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Cell-free seminal mRNA (cfs-mRNA) and microRNA (cfs-miRNA) have been found in human ejaculate and reported as promising noninvasive biomarkers for disorders of male reproductive organs and forensic identification. However, seminal plasma is particularly challenging for RNA extraction due to its complicated composition and high content of protein, DNA, and polysaccharide. Here, we report a novel, simple, and reliable method for the isolation of cfs-mRNA and cfs-miRNA from human semen based on our previous findings of their physical nature. Seminal microvesicles (0.1-0.5 µm in diameter), which contain the majority of cfs-mRNA, were enriched by a microfilter. Protein complexes, which most cfs-miRNA is bound with, were enriched by an ultrafilter. Harvesting the complexes or microvesicles, in which RNAs exist, avoided the influence of other components in human semen, thus favoring RNA isolation and purification. This new method can efficiently isolate cfs-mRNA and cfs-miRNA separately based on their physical nature, with high RNA purity, and low DNA contamination. It may also be applied or modified to isolate cell-free RNAs in other fluids.

Summary In this study, the ageing males' symptoms (AMS) scale was translated into Chinese following methodological recommendations for linguistic and cultural adaptation. This study aimed to confirm the reliability, validation and applicability of the simplified Chinese version of the scale (CN-AMS) in older Chinese men, a free health screening for men older than 40 years was conducted. All participants completed a health questionnaire, which consisted of personal health information, AMS scale, the generic quality of life (QoL) instrument SF36 and the Beck Depression Inventory (BDI). The fasting blood samples of participants were collected on the day of completing the health questionnaire. Serum total testosterone (TT), albumin and sex hormone-binding globulin levels were measured and the level of free testosterone was calculated (calculated free testosterone, CFT). A total of 244 men (mean age: 52 ± 7.3 years, range: 40–79 years) were involved in the investigation and provided informed consent before their participation. The reliability of CN-AMS was analysed as internal consistency reliability (Cronbach's alpha was 0.91) as well as a 4-week-interval test–retest stability (Pearson's correlation was 0.83) and found to be good. The validation of CN-AMS was analysed as the internal structure analysis (Pearson's correlation between total score and each item score r = 0.48–0.75), total-domain-correlation (among the three domains r = 0.47–0.68, p

Background Cell-free seminal mRNA (cfs-mRNA) exists in human ejaculate at high concentrations and with high stability, and contains many tissue-specific transcripts secreted from the male reproductive system. Owing to the sensitivity of RNA technology, cfs-mRNAs are ideal candidates for non-invasive biomarkers of physiopathological conditions. This study applied cfs-mRNA in identifying the presence of either germ cells or complete obstruction in men with azoospermia. Methods RT-PCR was performed to amplify the germ cell-specific (DDX4), seminal vesicle-specific (SEMG1) and prostate-specific (TGM4) mRNAs from cfs-mRNAs, which were isolated from the seminal plasma of men with non-obstructive azoospermia (NOA) or obstructive azoospermia (OA). The 39 patients with NOA, diagnosed by testicular biopsy, included 8 men with maturation arrest (MA), 3 men with incomplete sertoli cell only (iSCO) syndrome and 28 men with complete SCO (cSCO). The 29 patients with OA, confirmed by the presence of sperm in the testis or epididymis, included 8 men with congenital bilateral absence of the vas deferens (CBAVD) and 21 men with non-CBAVD. Healthy individuals and vasectomized men were enrolled as controls. Results TGM4 was detected in all participants. Consistent with their diagnosis, DDX4 was detected in all patients with MA or iSCO but was absent in most cases of cSCO (n = 21, 75.0%) or non-CBAVD (n = 18, 85.7%), and in all men with vasectomy or CBAVD. The presence of DDX4 in the other seven men with cSCO and three patients with non-CBAVD suggests the presence of germ cells in the testis, and incomplete obstruction, respectively. SEMG1 was undetectable in three patients with CBAVD with bilateral absence of the seminal vesicles, and in two non-CBAVD cases with low ejaculate volume. Conclusions These results suggest that, with high sensitivity and representativity, cfs-mRNA could be non-invasive biomarkers for identifying the presence of germ cells or complete obstruction in azoospermia.

Background The great interest in cell-free mRNA, microRNA (miRNA) as molecular biomarkers for clinical applications, and as ‘signaling’ molecules for intercellular communication highlights the need to reveal their physical nature. Here this issue was explored in human cell-free seminal mRNA (cfs-mRNA) and miRNA (cfs-miRNA). Methodology/Principal Findings Selected male reproductive organ-specific mRNAs, miRNAs, and piRNAs were quantified by quantitative real-time PCR in all experiments. While the stability of cfs-miRNA assessed by time-course analysis (up to 24 h at room temperature) was similar with cfs-mRNA, the reductive changes between cfs-miRNA and cfs-mRNA after filtration and Triton X-100 treatment on seminal plasma were very different, implying their different physical nature. Seminal microvesicles (SMVs) were then recovered and proportions of cfs-mRNA and cfs-miRNA within SMVs were quantified. The amounts of SMVs- sequestered cfs-mRNAs almost were the same as total cfs-mRNA, and were highly variable depending on the different sizes of SMVs. But most of cfs-miRNA was independent of SMVs and existed in the supernatant. The possible form of cfs-miRNA in the supernatant was further explored by filtration and protease K digestion. It passed through the 0.10-µm pore, but was degraded dramatically after intense protease K digestion. Conclusions/Significance The predominant cfs-mRNA is contained in SMVs, while most cfs-miRNA is bound with protein complexes. Our data explained the stability of extracellular RNAs in human semen, and shed light on their origins and potential functions in male reproduction, and strategy of developing them as biomarkers of male reproductive system.

Objective To evaluate the contraceptive ability of two B-cell epitopes in CatSper1. Design In vivo study with mice. Setting University laboratory animal service center. Animal(s) BALB/c mice. Intervention(s) Two predicted B-cell epitopes in the extracellular part of transmembrane domains and pore region of CatSper1 were synthesized to immunize male mice. Main Outcome Measure(s) Fertility, epididymal sperm function, and the presence of specific antibody in immunized males were investigated. Result(s) Significant reduction of fertility was observed in mating trial with no evident systemic illness or abnormal mating behavior. Epididymal sperm of epitope-immunized males exhibited impaired ability to fertilize eggs in vitro, and showed sperm agglutination in some animals, while presenting no changes in sperm viability or progressive motility. High titer of antibodies was induced in the sera, and the antibodies’ specificity was confirmed. The binding of the antibodies to epididymal sperm of epitope-immunized males was observed. Conclusion(s) CatSper members could be the effective and viable targets for immunocontraception. These two epitopes in CatSper1 share high identity between mouse and human and may be effective for fertility regulation in humans.

In particular, an early integration of performance specifications in the SDP has been recognized during the last few years as an effective approach to improve the overall quality of a software. Performance related problems are becoming more and more strategic in the software development, especially recently with the advent of Web Services and related business-oriented composition techniques (software as a service, Web 2.0, orchestration, choreography, etc.). The goal of our work is the definition of a software development process that integrates performance evaluation and prediction. The software performance engineering development process (SPEDP) we specify is focused on performance, which plays a key role driving the software development process, thus implementing a performance/QoS-driven (software) development process. More specifically, in this paper our aim is to formally define the SPEDP design process, posing particular interest on the basis, on the first step of SPEDP, the software/system architecture design, modeling and/or representation. We define both the diagrams to use and show how to model the structure of the software architecture, its behavior and performance requirements. This is the first mandatory step for the automation of the SPEDP into a specific tool.

Raman optical activity (ROA) spectroscopy is a chiral sensitive vibrational spectroscopic technique that measures the difference of molecular responses to either left or right circularly polarized photon radiation. As a structural sensitive probe, ROA has great potential applications in fabricating bio-molecular sensors due to its capability of capturing small changes in molecular structure. However, ROA is a weak effect on the order of 10 -3 or 10 -4 smaller than its parent spontaneous Raman. Extension of ROA to widely se nsing applications can be accelerated by combining it with surface plasmon resonance effect, surface-enhanced Raman sp ectroscopy (SERS), allowing for trace chemical detections. However, it has been shown to be very challenging to co mbine SERS with ROA to obtai n a reliable surface-enhanced Raman optical activity measurement by several independent ROA research groups. This is because of the complexity of interaction of chiral molecules with metal surfaces. In addition, another main factor for the difficulty is the lack of a depolarized ROA spectrometer which can remove effectively huge polarized Raman bands, generally the major sources of artifacts in ROA. In this paper, we will discuss the development of a depolarized Raman spectrometer. This newly developed depolarized Raman instrument is based on the BioTools’ Chiral RAMAN-2X scattered circular polarization (SCP) ROA spectrometer and has been modified into a dual circular po larization ROA spectrometer with the introducing circularity conversion in the incident radiation path. Combing the simultaneous detection of left and right circularly polarized scattering photons, newly developed Raman instrument are capable of modulate both incident and scattered light simultaneously resulting dual circular polarization (DCP) ROA measurements from depolarized parent Raman scattering. The performance of the Raman spectrometer has been initially evaluated with standard sample of small molecules for the proof-of-principle measurements of DCP-ROA, and its potential application in SEROA measurement has also been discussed. Keywords: ROA, DCP, SERS, SEROA, depolarized, sensor

Both laboratory and field evidence suggest that people tend to voluntarily incur costs to punish non-cooperators. While costly punishment typically reduces the average payoff as well as promotes cooperation. Why does the costly punishment evolve? We study the role of punishment in cooperation promotion within a two-level evolution framework of individual strategies and social norms. In a population with certain social norm, players update their strategies according to the payoff differences among different strategies. In a longer horizon, the evolution of social norm may be driven by the average payoffs of of all members of the society. Norms differ in whether they allow or do not allow for the punishment action as part of strategies, and, for the former, they further differ in whether they encourage or do not encourage the punishment action. The strategy dynamics are articulated under different social norms. It is found that costly punishment does contribute to the evolution toward cooperation. Not only does the attraction basin of cooperative evolutionary stable state (CESS) become larger, but also the convergence speed to CESS is faster. These two properties are further enhanced if the punishment action is encouraged by the social norm. This model can be used to explain the widespread existence of costly punishment in human society.

To investigate the origin of tumor blood vessel and blood supply during pulmonary carcinogenesis, and the relationship between vascular endothelial growth factor (VEGF), its receptor Flk-1 and angiogenesis.One hundred Wistar rats were instilled with 3-methylcholanthrene (MCA) and diethylinitrosamine (DEN) to induce pulmonary squamous cell carcinoma through left lower lobe bronchus. To acquire different pathological phase during the carcinogenesis, rats were killed in 15, 35, 55, 65, 75 days after instillation. Yellow and green silastics were respectively injected into the bronchial and pulmonary arteries of 30 rats in 55, 65, 75 days after instillation. Intertumor microvessel density (MVD) was marked by anti-von Willebrand factor monoantibody. VEGF and Flk-1 expression were examined by immunohistochemistry.In the tumor area the tumor blood vessels were yellow and connected with distorted bronchial artery and very few green incomplete branches of pulmonary artery were seen. Silastic particles could be seen in the disordered tumor blood vessels by microscope after bronchial artery perfusion. There was no silastic particles in the carcinoma interstitial blood vessels after pulmonary artery perfusion. MVD count significantly increased in carcinoma in situ (39.50±12.60) and infiltrative carcinoma (61.05±19.92) as compared to atypical hyperplasia (8.92±3.80)(both P0.01), and the increased vessels originated from bronchial artery, but not pulmonary artery. The expression of VEGF and Flk-1 increased during pulmonary carcinogenesis. The positive coefficients of VEGF and FLK-1 expressions became higher and higher from epithelial proliferation to squamous metaplasia, to atypical hyperplasia, to carcinoma in situ and finally to infiltrative carcinoma. There was significant correlation between MVD and VEGF expression (r=0.979 8, P0.005), as well as between MVD and Flk-1 expression (r=0.907 8, P0.05).Angiogenesis is the important phenomenon of the rat pulmonary carcinogenesis and the newly formed blood vessels in tumor connect with the branches of bronchial artery, but not pulmonary artery. This confirms that the blood supply of pulmonary carcinoma is from bronchial artery, not from pulmonary artery. VEGF and Flk-1 are closely related to angiogenesis of tumor.

To investigate the expression of cyclooxygenase-2 (COX-2) protein and inducible nitric oxide synthase (iNOS) protein during the experimental lung carcinogenesis in rats, as well as their association with microvessel density (MVD).Diethylinitrosamine and 3-methylcholanthrene were instilled into the left lobar bronchus to induce lung squamous cell carcinoma in 88 Wistar rats, and 10 nomal rats as controls. COX-2, iNOS expression and MVD count of the specimens obtained from the rats were examined by immunohistochemistry.A total of 155 specimens of various pathological phase during the carcinogenesis were obtained: 14 hyperplasia, 25 squamous metaplasia, 33 dysplasia, 12 carcinoma in situ, 54 infiltration carcinoma, and 17 metastasis. The immunohistochemical score (IHS) of COX-2 significantly increased in dysplasia, carcinoma in situ and metastasis (P0.01,P0.05,P0.01). IHS of iNOS significantly increased in hyperplasia and metastasis (P0.05,P0.01 ). Remarkably increased MVD was found in carcinoma in situ, infiltration carcinoma and metastasis (P0.01, P0.01, P0.01). There was a positive correlation between COX-2 and iNOS (r=0.601 6,P0.001) expression. Expression of COX-2 or iNOS were remarkably related to MVD count (P0.01,P0.01)COX-2 and iNOS may play important roles in the carcinogenesis of experimental rat lung squamous cell carcinoma as well as its progress, and it may be associated with stimulating angiogenesis.

The introduction of four transcription factors Oct4, Klf4, Sox2 and c-Myc by viral transduction can induce reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), but the use of iPSCs is hindered by the use of viral delivery systems. Chemical-induced reprogramming offers a novel approach to generating iPSCs without any viral vector-based genetic modification. Previous reports showed that several small molecules could replace some of the reprogramming factors although at least two transcription factors, Oct4 and Klf4, are still required to generate iPSCs from mouse embryonic fibroblasts. Here, we identify a specific chemical combination, which is sufficient to permit reprogramming from mouse embryonic and adult fibroblasts in the presence of a single transcription factor, Oct4, within 20 days, replacing Sox2, Klf4 and c-Myc. The iPSCs generated using this treatment resembled mouse embryonic stem cells in terms of global gene expression profile, epigenetic status and pluripotency both in vitro and in vivo. We also found that 8 days of Oct4 induction was sufficient to enable Oct4-induced reprogramming in the presence of the small molecules, which suggests that reprogramming was initiated within the first 8 days and was independent of continuous exogenous Oct4 expression. These discoveries will aid in the future generation of iPSCs without genetic modification, as well as elucidating the molecular mechanisms that underlie the reprogramming process.

White-collar workers are high-productivity people arisen in recent years, and they directly affect the economic development situation and development mode in the cities or the areas where they located in, and it is also considered of the change of such population migration. The article will research on the mode and the white-collar population flow in the domestic 30 big/medium cities through the attraction model for population flow established from wage, housing price etc. The result proves that the attraction factor we selected is a chief reason for white-collar to decide to migrate. The last part of the article forecasts the amount of white-collar in each city in the near future.

Electronic commerce (EC) can be defined as to use information technology for improving business relations between trading partners. The objectives of this work are to develop the reference model for IDEF0 based B2B electronic commerce and to implement a prototype system for its fundamental issues such as business process, information standards, and information system integration. The research scope is to develop standards for related business processes and their information exchange, to develop XML EDI as the standard exchange method, and to implement a prototype system.

Dynamic intracellular analysis has important applications in areas like biomedical research, defense and security and many others. Although, there are several methods for intracellular analysis, surface enhanced Raman scattering (SERS) is becoming a preferred transduction method for such applications, due to its narrow spectral bandwidth, large SERS enhancement factors and high sensitivity. In our laboratory, SERS-based immuno-nanosensors are being developed and optimized for real-time, dynamic, and multiplexed analysis of molecular interaction within individual living cells. These nanosensors are fabricated by drop coating silica nanospheres onto a microscope slide. A film of SERS active metal is deposited on the nanospheres to form metal film over nanospheres (MFON), which are then removed from the slide by mechanical processes. The MFONs are functionalized with antibodies that target specific proteins under investigation. Radiation induced cell perturbation is minimized by the use of a HeNe laser for excitation at 632.8 nm. To improve SERS enhancement, different types of metal deposited substrates have been studied with multilayer- MFON (MULTI-FON) substrates demonstrating ideal enhancement. This paper evaluates the SERS enhancement of MULTI-FONs with self-assembled monolayers (SAMs) spacers sandwiched between layers of the metal film. Monolayers with carboxylic acid tail groups and different chain lengths are used as spacers in order to evaluate the effect of spacer length and chain functionalities on the SERS enhancement. The paper also discusses the effect of solvent used for the monolayer formation on the sensitivity of the SAM MULTI-FON SERS substrates.

We present a multi-agent stock market model with different dynamic regimes including fundamental equilibrium, non-fundamental equilibrium, periodicity and chaos. The influence of traders' behavioral propensities such as mimetic propensity, price-chasing propensity and strategy-switching propensity to market dynamic regime is investigated by both analytical and numerical methods. The mimetic and price-chasing propensities play a more important role in determining market regimes, while the strategy-switching propensity determines the price deviation distance from fundamental value if the deviation exists.

Approaches of both theoretical analysis and computer simulation are used to study a stochastic multi-agent stock market model. Theoretical analysis provides the parameter settings for different dynamic regimes including fundamental equilibrium, non-fundamental equilibrium, periodicity and chaos. Agent-based computer simulations with those settings are performed to produce the price series. Statistical analysis of these data shows: markets of all regimes present stylized facts such as fat-tail, volatility clustering and long-term memory; the fundamental equilibrium regime has the most significant stylized facts, followed by periodicity and chaos, and non-fundamental equilibrium the least.

During the construction of cities in mountain area, plenty of human waste-dump is produced because of the topographic conditions. In order to solve the problem of site leveling and rock spoils placing, the dumping site construction become an unavoidably problem which concerns two main problems-the stability of rock filling and the drain of ravine. This paper selects the dumping site of the Huang Jia ravine (to be built) as a case, which is a rock filling in Badong county that is an emigratory city of the reservoir region. The source of materials in the spoil site comes from the waste of landslide prevention program. Considering the engineering geological condition, hydrological geological and the characteristics of the rock filling slope, the possible damage mode of the rock filling after filling is speculated. On that basis, the un-equilibrium pushing method is used to calculate the stability of rock filling slope . Consulting the principles of rockfill dam in hydropower engineering ,the comprehensive protection measures are adopted to ensure the stability of the rock filling which includes canal revetment rolling and supporting structure . So, the protection design for the rock filling is a new method and approach of urban planning and environmental protection for the emigratory towns in reservoir region. It also has an important social and economic benefit.

Expectations’ self-fulfillment and self-destroying co-exist in socio-economic systems, which makes the evolution of the systems seem to be mysterious to us. We regard these collective phenomenon arise from people’s ways to expect and act, namely, the expectation mode and action strategy, and propose a simple agent-based model in consideration of four direct combinations of general expectation modes and action strategies to detect the underlying mechanism. We find that the action strategy, or originally, the win-loss rule of the system, is indeed a crucial factor to determine whether the evolution of the systems would satisfy people’s expectations.

This paper put forward the realization of the self-automation role, which has leaning ability and dynamical acclimatization. First of all, BP algorithm of artificial neural net(ANN) is improved, the self-adjusted algorithm of all parameters has been proposed for the back-propagation learning, which can make the selection of hidden layer units and rate of studying easily in the course of training, reduce artificial influence and improve the adaptive ability of rate of studying and neural net. Secondly, Genetic Algorithms (GA) has been optimized from primitive colony, selective manipulation, intercross manipulation. At the same time, methodology of ANN was integrated with GA and self-learning models of NPC were created to control their behaviors. At last, the experimental results have shown that self-learning system of NPC provides artificial behaviors with more automation and intelligence.

Summary Human fibroblasts can be induced into pluripotent stem cells (iPSCs), but the reprogramming efficiency is quite low. Here, we screened a panel of candidate factors in the presence of OCT4 , SOX2 , KLF4 , and c-MYC in an effort to improve the reprogramming efficiency from human adult fibroblasts. We found that p53 siRNA and UTF1 enhanced the efficiency of iPSC generation up to 100-fold, even when the oncogene c-MYC was removed from the combinations.

Objective To explore the contraceptive potential of the CatSper1 transmembrane domains and pore region in vitro. Design In vitro study with human sperm and mouse fertilization. Setting Andrology laboratory of an academic research center. Patient(s) and Animal(s) Normozoospermia and viripotent BALB/c mice. Intervention(s) The specific binding of an anti-CatSper1 IgG antibody (H-300) to CatSper1 was confirmed by Western blot and immunofluorescence. Sperm from humans and mice were incubated with H-300. Main Outcome Measure(s) The effects of H-300 on human sperm progressive motility, abnormal acrosome, hyperactivated motility, and mouse in vitro fertilization rates were analyzed. Result(s) A significant decline in sperm progressive motility was observed after 1, 2, and 4 hours of incubation with H-300; the change was mainly ascribed to the decline of fast progressive motility. Significant inhibition of the hyperactivated motility was observed after 5 hours of incubation with H-300. The incubation of mouse sperm with H-300 before insemination reduced the in vitro fertilization rate to 28% of control levels (72% inhibition). Conclusion(s) CatSper1 may be a potential target for immunocontraception, and the antibody may be a tool to study the function of ion channels in sperm in which relatively fewer methods can be applied.

In this paper, we focus on the statistical features of runs which is defined as a sequence of consecutive gain/loss (rise/fall) stock returns. By studying daily data of the Dow Jones industrial average (DJIA), we get the following points: firstly, the distribution of the length and magnitude of stock return runs both follow an exponential law; and secondly, the positive runs and negative runs show a significant asymmetry in frequency distribution. We expect that the two properties may be new members in the family of stylized facts about stock returns.

Aim: To investigate the gene expression changes of urokinase plasminogen activator (uPA)/urokinase receptor (uPAR) in rat testes at postnatal stages and explore the effects of uPA/uPAR system on the rat spermatogenesis. Methods: The mRNAs of uPA and uPAR in rat testes were measured by using real-time quantitative polymerase chain reaction (PCR) at postnatal days 0, 5, 10, 15, 21, 28, 35, 42, 49 and 56, respectively. Results: The tendencies of uPA and uPAR mRNA expression were similar at most postnatal stages except for D0. The expression of uPAR mRNA in rats testes was relatively higher than that of uPA at postnatal D0, and both were decreased until D21, increased obviously at postnatal D28, reached a peak at postnatal D35, then declined sharply at postnatal D42 and retained at a low level afterwards. Conclusion: The uPA/uPAR system may be strongly linked to spermiation and spermatogenesis via regulating germ cell migration and proliferation, as well as promoting the spermiation and detached residual bodies from the mature spermatids. (Asian J Androl 2007 Sep; 9: 679–683)

A detailed study of SERS enhancements from novel multilayer gold film over nanoparticle (GFON) substrates is presented. These multilayer GFON substrates were optimized in terms of the number of metal layers, and the amounts of gold and silver oxide deposited. These multilayer GFON substrates were also structurally characterized in terms of surface roughness. No significant changes in the surface roughness of these multilayer GFON substrates, even with different layers of gold, have been observed, suggesting there is no direct correlation between the multilayer SERS enhancements and the surface roughness. UV-Vis reflectance spectra of these substrates were also characterized, indicating that the significant multilayer enhancements require the presence of silver oxide layers separating the continuous gold film layers.

A multilayer surface-enhanced Raman scattering (SERS) substrate geometry providing significantly greater SERS enhancements, longer active lifetimes, better reproducibility, and lower detection limits for trace chemical analysis than traditional SERS substrates has been developed. We have fabricated and characterized this novel class of multilayered metal film-based SERS substrates, which are capable of enhancing SERS signals over an order of magnitude relative to conventional metal film over nanostructure substrates. These multilayer enhanced metal film substrates are fabricated by repeated vapor deposition of metal films over nanometer sized structures. Different sizes of nanostructures were evaluated in order to obtain the optimal SERS enhancements. Meanwhile, different dielectric coatings were fabricated between silver layers, and SERS enhancements were evaluated for each type. Additionally, different metals, such as gold, were used to further optimize the stability and reproducibility of these novel substrates. Silver oxide layers produced at elevated temperatures were also investigated to accelerate the fabrication rate of these multilayer substrates. Finally, this paper also discusses the application of these novel multilayer substrates for trace detection of chemical agents and simulants.

Aim: To investigate the distribution of cation channel of sperm 1 (CATSPER1) protein and the presence of CATSPER1 mRNA in human testis and ejaculated spermatozoa. The influence of anti-human CATSPER1 antibody upon human sperm motility was used to evaluate the function of human CATSPER1 and to estimate its possible use as a target for immunocontraception. Methods: Human ejaculated sperm from normozoospermic donors (n = 12) and liquid nitrogen frozen human testis were used for the study of mRNA and protein expression of CATSPER1 by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. Spermatozoa from normozoospermic donors (n = 12) were individually processed using a swim-up procedure and were then incubated with CATSPER1 antibody at final concentrations of 20, 4 and 0.8 µg/mL. After 1, 2 and 6 h incubation, progressive motility and fast progressive motility were measured by means of computer-assisted semen analysis. Results: CATSPER1 transcript was detected in both human testis and each human ejaculated semen sample. CATSPER1 protein expressed in the membrane of spermatid and was localized in the principal piece of the sperm tail. The application of CATSPER1 antibody at all concentrations significantly inhibited both progressive motility and fast progressive motility after 1, 2 and 6 h incubation, and significant dose-dependent changes were observed. Conclusion: CATSPER1 is meiotically and post-meiotically expressed in human testis tissue. CATSPER1 mRNA in human ejaculated spermatozoa could be a more feasible target for study and infertility screening than testis biopsy. In addition, our results suggest that human CATSPER1 could be a possible target for immunocontraception. (Asian J Androl 2006 May; 8: 301–306)

Monitoring the presence, production and transport of proteins inside individual living cells can provide vital information about cellular signaling pathways and the overall biological response of an organism. For example, cellular response to external stimuli, such as biological warfare (BW) agents, can be monitored by measuring interleukin-II (IL-2) expression inside T-cells as well as other chemical species associated with T-cell activation. By monitoring such species, pre-symptomatic detection of exposure to BW agents can be achieved, leading to significantly increased post-exposure survival rates. To accomplish such monitoring, we have developed and optimized implantable nanosphere-based nanosensors for the intracellular analysis of specific proteins in a label-free fashion. These sensors consist of 300-520 nm diameter silica spheres that have been coated with silver and antibodies to allow for trace protein detection via surface enhanced Raman spectroscopy (SERS). They have been optimized for SERS response by evaluating the size of the nanospheres best suited to 632.8 nm laser excitation, as well as the various nanosensor fabrication steps (i.e., silver deposition process, antibody binding, etc.). During usage, the presence of the specific protein of interest is monitored by either directly measuring SERS signals associated with the protein and/or changes in the SERS spectrum of the antibodies resulting from conformational changes after antigen binding. In this work, human insulin was used as a model compound for initial studies into the sensitivity of these optimized nanosensors.

Novel dual layer and multilayer silver film over nanostructure (SFON) substrates have been developed that provide surface-enhanced Raman scattering (SERS) signal enhancements of greater than 1000% compared to conventional single layer SFON substrates. These substrates provide signal enhancement factors of 3.8 × 105 and greater for a variety of SERS active analytes. Substrate preparation is accomplished by vapor depositing a thick (∼100 nm) layer of silver on top of an underlying layer of alumina nanoparticles, followed by deposition of additional layers of silver with silver oxide layers between them. Unlike previous dual layer silver island based substrates that have been developed, these substrates do not rely on achieving an optimal morphology via deposition of silver. Instead, these substrates rely on the roughness being provided by the original under-layer, providing enhanced substrate homogeneity and more reproducible signals than either silver island substrates or colloidal substrates. In addition, the signal enhancement gives these substrates extended lifetimes compared to conventional single layer SFON substrates. Finally, this study also shows that geometric surface structure and surface roughness factors play little or no role in this enhancement process, allowing for this multilayer fabrication process to be applied to many different types of substrates achieving similar or even greater results.

The early detection of biological warfare (BW) agents before any symptoms are present is critical for saving lives and reducing cost of therapy. Protein expression in T-cells represents one of the earliest detectable cellular signaling events to occur in response to the exposure to various toxins or BW agents. In order to fully understand a cellular response to a particular BW agent, it is often necessary to monitor the expression of specific proteins. Therefore, we have developed a novel class of surface enhanced Raman scattering (SERS) immuno-nanosensors for the real-time monitoring of protein expression within individual living cells. In this work, we have developed and optimized novel nanosphere-based silver coated SERS nanosensors for the detection of proteins at cellular levels. SERS nanosensors were optimized in terms of nanosphere size, silver coating methods, number of silver layers, antibody binding and affinity. These nanosensors are capable of being inserted into individual cells and non-invasively positioned to the sub-cellular location of interest using optical tweezers. They were constructed from monodisperse silica nanospheres. These nanospheres were condensed from tetraalkoxysilanes in an alcoholic solution of water and ammonia. Accurate control of the silica nanospheres’ diameter was achieved by varying the reaction conditions. Nanosphere-based SERS immuno-nanosensors were then prepared by depositing multiple layers of silver on silica spheres, followed by binding of the antibody of interest to the silver. In binding the antibodies, different cross linker agents were characterized and compared. On one end, each of these cross linker agents contained sulfur or isothiocyanate groups which bound to the silver surface, while the other end contained a carboxylic or primary amine group which reacted readily with the antibodies. In order to improve sensitivity of these nanosensors, optimal silver surface coverage with crosslinkers was determined. Following binding of antibodies, evaluation of the nanosensors was performed by monitoring the SERS spectra of the nanosensors prior to and following exposure to the antigen of interest. These results showed reproducible differences in the SERS spectra upon exposure to the antigens confirming their ability to monitor trace amounts of antigen. In particular, these SERS-based nanosensors were shown successfully detect human insulin at trace levels.

We have developed and characterized novel multilayered metal film-based surface-enhanced Raman scattering (SERS) substrates capable of enhancing SERS signals over an order of magnitude compared to conventional single layer substrates. In addition to enhanced signal intensity, these multilayered metal film substrates also exhibit longer SERS active lifetimes, higher reproducibility and lower detection limits than single layer silver substrates. Multilayered metal film substrates were fabricated by repeated vapor deposition of metal films over nanometer sized silica spheres. Different sizes of silica spheres were evaluated in order to obtain the optimal SERS enhancements. Meanwhile, different coating methods, drop coating and spin coating, were applied to form silica sphere layers that provided the roughness for SERS enhancements. These two coating methods were also compared for various silica sphere sizes by investigating their effects on the SERS enhancements. By applying additional silver layers on top of silver film over silica sphere SERS substrates, multi-layer enhancements can be observed. Additionally, different metals, such as gold, were used to further optimize the stability and reproducibility of these novel substrates. In order to speed up the fabrication of these multiple metal layer SERS substrates, silver oxide layers produced in an oven were investigated, reducing fabrication time by a factor of 50.

Surface enhanced Raman scattering (SERS) is an extremely sensitive and selective spectroscopic technique commonly employed for trace environmental analyses. We are currently developing and optimizing novel SERS based immuno-nanosensors for the monitoring of protein expression within individual living cells. These sensors, based upon antibody bound, silver coated silica nanospheres, can be inserted into individual cells and non-invasively positioned to the subcellular location of interest using optical tweezers. Because a HeNe laser is used for excitation, both photodamage to cells and cellular autofluorescence are minimized. Fabrication of the nanosensors is performed by first depositing a thin layer of silver on silica nanospheres by either homogeneous chemical deposition or vapor deposition. This is followed by binding an antibody against the analyte of interest to the sensor’s surface. In this paper we have investigated and optimized the reduction conditions of a chemical deposition substrate fabrication method and have compared these substrates to three types of vapor deposited substrates: (1) single layer silver film over nanosphere (SFON), (2) dual layer silver film over nanosphere (DUAL-FON) substrates (produced by coating an additional layer of silver film over the SFON), and (3) multilayer silver film over nanosphere (MULTI-FON) substrates (prepared by repeated coatings of several silver films over a SFON). In the case of chemical deposition, parameters optimized included silver nitrate concentration, reaction temperature, and silver coating time of substrates. In general, we have found SFON substrates yield better signal-to-noise ratios (S/N) with less background than the optimized, chemically prepared substrates. Even greater SERS enhancements have been found using multilayered SERS substrates (i.e., DUAL-FON substrates and MULTI-FON substrates). Both the stability and S/N of these substrates are enhanced by using multiple silver layers compared to SFON substrates of similar thickness. From this work, SERS signal enhancements of over an order of magnitude can be predicted by combining the enhancements obtained from the multilayered geometry and that from optimized silver deposition parameters. This paper includes investigations into the mechanism of multi-layer enhancement by studying the SERS S/N changes after different amount of exposure time of underlayer silver films to air. From these studies, we suggest that silver oxide layers play an important role in this multilayered enhancement. To the best of our knowledge, this paper is the first report of this type of silver oxide layer production affecting and even enhancing the resulting SERS signals from continuous film SERS substrates. In this paper, the fabrication, characterization and comparison of these SERS substrates in terms of enhancement factor, and lifetime will be discussed.

The second order sampling was proposed by Kohnlenberg (1953), and Wang rebuilt it on a translation invariance view point. In this paper, we study the stability of this sampling scheme for band-pass signals, discuss some properties of sampling functions, and give the optimal phase shift to reduce the condition number.

To investigate the roles of p53 and K-ras gene in carcinogenesis and development of the lung carcinoma induced by 3-methylcholanthrene (MCA) and diethylinitrosamine (DEN) in Wistar rats, and to elucidate the relationships between the protein expression and gene mutation of p53 and K-ras.Microdissection was used to obtain pure cell populations of each phase in the carcinogenesis and development of lung carcinoma induced by MCA and DEN. DNA of the microdissected cell populations was extracted and used to analyze the mutations of p53 exons 5 approximately 8 and K-ras exons 1 approximately 2 by PCR-SSCP. The expressions of p53 and K-ras protein in each phase were detected by immunohistochemistry.No mutation and protein expression of p53 and K-ras was found in the 30 cases with normal bronchial epithelium. Mutation of p53 was detected in 3.1% of 18 hyperplasia and 14 squamous metaplasia cases, 28.6% of 21 dysplasia, 30.0% of 12 carcinomas in situ, 51.2% of 43 infiltration carcinomas, 52.9% of 17 metastases. The positive immunostaining rate of p53 protein was 0, 42.9%, 50.0%, 60.5% and 64.7% respectively. K-ras mutation rate was 0, 4.8%, 8.3%, 9.3%, 11.8% respectively, while the overexpression rate of K-ras protein was 15.6%, 19.0%, 25.0%, 41.9%, 52.9% respectively. p53 protein expression was closely related with p53 mutation (P0.005, Pearson's R = 0.599 6). There was no relationship between the protein expression and gene mutation of K-ras (P0.500).p53 gene mutation and K-ras overexpression were early events in the carcinogenesis and development of rat lung carcinoma induced by MCA and DEN, while K-ras mutation does not play any important role.

To investigate the expression of two inflammation related enzymes - cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) during the experimental rat lung carcinogenesis.Eighty Wistar rats were instilled with 3-methylcholanthrene (MCA) and diethylinitrosamine (DEN) into the left lobar branchus to induce lung squamous cell carcinoma. To obtain specimen in every pathological phase during the carcinogenesis, these rats were sacrificed at different intervals. The expression of COX-2 and iNOS in every pathological phase during the carcinogenesis were examined by immunohistochemical method. The immunohistochemical scores (IHS) were calculated by combining an estimate of the percentage of immunoreactive cells with that of the stain intensity.155 specimens of every pathological phase during the carcinogenesis showed: hyperplasia 14, squamous metaplasia 25, dysplasia 33, carcinoma in situ 12, infiltrating carcinoma 54 and metastasis 17. Inflammation and elevated expressions of COX-2 and iNOS were shown in the precancerous lesions. The COX-2 IHS was significantly increased in dysplasia, carcinoma in situ and metastasis (P0.01, P0.05, P0.01 respectively). The iNOS IHS significantly increased in hyperplasia and metastasis (P0.05, P0.01 respectively). There was a positive correlation between the expression of COX-2 and iNOS (gamma = 0.601 6, P0.001).COX-2 and iNOS, two inflammation related enzymes, playing important roles in the carcinogenesis of MCA and DEN, induce rat lung squamous cell carcinoma as well as its metastasis. The relation between inflammation and carcinogenesis may partly be explained by the elevated expression of these two enzymes. Nonsteroidal antiinflammatory drug (COX-2 inhibitors) and iNOS inhibitors may possess antitumor activities because of their prevention of bronchial dysplasia, carcinogenesis and metastasis.

To investigate the dynamic expression and its relation of gelatinase A (MMP-2), its natural inhibitor (TIMP-2) and DNA index (DI) changes during carcinogenesis, invasion and metastasis in Wistar rats.Squamous cell carcinoma of lung was induced with 3-methylcholanthrene (MCA) and diethyinitrosamine (DEN) in iodized oil by left intra-bronchial instillation in 80 Wistar rats. Immrno histochemistay (IHC) and in situ hybridigation were used in the monitor of MMP-2, TIMP-2 proteins and mRNA expression during invasion and metastasis of lung cancer in these rats, DNA index (DI) value was measured by guantitatove image analysis on feulgen stained sections.Along with the carcinogenis, the average poritive MNP-2 and TIMP-2 expressions increased, with positive rates of 8.5% - 85.7% and 6.4% - 35.7%. DI value also underwent the same changes (1.47 +/- 0.54) - (2.87 +/- 0.55). The difference of MMP-2 expression in carcinoma in situ versus early carcinoma and early carcinoma versus metastatic carcinoma are statistically significant (P0.05). Companing lung carcinome, the contrel group and non-cancerous lesions, the elevation of MNP-2 and TIMP-2 expressions were also sigmificant (P0.01). The DI elevation in carcinoma in situ and dysplasia were obviously significant (P0.05). Meanwhile a negative relation was noted in TINP-2 and MMP-2 expressions during carcinogenesis. There was a positive relation between MMP-2 expression and DNA poikiloidy (P0.01), which was related to the close relationship between MMP-2 and metastasis in advanced rat lung carcinoma (P0.05).The excess degradation and disruption of basement membranes by activated MMP-2 may be a key step in inducing lung cancer invasion and metastasis. The imbalance between MMP and TIMP may be a critical factor which affects biologic behavior of lung carcinogenesis, invasion and metastasis.

Argonaute proteins and Piwi proteins bind with microRNA (mRNA) and Piwi-interacting RNA (piRNA), respectively, to form functional complexes. Piwi proteins are mostly restricted to germ cells and stem cells, and the Piwi-piRNA pathway is required for normal spermatogenesis. Although piRNAs were also recently identified in mammalian oocytes, expression of Piwi proteins in the ovary has not been well characterized. Previous studies did not detect mRNA of Miwi, a murine homologue of Piwi proteins, in total RNA of mouse ovary tissue. We demonstrated herein the presence of Miwi in murine oocytes. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunofluorescence based on quantum dots immune labeling technique were used to investigate the expression profile of Miwi in oocytes of adult and neonatal females at 0, 1, 2, 3, and 4 weeks postpartum. Although RT-PCR was negative in total RNA of the adult ovary, both RT-PCR and Western blot detected Miwi in oocytes of adult mice, and ovaries of neonatal females. Miwi transcript and protein peaked at 1 and 2 weeks postpartum, respectively. Miwi mRNA was detectable in newborn mouse ovaries, implying its transcription was initiated at least in the primordial follicle. Its protein was strong in late primary and secondary follicles, but appeared to decrease as maturation proceeded. The exclusion of anti-Miwi immunofluorescence from some cytoplasmic granules was observed. Given that diverse biologic and molecular functions have been revealed for the Piwi-piRNA pathway in germline cells of many species, Miwi might be an important functional protein in murine folliculogenesis.