of the procedure., Results: OCT images verified the creation of the partial thickness subsurface scleral channels in the eyes of the in vivo rabbits. Comparison of pre- and postoperative IOP measurements in treated and control eyes revealed a reduction in the intraocular pressure due to the increased rate of AH outflow resulted in by the presence of the partial thickness scleral channels., Conclusions: The creation of partial thickness subsurface drainage channels was demonstrated in the sclera of in vivo rabbit eyes with a 1.7 microm wavelength femtosecond laser. Reduction in IOP achieved by the partial thickness channels suggests potential utility in the treatment of elevated IOP., (2010 Wiley-Liss, Inc.)

Purpose: Recent developments in nonlinear optical (NLO) imaging using femtosecond lasers provides a noninvasive method for detecting collagen fibers by imaging second harmonic-generated (SHG) signals. However, this technique is limited by the small field of view necessary to generate SHG signals. The purpose of this report is to review our efforts to greatly extend the field of view to assess the entire collagen structure using high-resolution macroscopic (HRMac) imaging., Methods: Intact human eyes were fixed under pressure, and the whole cornea (13-mm diameter) was excised and embedded in low-melting point agar for vibratome sectioning (200-300 microm). Sections were then optically scanned using a Zeiss LSM 510 Meta and Chameleon femtosecond laser (Carl Zeiss Microimaging Inc., Thornwood, NY) to generate SHG images. For each vibratome section, an overlapping series of three-dimensional data sets (466 x 466 x 150 microm) were taken, covering the entire tissue (15 mm x 6 mm area) using a motorized, mechanical stage. The three-dimensional data sets were then concatenated to generate an NLO-based tomograph., Results: The HRMac of the cornea yielded large macroscopic (80 megapixels per plane), three-dimensional tomographs with high resolution (0.81 microm lateral, 2.0 microm axial) in which individual collagen fibers (stromal lamellae) could be traced, segmented, and extracted. Three-dimensional reconstructions suggested that the anterior cornea comprises highly intertwined lamellae that insert into the anterior limiting lamina (Bowman's layer)., Conclusions: We conclude that HRMac using NLO-based tomography provides a powerful new tool to assess collagen structural organization within the cornea.

Background: Techniques for uterus transplantation (UTx) have been developed in rodent/domestic animals towards future clinical introduction of UTx to treat uterine factor infertility. The aim of this study was to extend the UTx research into a non-human primate species by developing surgical techniques for uterus retrieval and transplantation in the baboon., Methods: Female baboons (n = 15) underwent surgery, with the initial five animals used for studies of pelvic vascular anatomy. Retrieval surgery included isolation of the ovarian veins and the uterine arteries together with the anterior branches of the internal iliacs. The utero-tubal-ovarian specimen was removed, flushed and kept ex vivo for 2 h when the two arterial ends and two venous ends were anastomosed side-to-side to construct one arterial and one venous end. These were, at auto-transplantation, anastomosed end-to-side to the external iliacs and the animals (n = 10) were evaluated concerning cyclicity and later by laparoscopy/laparotomy., Results: The total duration of organ retrieval, backtable preparation and transplantation was around 6 h with an overall ischaemic time of the specimen of about 3 h. One animal died due to cardiomyopathy. Five out of the nine surviving animals resumed cyclicity, as a sign of re-established ovarian function. Only two out of these five animals exhibited resumed menstruation, indicating re-established ovarian and uterine function. Laparoscopy confirmed normal-sized uteri in these two animals., Conclusions: This study demonstrates the feasibility of UTx by vascular anastomosis in a non-human primate species. The low success rate demonstrates the complexity involved in UTx surgery and the need for further methodological developments.

Objective: To investigate the relationship between single nucleotide polymorphism (SNP) of hypoxia inducible factor-1alpha (HIF-1alpha) C1772T and genetic susceptibility to and clinical-pathological features of cervical cancers in Han population in Sichuan province of China., Methods: A case control study was undertaken in Sichuan province of China, with 97 patients with uterine cervical cancer as case group and 117 negative for intraepithelial lesion or malignancy (NILM) patients as control group. Their gene types in HIF-1alpha C1772T were identified with a combination of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)., Results: The distribution of the frequencies of T/T, T/C and C/C genotypes in the two groups differed significantly (P < 0.01); T allele frequency in the patients with cervical cancer was much higher than that in the controls (P < 0.01). There were no significant differences in the distributions of T/T, T/C and C/C genotypes among cervical cancer patients at different FIGO stages, pathological grading, stromal invasive depth, lymph node metastasis and vascular invasions (P > 0.05)., Conclusion: T/C and T/T genotypes of HIF-1alpha C1772T are genetic susceptibility factors for cervical cancer in Han population in Sichuan province of China. HIF-1alpha C1772T SNP probably has no relationship with clinical-pathological features of cervical cancer.

Mg/Ca (1 wt.%, 5 wt.%, 10 wt.% Ca) composites were prepared from pure magnesium and calcium powders using the powder metallurgy method, aiming to enlarge the addition of Ca content without the formation of Mg(2)Ca. The microstructures, mechanical properties and cytotoxicities of Mg/Ca composite samples were investigated. The corrosion of Mg/Ca composites in Dulbecco's modified Eagle's medium (DMEM) for various immersion intervals was studied by electrochemical impedance spectroscopy measurements and environmental scanning electron microscope, with the concentrations of released Mg and Ca ions in DMEM for various immersion time intervals being measured. It was shown that the main constitutional phases were Mg and Ca, which were uniformly distributed in the Mg matrix. The ultimate tensile strength (UTS) and elongation of experimental composites decreased with increasing Ca content, and the UTS of Mg/1Ca composite was comparable with that of as-extruded Mg-1Ca alloy. The corrosion potential increased with increasing Ca content, whereas the current density and the impedance decreased. It was found that the protective surface film formed quickly at the initial immersion stage. With increasing immersion time, the surface film became compact, and the corrosion rate of Mg/Ca composites slowed down. The surface film consisted mainly of CaCO(3), MgCO(3)x3H(2)O, HA and Mg(OH)(2) after 72 h immersion in DMEM. Mg/1Ca and Mg/5Ca composite extracts had no significant toxicity (p>0.05) to L-929 cells, whereas Mg/10Ca composite extract induced approximately 40% reduced cell viability., (Copyright (c) 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Objective: Our goal was to test the hypothesis that specific integrin receptors regulate chondrocyte biosynthetic response to dynamic compression at early times in 3D gel culture, during initial evolution of the pericellular matrix, but prior to significant accumulation of further-removed matrix. The study was motivated by increased use of dynamic loading, in vitro, for early stimulation of tissue engineered cartilage, and the need to understand the effects of loading, in vivo, at early times after implantation of constructs., Methods: Bovine articular chondrocytes were seeded in 2% agarose gels (15x10(6)cells/mL) and incubated for 18 h with and without the presence of specific integrin blockers (small-molecule peptidomimetics, function-blocking antibodies, and RGD-containing disintegrins). Samples were then subjected to a 24-h dynamic compression regime found previously to stimulate chondrocyte biosynthesis in 3D gel as well as cartilage explant culture (1 Hz, 2.5% dynamic strain amplitude, 7% static offset strain). At the end of loading, proteoglycan (PG) synthesis ((35)S-sulfate incorporation), protein synthesis ((3)H-proline incorporation), DNA content (Hoechst dye 33258) and total glycosaminoglycan (GAG) content (dimethyl methylene blue (DMMB) dye binding) were assessed., Results: Consistent with previous studies, dynamic compression increased PG synthesis and total GAG accumulation compared to free-swelling controls. Blocking alphavbeta3 abolished this response, independent of effects on controls, while blocking beta1 abolished the relative changes in synthesis when changes in free-swelling synthesis rates were observed., Conclusions: This study suggests that both alphavbeta3 and beta1 play a role in pathways that regulate stimulation of PG synthesis and accumulation by dynamic compression, but through distinct complementary mechanisms., (Copyright 2009 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Inhibition of mitotic kinesins represents a novel approach for the discovery of a new generation of anti-mitotic cancer chemotherapeutics. We report here the discovery of the first potent and selective inhibitor of centromere-associated protein E (CENP-E) 3-chloro-N-{(1S)-2-[(N,N-dimethylglycyl)amino]-1-[(4-{8-[(1S)-1-hydroxyethyl]imidazo[1,2-a]pyridin-2-yl}phenyl)methyl]ethyl}-4-[(1-methylethyl)oxy]benzamide (GSK923295; 1), starting from a high-throughput screening hit, 3-chloro-4-isopropoxybenzoic acid 2. Compound 1 has demonstrated broad antitumor activity in vivo and is currently in human clinical trials.

Background: Array genomic hybridization is being used clinically to detect pathogenic copy number variants in children with intellectual disability and other birth defects. However, there is no agreement regarding the kind of array, the distribution of probes across the genome, or the resolution that is most appropriate for clinical use., Results: We performed 500 K Affymetrix GeneChip array genomic hybridization in 100 idiopathic intellectual disability trios, each comprised of a child with intellectual disability of unknown cause and both unaffected parents. We found pathogenic genomic imbalance in 16 of these 100 individuals with idiopathic intellectual disability. In comparison, we had found pathogenic genomic imbalance in 11 of 100 children with idiopathic intellectual disability in a previous cohort who had been studied by 100 K GeneChip array genomic hybridization. Among 54 intellectual disability trios selected from the previous cohort who were re-tested with 500 K GeneChip array genomic hybridization, we identified all 10 previously-detected pathogenic genomic alterations and at least one additional pathogenic copy number variant that had not been detected with 100 K GeneChip array genomic hybridization. Many benign copy number variants, including one that was de novo, were also detected with 500 K array genomic hybridization, but it was possible to distinguish the benign and pathogenic copy number variants with confidence in all but 3 (1.9%) of the 154 intellectual disability trios studied., Conclusion: Affymetrix GeneChip 500 K array genomic hybridization detected pathogenic genomic imbalance in 10 of 10 patients with idiopathic developmental disability in whom 100 K GeneChip array genomic hybridization had found genomic imbalance, 1 of 44 patients in whom 100 K GeneChip array genomic hybridization had found no abnormality, and 16 of 100 patients who had not previously been tested. Effective clinical interpretation of these studies requires considerable skill and experience.

This report describes a 4 year-old girl with history of hypotonia, developmental delay, and failure to thrive in infancy. She has cognitive impairment and multiple congenital anomalies, including Duane anomaly, Mondini malformation with associated deafness, external ear malformations, and atrial and ventricular septal defects. Array comparative genomic hybridization demonstrated a de novo tandem 6.9 Mb duplication of at least 15 genes in chromosome 8q12, inclusive of CHD7, with breakpoints at 58,388,614 bp and 65,306,097 bp (NCBI build 36.1). Loss of CHD7 by microdeletion or intragenic mutation causes CHARGE syndrome. There is one previous report of an individual with microduplication of 8q12 involving CHD7. He also had early hypotonia, cognitive impairment, Duane anomaly, sensorineural deafness and a congenital heart defect. This rather specific recurrent pattern of congenital anomalies associated with overlapping duplications of the genomic region containing CHD7 suggests that the phenotype in these two patients may be the result of abnormal CHD7 dosage.