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In avians, yolk synthesis is regulated by incorporation of portomicrons from the diet, transport of lipoproteins from the liver, and release of lipids from adipose tissue; however, the extent to which lipolysis in adipose tissue contributes to yolk synthesis and egg production has yet to be elucidated. G0/G1 switch gene 2 (G0S2) is known to bind and inhibit adipose triglyceride lipase (ATGL), the rate-limiting enzyme in lipolysis. The objective of this study was to determine whether overexpression of the G0S2 gene in adipose tissue could successfully inhibit endogenous ATGL activity associated with egg laying. Two independent lines of transgenic quail overexpressing G0S2 had delayed onset of egg production and reduced number of eggs over a six-week period compared to non-transgenic quail. Although no differences in measured parameters were observed at the pre-laying stage (5 weeks of age), G0S2 transgenic quail had significantly larger interclavicular fat pad weights and adipocyte sizes and lower NEFA concentrations in the serum at early (1 week after laying first egg) and active laying (5 weeks after laying first egg) stages. Overexpression of G0S2 inhibited lipolysis during early and active laying, which drastically shifted the balance towards a net accumulation of triacylglycerols and increased adipose tissue mass. Thereby, egg production was negatively affected as less triacylglycerols were catabolized to produce lipids for the yolk.

Bioorthogonal chemical reactions are a thriving area of chemical research in recent years as an unprecedented technique to dissect native biological processes through chemistry-enabled strategies. However, current concepts of bioorthogonal chemistry have largely centered on 'bond formation' reactions between two mutually reactive bioorthogonal handles. Recently, in a reverse strategy, a collection of 'bond cleavage' reactions has emerged with excellent biocompatibility. These reactions have expanded our bioorthogonal chemistry repertoire, enabling an array of exciting new biological applications that range from the chemically controlled spatial and temporal activation of intracellular proteins and small-molecule drugs to the direct manipulation of intact cells under physiological conditions. Here we highlight the development and applications of these bioorthogonal cleavage reactions. Furthermore, we lay out challenges and propose future directions along this appealing avenue of research.

Background: Mechanical thrombectomy with stent-retrievers results in higher recanalization rates compared with previous devices. Despite successful recanalization rates (Thrombolysis in Cerebral Infarction (TICI) score ≥2b) of 70-83%, good outcomes by 90-day modified Rankin Scale (mRS) score ≤2 are achieved in only 40-55% of patients. We evaluated predictors of poor outcomes (mRS >2) despite successful recanalization (TICI ≥2b) in the North American Solitaire Stent Retriever Acute Stroke (NASA) registry., Methods: Logistic regression was used to evaluate baseline characteristics and recanalization outcomes for association with 90-day mRS score of 0-2 (good outcome) vs 3-6 (poor outcome). Univariate tests were carried out for all factors. A multivariable model was developed based on backwards selection from the factors with at least marginal significance (p≤0.10) on univariate analysis with the retention criterion set at p≤0.05. The model was refit to minimize the number of cases excluded because of missing covariate values; the c-statistic was a measure of predictive power., Results: Of 354 patients, 256 (72.3%) were recanalized successfully. Based on 234 recanalized patients evaluated for 90-day mRS score, 116 (49.6%) had poor outcomes. Univariate analysis identified an increased risk of poor outcome for age ≥80 years, occlusion site of internal carotid artery (ICA)/basilar artery, National Institute of Health Stroke Scale (NIHSS) score ≥18, history of diabetes mellitus, TICI 2b, use of rescue therapy, not using a balloon-guided catheter or intravenous tissue plasminogen activator (IV t-PA), and >30 min to recanalization (p≤0.05). In multivariable analysis, age ≥80 years, occlusion site ICA/basilar, initial NIHSS score ≥18, diabetes, absence of IV t-PA, ≥3 passes, and use of rescue therapy were significant independent predictors of poor 90-day outcome in a model with good predictive power (c-index=0.80)., Conclusions: Age, occlusion site, high NIHSS, diabetes, no IV t-PA, ≥3 passes, and use of rescue therapy are associated with poor 90-day outcome despite successful recanalization., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

In recent years, bioorthogonal reactions have emerged as a powerful toolbox for specific labeling and visualization of biomolecules, even within the highly complex and fragile living systems. Among them, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is one of the most widely studied and used biocompatible reactions. The cytotoxicity of Cu(I) ions has been greatly reduced due to the use of Cu(I) ligands, which enabled the CuAAC reaction to proceed on the cell surface, as well as within an intracellular environment. Meanwhile, other transition metals such as ruthenium, rhodium and silver are now under development as alternative sources for catalyzing bioorthogonal cycloadditions. In this review, we summarize the development of CuAAC reaction as a prominent bioorthogonal reaction, discuss various ligands used in reducing Cu(I) toxicity while promoting the reaction rate, and illustrate some of its important biological applications. The development of additional transition metals in catalyzing cycloaddition reactions will also be briefly introduced.

MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nucleotides, which negatively regulate the gene expression at the post-transcriptional level. This study describes an update of the miRTarBase (http://miRTarBase.mbc.nctu.edu.tw/) that provides information about experimentally validated miRNA-target interactions (MTIs). The latest update of the miRTarBase expanded it to identify systematically Argonaute-miRNA-RNA interactions from 138 crosslinking and immunoprecipitation sequencing (CLIP-seq) data sets that were generated by 21 independent studies. The database contains 4966 articles, 7439 strongly validated MTIs (using reporter assays or western blots) and 348 007 MTIs from CLIP-seq. The number of MTIs in the miRTarBase has increased around 7-fold since the 2014 miRTarBase update. The miRNA and gene expression profiles from The Cancer Genome Atlas (TCGA) are integrated to provide an effective overview of this exponential growth in the miRNA experimental data. These improvements make the miRTarBase one of the more comprehensively annotated, experimentally validated miRNA-target interactions databases and motivate additional miRNA research efforts., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The oxidative pentose phosphate pathway (PPP) contributes to tumour growth, but the precise contribution of 6-phosphogluconate dehydrogenase (6PGD), the third enzyme in this pathway, to tumorigenesis remains unclear. We found that suppression of 6PGD decreased lipogenesis and RNA biosynthesis and elevated ROS levels in cancer cells, attenuating cell proliferation and tumour growth. 6PGD-mediated production of ribulose-5-phosphate (Ru-5-P) inhibits AMPK activation by disrupting the active LKB1 complex, thereby activating acetyl-CoA carboxylase 1 and lipogenesis. Ru-5-P and NADPH are thought to be precursors in RNA biosynthesis and lipogenesis, respectively; thus, our findings provide an additional link between the oxidative PPP and lipogenesis through Ru-5-P-dependent inhibition of LKB1-AMPK signalling. Moreover, we identified and developed 6PGD inhibitors, physcion and its derivative S3, that effectively inhibited 6PGD, cancer cell proliferation and tumour growth in nude mice xenografts without obvious toxicity, suggesting that 6PGD could be an anticancer target.

Hyperplastic growth and hypertrophic growth within adipose tissue is tightly associated with cell cycle activity. In this study, CCNG2 and CDKN2C were found to be correlated with cell cycle inhibition during fat cell differentiation, whereas CCND3, CCNA1, and ANAPC5 were positively associated with cell cycle activity during fat cell proliferation after selection based on GEO datasets available on the NCBI website. The findings were validated through comparison of expressions of these genes among different tissues/fractions in broiler chickens and time points during primary cell culture using quantitative real-time PCR. Development of broiler subcutaneous adipose tissue was investigated on embryonic days 15 and 17 and on post-hatch days 0, 5, 11, and 33 using H&E staining and PCNA immunostaining with DAPI counter stain. In addition, mRNA expressions of five cell cycle regulators as well as precursor cell and adipocyte markers were measured at those time points. The results suggest that cellular proliferation activity decreased as the fat pad grows, but a population of precursor cells seemed to be maintained until post-hatch day 5 despite increasing differentiation activity. Hypertrophic growth gradually intensified despite a slight cessation on post-hatch day 0 due to increased energy expenditure during hatching and delayed food access. From post-hatch day 5 to day 11, most of the precursor cells may become differentiated. After post-hatch day 11, hyperplastic growth seemed to slow, while hypertrophic growth may become dominant. This study provides further understanding about broiler fat tissue development which is imperative for effective control of fat deposition.

A growing body of evidence indicates that both inhibition and decision-making deficits play essential roles in the development and maintenance of Internet gaming disorder (IGD). Clarifying whether impaired decision-making among individuals with IGD is related to poor inhibition will advance our understanding of IGD and contribute to intervention development. However, the relationship between these two functions remains unclear. In this study, we sought to systemically examine inhibitory processes, decision-making and the relationship between the two among individuals with IGD. Thirty-four individuals with IGD and 32 matched healthy controls (HCs) were recruited. In comparison to HCs, IGD subjects demonstrated inhibition deficits during performance of the gaming-related Go/No-Go task and impaired decision-making under risk. In addition, errors on No-Go trials during the gaming-related Go/No-Go task were positively associated with decision-making impairments under risk but not under ambiguity among IGD subjects. These results suggest individuals with IGD are impaired in some aspects of inhibition and decision-making functions, and that decision-making deficits under risk are linked to poor inhibition specifically related to gaming cues, which has implications for the development of novel intervention., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background and Purpose: Failure to recanalize predicts mortality in acute ischemic stroke. In the North American Solitaire Acute Stroke registry, we investigated parameters associated with mortality in successfully recanalized patients., Methods: Logistic regression was used to evaluate baseline characteristics and recanalization parameters for association with 90-day mortality. A multivariable model was developed based on backward selection with retention criteria of P<0.05 from factors with at least marginal significance (P≤0.10), then refit to minimize the number of excluded cases (missing data)., Results: Successfully recanalized patients had lower mortality (25.2% [59/234] versus 46.9% [38/81] P<0.001). There was no difference in symptomatic intracranial hemorrhage between patients with successful versus failed recanalization (9% [21/234] versus 14% [11/79]; P=0.205). However, mortality was significantly higher in patients with symptomatic intracranial hemorrhage (72% [23/32] versus 26% [73/281]; P<0.001). Proximal occlusion (internal carotid artery or vertebrobasilar), initial National Institutes of Health Stroke Scale≥18, use of rescue therapy (P<0.05), and 3+ passes (P<0.10) were associated with mortality in recanalized patients. In the multivariate model with good predictive power (c index=0.72), proximal occlusion, initial National Institutes of Health Stroke Scale≥18, and use of rescue therapy remained significant independent predictors of 90-day mortality., Conclusions: Failure to recanalize and presence of symptomatic intracranial hemorrhage resulted in increased mortality. Despite successful recanalization, proximal occlusion, high National Institutes of Health Stroke Scale, and need for rescue therapy were predictors of mortality., (© 2015 American Heart Association, Inc.)

Heme plays pivotal roles in various cellular processes as well as in iron homeostasis in living systems. Here, we report a genetically encoded fluorescence resonance energy transfer (FRET) sensor for selective heme imaging by employing a pair of bacterial heme transfer chaperones as the sensory components. This heme-specific probe allows spatial-temporal visualization of intracellular heme distribution within living cells.

A common surgical complication of clipping aneurysms with a calcified neck is the calcified atheroma compromising the parent arteries after clipping the neck. Clips can slip downward at the calcified neck or cause calcified atheroma encroaching the parent arteries. This video demonstrates a reconstructive clip technique to avoid these issues. A fenes-trated clip is placed first to reconstruct the distal parent artery-aneurysm neck with the fenestrated ring over the thickest calcification. Then, a straight clip reconstructs the proximal artery-aneurysm junction, leaving the thickest point of calcified walls pinching together by themselves to achieve aneurysm occlusion while preserving the parent arteries. The video can be found here: http://youtu.be/9CM3o5&#95;qlNQ.

Objectives/hypothesis: The purpose of the study was to investigate whether cochlear implantation using the round window approach provided better preservation of residual hearing than the cochleostomy approach., Study Design: Case-control study., Methods: We designed a case-control study including 40 patients from a tertiary referral center who underwent cochlear implantation surgeries using devices from MED-EL Co., Innsbruck, Austria. Between November 2013 and July 2014, we prospectively enrolled 20 subjects for cochlear implantation surgery using the round window insertion approach. In addition, 20 age- and sex-matched control subjects from the database of cochlear implantees treated using the cochleostomy approach between January 2008 and October 2013 were retrospectively enrolled. The residual hearing of the operated ear was measured before and after surgery. The variables analyzed were the pure-tone average threshold at 250, 500, and 1,000 Hz and the residual hearing at frequencies of 250 to 8,000 Hz. The residual hearing was considered as preserved when the audiometric changes were <10 dB hearing loss for each variable. The audiological results of the two groups were compared., Results: No statistically significant difference in the preservation of residual hearing was found in the two groups (P > .05 for all of the variables)., Conclusions: The round window and cochleostomy approaches for cochlear implant surgery may preserve residual hearing at similar rates across a range of frequencies., (© 2014 The American Laryngological, Rhinological and Otological Society, Inc.)

Coupling genetically encoded peptide tags or unnatural amino acids (UAAs) with bioorthogonal reactions allows for precise control over the protein-labeling sites as well as the wide choice of labeling dyes. However, the value of these site-specific protein labeling strategies in a real biology setting, particularly their advantages over conventional labeling methods including fluorescent proteins (FPs), remains to be fully demonstrated. In this tutorial review, we first introduce various strategies for site-specific protein labeling that utilize artificial peptide sequences or genetically encoded UAAs as the labeling handle. Emphasis will be placed on introducing the advantages of protein site-specific labeling techniques as well as their applications in solving biological problems, particularly as to why a site-specific protein labeling approach is needed. Finally, beyond the widely used single site-specific labeling methods, the recently emerged dual site-specific protein labeling strategies will be introduced together with their fast-growing potential in illustrating biological processes.

Objectives: Stenotrophomonas maltophilia is a bacterial pathogen associated with health-care associated infections, particularly in immunocompromised patients. Members of the fluoroquinolone drug class are frequently used to treat S. maltophilia infection; however, S. maltophilia resistance to fluoroquinolones, especially levofloxacin, has been increasing., Methods: We sought to identify risk factors associated with levofloxacin resistance using a case-control study. We examined sputum from 76 S. maltophilia-positive patients admitted to our hospital between January 1, 2010 and June 30, 2011. Case groups were defined as patients who had S. maltophilia infections resistant to levofloxacin, and control groups were defined as patients who had S. maltophilia infections susceptible to levofloxacin treatment. Patient information including demographics, previous antibiotic use, and other traits were recorded. In addition, S. maltophilia isolates from patient sputum were assessed for antibiotic resistance as well as for the presence of genes associated with drug resistance., Results: Previous antibiotic treatment with first- or second-generation cephalosporin was found more often in the levofloxacin-susceptible group; by contrast, previous piperacillin/tazobactam treatment occurred more often in the levofloxacin-resistant group. Three genes associated with drug resistance, including SmeA, SmeD, and SpgM were not significantly different between these groups., Conclusion: Piperacillin/tazobactam treatment is associated with subsequent isolation of levofloxacin-resistant S. maltophilia from the respiratory tract., (Copyright © 2013. Published by Elsevier B.V.)

Background: Delayed cerebral ischemia (DCI) is a major cause of morbidity and mortality in aneurysmal subarachnoid hemorrhage. Endovascular management of this condition offers a new hope in preventing adverse outcome; however, a uniform standard of practice is lacking owing to a paucity of clinical trials. We conducted an international survey on the use of investigative and endovascular techniques in the treatment of DCI to assess the variability of current practice., Methods: Neurovascular neurosurgeons and neuroradiologists were contacted through professional societies from America, United Kingdom, Europe, and Australasia. Members were invited to complete a 13-item questionnaire regarding screening techniques, first-line and second-line therapies in endovascular intervention, and the role of angioplasty. Answers were compared using χ(2) testing for nonparametric data., Results: Data from 344 respondents from 32 countries were analyzed: 167 non-United States and 177 U.S., Respondents: More than half of all clinicians had 10+ years of experience in units with a mixture of higher and lower case volumes. Daily transcranial Doppler ultrasonography was the most commonly used screening technique by both U.S. (70%) and non-U.S. (53%) practitioners. Verapamil was the most common first-line therapy in the United States, whereas nimodipine was most popular in non-U.S. countries. Angioplasty was performed by 83% of non-U.S. and 91% of U.S. clinicians in the treatment of vasospasm; however, more U.S. clinicians reported using angioplasty for distal vasospasm., Conclusions: Treatment practices for DCI vary considerably, with the greatest variability in the choice of agent for intra-arterial therapy. Our data demonstrate the wide variation of approaches in use at present. However, without further clinical trials and development of a uniform standard of best practice, variability in treatment and outcome for DCI is likely to continue., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: Following aneurysmal subarachnoid hemorrhage, cerebral vasospasm/delayed cerebral ischemia accounts for significant morbidity and mortality. In this paper we provide the first glimpse of actual practice in the management of cerebral vasospasm in the USA., Methods: All active members of the Joint American Association of Neurological Surgeons (AANS)/Congress of Neurological Surgeons (CNS) Cerebrovascular Section were emailed the survey. The responses were collected anonymously., Results: The response rate for this survey was 44% (177 responses/400 total members). Non-responders were characterized by the fact that multiple responders were not obtained from practices that had multiple providers with uniform practice. Both high-volume and low-volume centers were equally represented. Optimizing medical management is first-line treatment in practice. Although there is some variability regarding screening methods, the greatest variability occurs with regard to the choice of intra-arterial treatment drug and dose. There is also considerable variability in the perceived effectiveness of endovascular treatment for vasospasm., Conclusions: In this preliminary glimpse of actual cerebral vasospasm management practice in the USA, two salient points emerge: (1) there is considerable variability in intra-arterial therapies for vasospasm; and (2) there are major differences in the perceived effectiveness of these therapies. Standardization of intra-arterial therapies may contribute to improved outcomes. A prospective randomized trial evaluating endovascular treatment for cerebral vasospasm is needed., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

We herein report a chemical decaging strategy for the in situ generation of neuramic acid (Neu), a unique type of sialic acid, on live cells by the use of a palladium-mediated bioorthogonal elimination reaction. Palladium nanoparticles (Pd NPs) were found to be a highly efficient and biocompatible depropargylation catalyst for the direct conversion of metabolically incorporated N-(propargyloxycarbonyl)neuramic acid (Neu5Proc) into Neu on cell-surface glycans. This conversion chemically mimics the enzymatic de-N-acetylation of N-acetylneuramic acid (Neu5Ac), a proposed mechanism for the natural occurrence of Neu on cell-surface glycans. The bioorthogonal elimination was also exploited for the manipulation of cell-surface charge by unmasking the free amine at C5 to neutralize the negatively charged carboxyl group at C1 of sialic acids., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Aberrant insulin-like growth factor-binding protein 7 (IGFBP-7) expression has been found in various cancers such as prostate, breast, and colon. IGFBP-7 induced the apoptosis of tumor and potentially predicted the clinical outcome in some cancers is further demonstrated. This study investigates the causes and underlying mechanisms of aberrant IGFBP-7 expression in unravelling head and neck squamous cell carcinoma (HNSCC)., Methods: A total of 47 oral tongue cancer patient samples were primarily analyzed for the methylation status in 5' region of IGFBP-7 by methylation-specific PCR (MS-PCR). Subsequently the invasion, overexpression, and knockdown of IGFBP-7 in the HNSCC A253 invasive subpopulation were employed to examine the effect of IGFBP-7. The epithelial-mesenchymal transition (EMT) marker genes and AKT/GSK3β/β-catenin signaling were further evaluated by Western blot for the understanding the role of aberrant IGFBP-7 expression and thereof putative mechanism., Results: EMT expressed in the invasive subpopulation of HNSCC cell lines (A253 and RPMI 2650) was contemporary with the down-regulation of IGFBP-7. After treatment with 5-AZA-2' deoxycytidine, the de-methylated CpG sites in the 5' region of IGFBP-7 were observed and IGFBP-7 mRNA expression was also restored. Accordingly, re-expression IGFBP-7 in invasive subpopulation of A253 could induce the mesenchymal-epithelial transition (MET) and concurrently inhibited the cell invasion. Moreover, IGFBP-7 methylation status of 47 oral tongue tumors showed a positive correlation to invasive depth of the tumor, loco-regional recurrence, and cancer sequence., Conclusions: IGFBP-7 can alter EMT relative marker genes and suppress cell invasion in A253 cell through AKT/GSK3β/β-catenin signaling. The epigenetic control of IGFBP-7 in the invasion and metastasis of HNSCC was reported, suggesting that IGFBP-7 could be a prognostic factor for the probability of invasion and a therapeutic remedy.

Background: The proximal lateral lower leg flap is a flap suited for the reconstruction of small and thin defects. The purpose of this study was to map the position and consistency of the perforator vessels and to review its reliability and technical considerations clinically., Methods: The location, number, and size of perforator vessels in the proximal third of the lateral lower leg were investigated in 20 fresh frozen cadaveric lower limbs. This was analyzed together with 22 clinical cases., Results: Cadaveric dissection showed that there were 1-2 perforators in the proximal third of the lateral lower leg and these perforator vessels were found to be 63% septocutaneous and 37% musculocutaneous. The source vessel of the perforators was variable. Clinically the recipient site consisted of the head and neck in 8 cases, the foot and ankle region in 13 cases, and 1 case in the hand. The mean thickness of this flap was 5.8 ± 0.8 mm. Vascular pedicle length ranged from 5 to 8.5 cm. The mean diameter of flap artery was 1.3 ± 0.3 mm. One flap failure was seen due to arterial thrombosis. The overall flap survival rate was 95%., Conclusions: The proximal lateral lower leg flap has the advantages of being thin and pliable, quick to harvest with no major arteries sacrificed. There is minimal donor site morbidity and primary closure of the donor site is possible in the majority of cases., (© 2014 Wiley Periodicals, Inc.)

Individuals with Internet gaming disorder (IGD) tend to exhibit disadvantageous risky decision-making not only in their real life but also in laboratory tasks. Decision-making is a complex multifaceted function and different cognitive processes are involved in decision-making for gains and losses. However, the relationship between impaired decision-making and gain versus loss processing in the context of IGD is poorly understood. The main aim of the present study was to separately evaluate decision-making for risky gains and losses among college students with IGD using the Cups task. Additionally, we further examined the effects of outcome magnitude and probability level on decision-making related to risky gains and losses respectively. Sixty college students with IGD and 42 matched healthy controls (HCs) participated. Results indicated that IGD subjects exhibited generally greater risk taking tendencies than HCs. In comparison to HCs, IGD subjects made more disadvantageous risky choices in the loss domain (but not in the gain domain). Follow-up analyses indicated that the impairment was associated to insensitivity to changes in outcome magnitude and probability level for risky losses among IGD subjects. In addition, higher Internet addiction severity scores were associated with percentage of disadvantageous risky options in the loss domain. These findings emphasize the effect of insensitivity to losses on disadvantageous decisions under risk in the context of IGD, which has implications for future intervention studies.

Extramedullary hematopoiesis (EMH) is a pathological process secondary to underlying bone marrow (BM) insufficiency in adults. It is characterized by the emergence of multipotent hematopoietic progenitors scattered around the affected tissue, most likely in the spleen, liver, and lymph node, etc. EMH in patients frequently receives less medical attention and is neglected unless a compressive or obstructive hematopoietic mass appears to endanger the patient's life. However, on a biological basis, EMH reflects the alteration of relationships among hematopoietic stem and progenitor cells (HSPCs) and their original and new microenvironments. The ability of hematopoietic stem cells (HSCs) to mobilize from the bone marrow and to accommodate and function in extramedullary tissues is rather complicated and far from our current understanding. Fortunately, many reports from the studies of drugs and genetics using animals have incidentally found EMH to be involved. Thereby, the molecular basis of EMH could further be elucidated from those animals after cross-comparison. A deeper understanding of the extramedullary hematopoietic niche could help expand stem cells in vitro and establish a better treatment in patients for stem cell transplantation.

The biological characteristics of rooster sperm that has undergone freezing treatment remain elusive. This study analyzed the change in sperm proteins after freezing-thawing treatment by using a proteomic approach. Semen from three 36-wk-old L2 strain Taiwan country chickens were used. A qualifying ejaculate containing more than 80% motility and volume 200μL was used for cryopreservation. The proteomic analysis explored 55 protein spots that differed significantly before and after freezing-thawing treatment (P<0.05). Among the 55 protein spots, expression levels of 19 proteins decreased after treatment. Forty-five differentially expressed protein spots were identified and belong to 33 proteins. Results of gene ontology analysis revealed that most differentially expressed proteins were involved in molecular function of the cellular metabolism process (28%) and cellular carbohydrate metabolism process (15%), and were associated with molecular function of oxidoreductase activity (19%) and protein binding (18%). The differentially expressed proteins before and after freezing-thawing treatment, including fructose-bisphosphate aldolase C, triosephosphate isomerase, aconitate hydratase, tubulin and outer dense-fiber protein, are associated with sperm energy metabolism and flagellum structure. In conclusion, freezing-thawing treatment significantly affects the expression of proteins related to sperm metabolism and structure in chicken spermatozoa. The differing levels of these proteins could be valuable for further enhancing the fertility of frozen-thawed chicken spermatozoa., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We achieved single-molecule imaging and tracking of the transforming growth factor type II receptor (TβRII) that was labeled by an organic dye via a genetically encoded unnatural amino acid (UAA) and the copper-free click chemistry. The stoichiometry, mobility and dimerization kinetics of individual TβRII molecules were determined.

As the number of cardiac implantable electronic devices (CIEDs) is increasing annually, CIED-related complications are becoming increasingly important. The aim of the study was to assess the risks associated with CIEDs by a nationwide database. Patients were selected from the Taiwan National Health Insurance Database. Admissions for CIED implantation, replacement, and revision were evaluated and the evaluation period was 14 years. Endpoints included CIED-related infection, pneumothorax, and heart perforation. The study included 40,608 patients with a mean age of 71.8 ± 13.3 years. Regarding infection, the incidence rate was 2.45 per 1000 CIED-years. Male gender, younger age, device replacement, and previous infection were risks for infection while old age and high-volume centers (>200 per year) were protectors. The incidence of pneumothorax was 0.6%, with an increased risk in individuals who had chronic obstructive lung disease (COPD) and cardiac resynchronized therapy (CRT). The incidence of heart perforation was 0.09%, with an increased risk in individuals who had pre-operation temporal pacing and steroid use. High-volume center was found to decrease infection rate while male gender, young people, and individuals who underwent replacements were associated with an increased risk of infection. Additionally, pre-operation temporal pacing and steroid use should be avoided if possible. Furthermore, COPD patients or those who accept CRTs should be monitored closely.

Small molecules that specifically activate an intracellular protein of interest are highly desirable. A generally applicable strategy, however, remains elusive. Herein we describe a small molecule-triggered bioorthogonal protein decaging technique that relies on the inverse electron-demand Diels-Alder reaction for eliminating a chemically caged protein side chain within living cells. This method permits the efficient activation of a given protein (for example, an enzyme) in its native cellular context within minutes.

Background and Purpose: The Solitaire With the Intention for Thrombectomy (SWIFT) and thrombectomy revascularization of large vessel occlusions in acute ischemic stroke (TREVO 2) trial results demonstrated improved recanalization rates with mechanical thrombectomy; however, outcomes in the elderly population remain poorly understood. Here, we report the effect of age on clinical and angiographic outcome within the North American Solitaire-FR Stent-Retriever Acute Stroke (NASA) Registry., Methods: The NASA Registry recruited sites to submit data on consecutive patients treated with Solitaire-FR. Influence of age on clinical and angiographic outcomes was assessed by dichotomizing the cohort into ≤80 and >80 years of age., Results: Three hundred fifty-four patients underwent treatment in 24 centers; 276 patients were ≤80 years and 78 were >80 years of age. Mean age in the ≤80 and >80 cohorts was 62.2±13.2 and 85.2±3.8 years, respectively. Of patients >80 years, 27.3% had a 90-day modified Rankin Score ≤2 versus 45.4% ≤80 years (P=0.02). Mortality was 43.9% and 27.3% in the >80 and ≤80 years cohorts, respectively (P=0.01). There was no significant difference in time to revascularization, revascularization success, or symptomatic intracranial hemorrhage between the groups. Multivariate analysis showed age >80 years as an independent predictor of poor clinical outcome and mortality. Within the >80 cohort, National Institutes of Health Stroke Scale (NIHSS), revascularization rate, rescue therapy use, and symptomatic intracranial hemorrhage were independent predictors of mortality., Conclusion: Greater than 80 years of age is predictive of poor clinical outcome and increased mortality compared with younger patients in the NASA registry. However, intravenous tissue-type plasminogen activator use, lower NIHSS, and shorter revascularization time are associated with better outcomes. Further studies are needed to understand the endovascular therapy role in this cohort compared with medical therapy., (© 2014 American Heart Association, Inc.)

Background: Video-assisted thoracoscopic surgery (VATS) is a minimally invasive alternative to conventional surgery (CS). We aimed to estimate the short-term cost-effectiveness of VATS vs. CS for clinical stage I non-small cell lung cancer (NSCLC-c-stage-I) patients from the payer's perspective (National Health Insurance)., Methods: We identified NSCLC-c-stage-I patients diagnosed and received surgery within 2007-2009 through a comprehensive population-based database containing cancer and death registries, and reimbursement data. The duration of interest was 1 year. We included potential confounding covariables through literature searching and our own experience, and used a propensity score to construct a 1:1 population for adjustment., Results: Our study population constituted 966 patients. The mean hospital stay [days, standard deviation (SD)] were 14.4 [7] and 16.1 (7.7) for VATS and CS respectively (P=0.002). The mean cost (2013 USD) and survival (year) was $22,316 vs. $21,976 and 0.98 vs. 0.974 for VATS vs. CS. The probability for VATS to be cost-effective (i.e., positive net benefit) was 0.49 & 0.56 at willingness-to-pay (WTP) 50,000 & 100,000 USD/life-year, respectively., Conclusions: We provide the first empirical evidence that when compared to CS, VATS was potentially cost-effective in the short term (1 year) within the common WTP levels in Taiwan.

Internet gaming addiction (IGA) is an increasing mental health issue worldwide. Previous studies have revealed decision-making impairments in excessive Internet gamers (EIGs) with high symptoms of IGA. However, the role of feedback processing in decision-making deficits among EIGs remains unknown. The present study aimed to investigate the effect of feedback processing on decision-making deficits under risk among EIGs, using the Game of Dice Task (GDT) and a modified version of the GDT in which no feedback was provided. Twenty-six EIGs and 26 matched occasional Internet gamers (OIGs) were recruited. The results showed: (a) OIGs performed better on the original GDT than on the modified GDT (no feedback condition); however, EIGs performed similarly on both tasks; (b) EIGs and OIGs performed equally on the modified GDT; however, EIGs chose more disadvantageous options than OIGs on the original GDT; (c) EIGs utilized feedback less frequently on the original GDT relative to OIGs. These results suggest that EIGs are not able to utilize feedback to optimize their decisions, which could underlie their poor decision-making under risk., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: Thoracic endovascular aortic repair (TEVAR) is becoming increasingly popular due to reduced perioperative morbidity and mortality compared with open surgical repair. However, complications can occur when the left subclavian artery is involved. When performing TEVAR with left carotid-subclavian artery bypass the stent graft will extend to the left common carotid artery. We herein present the case of a patient with a type B aortic dissection with an acute intramural hematoma. Chylothorax was noted after TEVAR with left carotid-subclavian artery bypass., Case Report: A 66-year-old female with descending aortic dissection that was treated conservatively developed the sudden onset of back pain. Aortic computed tomography (CT) showed a type B intramural aortic dissection. TEVAR with left carotid-subclavian artery bypass was performed. Left chylothorax was noted after surgery with drainage of up to 1000 mL per day. Conservative management was ineffective. Thoracoscopic ligation of the thoracic duct was performed with resolution of the chyle leakage., Conclusion: Chylothorax can occur after TEVAR with carotid-subclavian artery bypass and likely results from thoracic duct injury. When conservative treatments fail, ligation of the thoracic duct cephalad to aortic hiatus can resolve the chyle leakage.

Objective: Analyze the reliability, complications, and donor site morbidity of the proximal lateral leg flap when applied to head and neck reconstruction., Study Design: Case series and chart review., Setting: Tertiary care teaching hospital., Subjects and Methods: Nineteen patients who underwent reconstruction of various head and neck defects with this flap were analyzed. The patient demographics, flap characteristics, method of donor site closure, scars of the donor area, complication rates, as well as functional results at the recipient site were assessed., Results: The flap size ranged from 4 × 4 cm to 11 × 8 cm. Vascular pedicle length ranged from 5 to 9 cm. The mean distance of the perforator from the fibula head was 9.2 cm. The mean thickness of this flap was 5.5 mm. All the donor wounds were closed primarily. The flap survival rate was 100%., Conclusion: This flap has the advantages of thinness, short harvesting time, minimal donor site morbidity, and primary closure at the donor site when the flap width is less than 6 cm. This flap may be useful for reconstruction in selected patients with small and thin heads and neck defects., (© American Academy of Otolaryngology-Head and Neck Surgery Foundation 2014.)

Background: Limited post-marketing data exist on the use of the Solitaire FR device in clinical practice. The North American Solitaire Stent Retriever Acute Stroke (NASA) registry aimed to assess the real world performance of the Solitaire FR device in contrast with the results from the SWIFT (Solitaire with the Intention for Thrombectomy) and TREVO 2 (Trevo versus Merci retrievers for thrombectomy revascularization of large vessel occlusions in acute ischemic stroke) trials., Methods: The investigator initiated NASA registry recruited North American sites to submit retrospective angiographic and clinical outcome data on consecutive acute ischemic stroke (AIS) patients treated with the Solitaire FR between March 2012 and February 2013. The primary outcome was a Thrombolysis in Myocardial Ischemia (TIMI) score of ≥2 or a Treatment in Cerebral Infarction (TICI) score of ≥2a. Secondary outcomes were 90 day modified Rankin Scale (mRS) score, mortality, and symptomatic intracranial hemorrhage., Results: 354 patients underwent treatment for AIS using the Solitaire FR device in 24 centers. Mean time from onset to groin puncture was 363.4±239 min, mean fluoroscopy time was 32.9±25.7 min, and mean procedure time was 100.9±57.8 min. Recanalization outcome: TIMI ≥2 rate of 83.3% (315/354) and TICI ≥2a rate of 87.5% (310/354) compared with the operator reported TIMI ≥2 rate of 83% in SWIFT and TICI ≥2a rate of 85% in TREVO 2., Clinical Outcome: 42% (132/315) of NASA patients demonstrated a 90 day mRS ≤2 compared with 37% (SWIFT) and 40% (TREVO 2). 90 day mortality was 30.2% (95/315) versus 17.2% (SWIFT) and 29% (TREVO 2)., Conclusions: The NASA registry demonstrated that the Solitaire FR device performance in clinical practice is comparable with the SWIFT and TREVO 2 trial results., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Considerable attention has been focused on improving the biocompatibility of Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), a hallmark of bioorthogonal reaction, in living cells. Besides creating copper-free versions of click chemistry such as strain promoted azide-alkyne cycloaddition (SPAAC), a central effort has also been made to develop various Cu(I) ligands that can prevent the cytotoxicity of Cu(I) ions while accelerating the CuAAC reaction. Meanwhile, additional transition metals such as palladium have been explored as alternative sources to promote a bioorthogonal conjugation reaction on cell surface, as well as within an intracellular environment. Furthermore, transition metal mediated chemical conversions beyond conjugation have also been utilized to manipulate protein activity within living systems. We highlight these emerging examples that significantly enriched our protein chemistry toolkit, which will likely expand our view on the definition and applications of bioorthogonal chemistry.

Bioorthogonal reactions, especially the Cu(I)-catalysed azide-alkyne cycloaddition, have revolutionized our ability to label and manipulate biomolecules under living conditions. The cytotoxicity of Cu(I) ions, however, has hindered the application of this reaction in the internal space of living cells. By systematically surveying a panel of Cu(I)-stabilizing ligands in promoting protein labelling within the cytoplasm of Escherichia coli, we identify a highly efficient and biocompatible catalyst for intracellular modification of proteins by azide-alkyne cycloaddition. This reaction permits us to conjugate an environment-sensitive fluorophore site specifically onto HdeA, an acid-stress chaperone that adopts pH-dependent conformational changes, in both the periplasm and cytoplasm of E. coli. The resulting protein-fluorophore hybrid pH indicators enable compartment-specific pH measurement to determine the pH gradient across the E. coli cytoplasmic membrane. This construct also allows the measurement of E. coli transmembrane potential, and the determination of the proton motive force across its inner membrane under normal and acid-stress conditions.

We have developed a genetically encoded, selenium-based cleavable photo-cross-linker that allows for the separation of bait and prey proteins after protein photo-cross-linking. We have further demonstrated the efficient capture of the in situ generated selenenic acid on the cleaved prey proteins. Our strategy involves tagging the selenenic acid with an alkyne-containing dimethoxyaniline molecule and subsequently labeling with an azide-bearing fluorophore or biotin probe. This cleavage-and-capture after protein photo-cross-linking strategy allows for the efficient capture of prey proteins that are readily accessible by two-dimensional gel-based proteomics and mass spectrometry analysis.

Although the oxidative pentose phosphate pathway is important for tumor growth, how 6-phosphogluconate dehydrogenase (6PGD) in this pathway is upregulated in human cancers is unknown. We found that 6PGD is commonly activated in EGF-stimulated cells and human cancer cells by lysine acetylation. Acetylation at K76 and K294 of 6PGD promotes NADP(+) binding to 6PGD and formation of active 6PGD dimers, respectively. Moreover, we identified DLAT and ACAT2 as upstream acetyltransferases of K76 and K294, respectively, and HDAC4 as the deacetylase of both sites. Expressing acetyl-deficient mutants of 6PGD in cancer cells significantly attenuated cell proliferation and tumor growth. This is due in part to reduced levels of 6PGD products ribulose-5-phosphate and NADPH, which led to reduced RNA and lipid biosynthesis as well as elevated ROS. Furthermore, 6PGD activity is upregulated with increased lysine acetylation in primary leukemia cells from human patients, providing mechanistic insights into 6PGD upregulation in cancer cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We have developed a dual-site click labeling strategy for the simultaneous installation of a FRET donor-acceptor pair onto the extracellular domains of epidermal growth factor receptor (EGFR) on living cells. Our method integrates the genetic code expansion strategy, enzyme-mediated protein labeling, and ligand-assisted Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) into a tri-step labeling procedure. This enabled cis-membrane FRET imaging of EGFR under living conditions. This procedure might be generally applicable for dual-site labeling and cis-membrane FRET analysis of the domain-domain dynamics of important mammalian cell-surface receptors., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Estradiol plays an important role in the regulation of collagen metabolism. Deficiency of estradiol has been reported to be associated with the degeneration of many connective tissues. However, the association of estradiol and hypertrophy of the ligamentum flavum was seldom explored. Therefore, we studied the effects of estradiol on cultured cells from the ligamentum flavum., Methods: Primary cultures of human ligamentum flavum cells obtained from surgical specimens of 14 patients undergoing spinal surgery were used to investigate the effect of estradiol on cell proliferation and the expression of collagen, elastin, and matrix metalloproteinases. Downstream pathways of estrogen receptor underlying the regulation of metalloproteinases were also investigated., Results: In our study, we revealed the existence of estrogen receptors on both female and male ligamentum flavum cells with a gender difference. 17β-estradiol increased early (24 hours) proliferation of ligamentum flavum cells in a dose dependent manner and the effect could not be seen when the cell density increased. Estradiol with a concentration of 10(-9) M decreased collagen levels and increased the expression of MMP-13. Adding an antagonist of PI3K downstream pathway could reverse the expression of MMP-13 caused by estradiol., Conclusions: The results implied estradiol regulated the expression of MMP-13 via PI3K pathway and contributed to the homeostasis of extracellular matrix in the ligamentum flavum.

It is generally considered difficult and risky to treat a small aneurysm (less than 4 mm) by use of coil embolization. The main issues are related to the following factors: frequently broad-based aneurysm with shallow dome, complex in shape, difficult to maintain microcatheter stability in an aneurysm, difficult to achieve complete coil packing, and higher risk of intra-procedure rupture hemorrhage. However, routine use of balloon-assisted coiling technique will enable us to achieve successful aneurysm embolization with safety. This video demonstrates some key nuances in the balloon-assisted coil embolization of small aneurysms via three case examples. The video can be found here: http://youtu.be/E1il3oPnUD8 .

Coupling the genetic code expansion technique with bioorthogonal reactions enables precise control over the conjugation site as well as the choice of fluorescent probes during protein labeling. However, the advantages of this strategy over bulky and rigid fluorescent proteins (FPs) remain to be fully explored. Here we applied site-specific bioorthogonal labeling on anthrax lethal factor (LF) to visualize its membrane translocation inside live cells. In contrast to the previously reported FP tags that significantly perturbed LF's membrane trafficking, our precisely and quantitatively labeled LF exhibited an endocytic activity comparable to wild-type LF. This allowed time-lapse imaging of LF's natural translocation process from host cell membrane to cytosol, which revealed molecular details of its virulence mechanism. Our strategy is generally applicable for monitoring intracellular protein membrane translocation that is difficult to access using conventional protein labeling methodologies., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Transdiaphragmatic intercostal hernia (TIH) caused by violent coughing is a rare clinical diagnosis. Most patients diagnosed with TIH have a chronic condition consisting of a hernia that can be reduced completely by surgical intervention. Our patient presented with acute abdomen resulting from mechanical bowel obstruction secondary to an incarcerated hernia. Acute TIH presents a diagnostic challenge because of its rarity and lack of specific signs or symptoms in the differential diagnosis of acute abdomen. We recommend performing diagnostic computed tomography (CT) early if there is suspicion of TIH. Surgical intervention is always needed. Surgical intervention was complicated in this case, necessitating both transthoracic and abdominal exposure to resect the ischemic bowel segment. Nonetheless, the patient recovered uneventfully., (Copyright © 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Profound idiopathic sudden sensorineural hearing loss is thought to have a poor prognosis, but few studies have focused on this condition. We aimed to assess the impact of patient factors, audiologic parameters, and salvage intratympanic steroid injection therapy on the prognosis of profound idiopathic sudden sensorineural hearing loss. The demographic, clinical, and audiologic data, degree of hearing recovery, and efficacy of intratympanic steroid injection therapy in 576 patients with profound idiopathic sudden sensorineural hearing loss (mean age 56.2 ± 14.9 years) who had been admitted at four tertiary referral centers between 2000 and 2011 were retrospectively reviewed. The mean hearing level at the initial presentation was 108.1 ± 9.5 dB. Many patients experienced vertigo (52.1%) and tinnitus (77.4%). At the 2-month follow-up, 172 (29.8%) patients showed some degree of hearing recovery, but only 21 (3.6%) patients recovered normal hearing. Further, the 116 patients who had received salvage intratympanic steroid injections showed a better audiologic outcome (improvement, 26.1 ± 24.3 vs. 15.7 ± 22.1 dB; P = 0.000) than those who had not (n = 429). In conclusion, a higher degree of hearing loss at the initial presentation indicates a poorer prognosis. Salvage intratympanic steroid injection therapy may improve the hearing of patients with profound idiopathic sudden sensorineural hearing loss after the failure of systemic steroid therapy.

Background and Purpose: Previous work that predated the availability of the safer stent-retriever devices has suggested that general anesthesia (GA) may have a negative impact on outcomes in patients with acute ischemic stroke undergoing endovascular therapy., Methods: We reviewed demographic, clinical, procedural (GA versus local anesthesia [LA], etc), and site-adjudicated angiographic and clinical outcomes data from consecutive patients treated with the Solitaire FR device in the investigator-initiated North American SOLITAIRE Stent-Retriever Acute Stroke (NASA) Registry. The primary outcomes were 90-day modified Rankin Scale, mortality, and symptomatic intracranial hemorrhage., Results: A total of 281 patients from 18 centers were enrolled. GA was used in 69.8% (196/281) of patients. Baseline demographic and procedural factors were comparable between the LA and GA groups, except the former demonstrated longer time-to-groin puncture (395.4±254 versus 337.4±208 min; P=0.04), lower National Institutes of Health Stroke Scale (NIHSS; 16.2±5.8 versus 18.8±6.9; P=0.002), lower balloon-guide catheter usage (22.4% versus 49.2%; P=0.0001), and longer fluoroscopy times (39.5±33 versus 28±22.8 min; P=0.008). Recanalization (thrombolysis in cerebral infarction ≥2b; 72.94% versus 73.6%; P=0.9) and rate of symptomatic intracranial hemorrhage (7.1% versus 11.2%; P=0.4) were similar but modified Rankin Scale ≤2 was achieved in more LA patients, 52.6% versus 35.6% (odds ratio, 1.4 [1.1-1.8]; P=0.01). In multivariate analysis, hypertension, NIHSS, unsuccessful revascularization, and GA use (odds ratio, 3.3 [1.6-7.1]; P=0.001) were associated with death. When only anterior circulation and elective GA patients were included, there was a persistent difference in good outcomes in favor of LA patients (50.7% versus 35.5%; odds ratio, 1.3 [1.01-1.6]; P=0.04)., Conclusions: The NASA Registry has demonstrated that clinical outcomes and survival are significantly better in patients treated with LA, without increased symptomatic intracranial hemorrhage risk. Future trials should prospectively evaluate the effect of GA on outcomes.

We report the engineering of zinc-finger-like motifs containing the unnatural amino acid (2,2'-bipyridin-5-yl)alanine (Bpy-Ala). A phage-display library was constructed in which five residues in the N-terminal finger of zif268 were randomized to include both canonical amino acids and Bpy-Ala. Panning of this library against a nine-base-pair DNA binding site identified several Bpy-Ala-containing functional Zif268 mutants. These mutants bind the Zif268 recognition site with affinities comparable to that of the wild-type protein. Further characterization indicated that the mutant fingers bind low-spin Fe(II) rather than Zn(II) . This work demonstrates that an expanded genetic code can lead to new metal ion binding motifs that can serve as structural, catalytic, or regulatory elements in proteins., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Employing small molecules or chemical reagents to modulate the function of an intracellular protein, particularly in a gain-of-function fashion, remains a challenge. In contrast to inhibitor-based loss-of-function approaches, methods based on a gain of function enable specific signalling pathways to be activated inside a cell. Here we report a chemical rescue strategy that uses a palladium-mediated deprotection reaction to activate a protein within living cells. We identify biocompatible and efficient palladium catalysts that cleave the propargyl carbamate group of a protected lysine analogue to generate a free lysine. The lysine analogue can be genetically and site-specifically incorporated into a protein, which enables control over the reaction site. This deprotection strategy is shown to work with a range of different cell lines and proteins. We further applied this biocompatible protection group/catalyst pair for caging and subsequent release of a crucial lysine residue in a bacterial Type III effector protein within host cells, which reveals details of its virulence mechanism.

Deregulated microRNAs (miRNAs) are known to be involved in the tumorigenesis of nasopharyngeal carcinoma (NPC). However, the role of miRNA expression in tumor recurrence is not yet understood. We found distinctive miRNA expression in repeated recurrent tumors using miRNA microarray, and verified this using quantitative real-time RT-PCR and miRNA in situ hybridization analysis. Computational analysis and immunohistochemistry further revealed that differentially expressed miRNAs may work in concert to modulate a multitude of biological pathways. The results not only indicate differential miRNA expression during tumor relapse, but imply the potential use of miRNAs to classify repeated recurrence of NPC beyond the histological approach., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Two water-soluble triscyclometalated organoiridium complexes, 1 and 2, with polar side chains that form nanoparticles emitting bright-red phosphorescence in water were synthesized. The optimal emitting properties are related to both the triscyclometalated structure and nanoparticle-forming ability in aqueous solution. Nonlinear optical properties are also observed with the nanoparticles. Because of their proper cellular uptake in addition to high emission brightness and effective two-photon absorbing ability, cell imaging can be achieved with nanoparticles of 2 bearing quaternary ammonium side chains at ultra-low effective concentrations using NIR incident light via the multiphoton excitation phosphorescence process.

Fibroblast growth factor 9 (FGF9) is a multifunctional polypeptide belonging to the FGF family and has functions related to bone formation, lens-fiber differentiation, nerve development, gap-junction formation and sex determination. In a previous study, we demonstrated that FGF9 stimulates the production of testosterone in mouse Leydig cells. In the present study, we used both primary mouse Leydig cells and MA-10 mouse Leydig tumor cells to further investigate the molecular mechanism of FGF9-stimulated steroidogenesis. Results showed that FGF9 significantly activated steroidogenesis in both mouse primary and tumor Leydig cells (p<0.05). Furthermore, FGF9 significantly induced the expression of phospho-Akt at 0.5 and 24 hr, phospho-JNK at 0.25, 0.5, and 24 hr, phospho-p38 at 0.5 hr, and phospho-ERK1/2 from 0.25 to 24 hr in primary Leydig cells (p<0.05). Also, FGF9 significantly up-regulated the expression of phospho-Akt at 3 hr, phospho-JNK at 0.25 hr, and phospho-ERK1/2 at 1 and 3 hr in MA-10 cells (p<0.05). Using specific inhibitors of Akt, JNK, p38, and ERK1/2, we further demonstrated that the inhibitors of Akt and ERK1/2 significantly suppressed the stimulatory effect of FGF9 on steroidogenesis in mouse Leydig cells. In conclusion, FGF9 specifically activated the Akt and ERK1/2 in normal mouse Leydig cells and the Akt, JNK and ERK1/2 in MA-10 mouse Leydig tumor cells to stimulate steroidogenesis.