Sperm cells manipulation employing dielectrophoresis

Infertility studies are an important growing field, where new methods for the manipulation, enrichment and selection of sperm cells are required. Microfluidic techniques offer attractive advantages such as requirement of low sample volume and short processing times in the range of second or minutes. Presented here is the application of insulator-based dielectrophoresis (iDEP) for the enrichment and separation of mature and spermatogenic cells by employing a microchannel with cylindrical insulating structures with DC electric potentials in the range of 200–1500 V. The results demonstrated that iDEP has the potential to concentrate sperm cells and distinguish between mature and spermatogenic cells by exploiting the differences in shape which lead to differences in electric polarization. Viability assessments revealed that a significant percentage of the cells are viable after the dielectrophoretic treatment, opening the possibility for iDEP to be developed as a tool in infertility studies.     

Kinetic and thermodynamic characterization of single-mismatch discrimination using single-molecule imaging

A single-molecule detection setup based on total internal reflection fluorescence (TIRF) microscopy has been used to investigate association and dissociation kinetics of unlabeled 30mer DNA strands. Single-molecule sensitivity was accomplished by letting unlabeled DNA target strands mediate the binding of DNA-modified and fluorescently labeled liposomes to a DNA-modified surface. The liposomes, acting as signal enhancer elements, enabled the number of binding events as well as the residence time for high affinity binders (K_d < 1 nM, k_off < 0.01 s⁻¹) to be collected under equilibrium conditions at low pM concentrations. The mismatch discrimination obtained from the residence time data was shown to be concentration and temperature independent in intervals of 1–100 pM and 23–46°C, respectively. This suggests the method as a robust means for detection of point mutations at low target concentrations in, for example, single nucleotide polymorphism (SNP) analysis.     

Mycobacterium tuberculosis Glucosyl-3-Phosphoglycerate Synthase: Structure of a Key Enzyme in Methylglucose Lipopolysaccharide Biosynthesis

Tuberculosis constitutes today a serious threat to human health worldwide, aggravated by the increasing number of identified multi-resistant strains of Mycobacterium tuberculosis, its causative agent, as well as by the lack of development of novel mycobactericidal compounds for the last few decades. The increased resilience of this pathogen is due, to a great extent, to its complex, polysaccharide-rich, and unusually impermeable cell wall. The synthesis of this essential structure is still poorly understood despite the fact that enzymes involved in glycosidic bond synthesis represent more than 1% of all M. tuberculosis ORFs identified to date. One of them is GpgS, a retaining glycosyltransferase (GT) with low sequence homology to any other GTs of known structure, which has been identified in two species of mycobacteria and shown to be essential for the survival of M. tuberculosis. To further understand the biochemical properties of M. tuberculosis GpgS, we determined the three-dimensional structure of the apo enzyme, as well as of its ternary complex with UDP and 3-phosphoglycerate, by X-ray crystallography, to a resolution of 2.5 and 2.7 Å, respectively. GpgS, the first enzyme from the newly established GT-81 family to be structurally characterized, displays a dimeric architecture with an overall fold similar to that of other GT-A-type glycosyltransferases. These three-dimensional structures provide a molecular explanation for the enzyme''s preference for UDP-containing donor substrates, as well as for its glucose versus mannose discrimination, and uncover the structural determinants for acceptor substrate selectivity. Glycosyltransferases constitute a growing family of enzymes for which structural and mechanistic data urges. The three-dimensional structures of M. tuberculosis GpgS now determined provide such data for a novel enzyme family, clearly establishing the molecular determinants for substrate recognition and catalysis, while providing an experimental scaffold for the structure-based rational design of specific inhibitors, which lay the foundation for the development of novel anti-tuberculosis therapies.     

Quadruplex priming amplification combined with nicking enzyme for diagnostics

Quadruplex priming amplification (QPA) is a straightforward assay that allows isothermal amplification of DNA and possesses an intrinsic real-time detection mechanism. QPA can be employed as a diagnostic tool for both linear and exponential signal amplification. The linear QPA, which is less prone to background activity characteristics of exponential systems, suffers from low sensitivity. To increase the sensitivity, here we introduce specific probe molecules that are designed for combined activities of Bst 2.0 polymerase and Nt.BstNBI nicking enzyme. The current assay, which is suitable for single-tube isothermal signal amplification, has increased sensitivity of plain linear QPA by three orders of magnitude to levels of low femtomolar concentration of target molecules.     

Threshold-dependence as a desirable attribute for discrimination assessment: implications for the evaluation of species distribution models

Species distribution modelling has become a common approach in ecology in the last decades. As in any modelling exercise, evaluation of the predicted suitability surfaces is a key process, and the area under the receiver operating characteristic (ROC) curve (AUC) has become the most popular statistic for this purpose. A close covariation between the AUC and threshold-dependent discrimination measures (sensitivity Se and specificity Sp) raises into question the advantage of the threshold-independence of the AUC. In this study, the relationship between the AUC and several threshold-dependent discrimination measures is characterized in detail, and the sensitivity of the pattern to variations in the shape of the ROC curve is assessed. Hypothetical suitability values, coming from normal and skew-normal distributions, were simulated for both instances of presence and absence. The flexibility of the skew-normal distribution allowed for the simulation of a wide range of ROC curve configurations. The relationship between the AUC and threshold-dependent measures was graphically assessed; independently of the ROC curve shape, a nonlinear asymptotic relationship between the AUC and Se (and Sp) was obtained after applying the threshold that makes Se = Sp. A nonlinear asymptotic relationship between the AUC and the Youden index was also reported. These results imply that the AUC does not appropriately measure changes in the discrimination of models, and it is especially incapable of distinguishing between models with high discrimination capacity. Se or Sp derived from the application of the threshold that makes them equal is a preferred measure of discrimination power. Together with the rate of false positives and negatives, and with the prevalence of the species, these statistics provide more information about the discrimination capacity of the models than the AUC.     

Preimplantation Mouse Embryo Selection Guided by Light-Induced Dielectrophoresis

Selection of optimal quality embryos for in vitro fertilization (IVF) transfer is critical to successful live birth outcomes. Currently, embryos are chosen based on subjective assessment of morphologic developmental maturity. A non-invasive means to quantitatively measure an embryo''s developmental maturity would reduce the variability introduced by the current standard. We present a method that exploits the scaling electrical properties of pre-transfer embryos to quantitatively discern embryo developmental maturity using light-induced dielectrophoresis (DEP). We show that an embryo''s DEP response is highly correlated with its developmental stage. Uniquely, this technique allows one to select, in sequence and under blinded conditions, the most developmentally mature embryos among a mixed cohort of morphologically indistinguishable embryos cultured in optimized and sub-optimal culture media. Following assay, embryos continue to develop normally in vitro. Light-induced dielectrophoresis provides a non-invasive, quantitative, and reproducible means to select embryos for applications including IVF transfer and embryonic stem cell harvest.     

Aspergillus Collagen-Like Genes (acl): Identification,Sequence Polymorphism,and Assessment for PCR-Based Pathogen Detection

The genus Aspergillus is a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection of Aspergillus colonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of the Aspergillus collagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of the Aspergillus genus, including Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. Genetic polymorphism and phylogenetic analysis of the aclF1 gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination between Aspergillus species. Overall, this study demonstrated that Aspergillus acl genes could be used as PCR targets to discriminate between clinically relevant Aspergillus species. Future studies aim to utilize the detection of Aspergillus acl genes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification of Aspergillus colonization and invasive aspergillosis in immunocompromised subjects.     

Polyaniline-carbon nanotube composite film for cholesterol biosensor

Nanocomposite film composed of polyaniline (PANI) and multiwalled carbon nanotubes (MWCNT), prepared electrophoretically onto indium tin oxide (ITO)-coated glass plate, was used for covalent immobilization of cholesterol oxidase (ChOx) via N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry. Results of linear sweep voltammetric measurements reveal that ChOx/PANI-MWCNT/ITO bioelectrode can detect cholesterol in the range of 1.29 to 12.93 mM with high sensitivity of 6800 nA mM⁻¹ and a fast response time of 10 s. Photometric studies for ChOx/PANI-MWCNT/ITO bioelectrode indicate that it is thermally stable up to 45 °C and has a shelf life of approximately 12 weeks when stored at 4 °C. The results of these studies have implications for the application of this interesting matrix (PANI-MWCNT) toward the development of other biosensors.     

Cytoplasm Resistivity of Mammalian Atrial Myocardium Determined by Dielectrophoresis and Impedance Methods

Many cardiac arrhythmias are caused by slowed conduction of action potentials, which in turn can be due to an abnormal increase of intracellular myocardial resistance. Intracellular resistivity is a linear sum of that offered by gap junctions between contiguous cells and the cytoplasm of the myocytes themselves. However, the relative contribution of the two components is unclear, especially in atrial myocardium, as there are no precise measurements of cytoplasmic resistivity, R_c. In this study, R_c was measured in atrial tissue using several methods: a dielectrophoresis technique with isolated cells and impedance measurements with both isolated cells and multicellular preparations. All methods yielded similar values for R_c, with a mean of 138 ± 5 Ω·cm at 23°C, and a Q₁₀ value of 1.20. This value is about half that of total intracellular resistivity and thus will be a significant determinant of the actual value of action potential conduction velocity. The dielectrophoresis experiments demonstrated the importance of including divalent cations (Ca²⁺ and Mg²⁺) in the suspension medium, as their omission reduced cell integrity by lowering membrane resistivity and increasing cytoplasm resistivity. Accurate measurement of R_c is essential to develop quantitative computational models that determine the key factors contributing to the development of cardiac arrhythmias.     

Hydrazo Pyrazole-Pyridone Fluorescent tag for NLO,Live cell imaging,LFPs visualization,Photophysical probing,and Electrochemical sensor for Dopamine detection

The present communication reports on the synthesis of a novel methyl-pyridone azo fluorescent tag (MPAFT) were proven through ¹H (NMR), FT-IR, UV–vis, and high-resolution mass spectrometry. The quantum chemical parameters of MPAFT were evaluated using density functional theory (DFT) analysis. It was further investigated for its latent fingerprint (LFPs) in various surfaces and anticounterfeiting applications. By exposing Level I–Level III, ridge features to UV light with a wavelength of 365 nm, a bioimaging investigation has also demonstrated the potential of MPAFT's emission behaviour. The cyclic voltammetry (CV) and linear sweep voltammetry (LSV) at MPAFT/MGCE (modified glassy carbon electrode) were used to explore the electrochemical sensitivity and reliable detection of dopamine (DA) in neutral PBS (pH 7) electrolyte solution, and the results show good sensitivity and detection. The lower detection limit for LSV was 0.81 μM under optimum conditions.     

Real Time PCR to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus subspecies venerealis

Bovine venereal campylobacter infection, caused by Campylobacter fetus venerealis, is of significant economic importance to the livestock industry. Unfortunately, the successful detection and discrimination of C. fetus venerealis from C. fetus fetus continue to be a limitation throughout the world. There are several publications warning of the problem with biotyping methods as well as with recent molecular based assays. In this study, assessed on 1071 isolates, we report on the successful development of two Real Time SYBR® Green PCR assays that will allow for the detection and discrimination of C. fetus fetus and C. fetus venerealis. The sensitivity reported here for the C. fetus (CampF4/R4) and the C. fetus venerealis (CampF7/R7) specific PCR assays are 100% and 98.7% respectively. The specificity for these same PCR assays are 99.6% and 99.8% respectively.     

Diet of Water Striders (Gerris lacustris L. 1758) in a Rice Field Near Seoul,Korea

Diet composition of Gerris lacustris in a rice-field was compared with the composition of insect fauna from sweep nets conducted in the same habitat. Diptera was the most common prey among the insects potentially available to waterstriders. Water striders consumed Diptera, Homoptera and Hymenoptera in larger proportions than proportions of these orders in the sweep samples. There was no indication of prey size preferences in water striders as they appeared to capture prey of various size classes in frequencies corresponding to the frequencies in sweep samples. This is the first report of the diet of G. lacustris and the second report of the diet composition of any Gerris sp. water strider.     

Effects of smoking,schizotypy, and eyes open/closed conditions on the γ1 rhythm phase synchrony of the electroencephalogram

This study aims at discrimination between various subject conditions and states on the basis of their electroencephalogram (EEG). For the purpose of such discrimination, phase synchrony (PS) coefficients were evaluated for two groups of participants. Statistical tests – such as two-factor ANOVA and Kruskal–Wallis – were used to select sets of EEG electrodes producing statistically different PS coefficients between two groups. The coefficients selected were used by Euclidean, weighted Euclidean, and Mahalanobis distance-based classifiers, as well as by the classifier using a quadratic discriminant function, to test their suitability for reliable discrimination. The method above has been tested on the EEG of smokers and non-smokers, of high and low schizotypy individuals, and for the experimental conditions of eyes open and eyes closed.We have observed that the average γ₁ rhythm PS is higher for high schizotypy non-smokers than for high schizotypy smokers and also higher for high schizotypy non-smokers than for low schizotypy non-smokers. Statistical tests showed that the differences between high schizotypy smokers and high schizotypy non-smokers are significant – at the 99% significance level – for a number of electrode pairs. The differences between the average γ₁ rhythm PS of high schizotypy non-smokers and all other participants are also significant for a number of electrode pairs. On the other hand, the differences between the average phase synchrony of high schizotypy smokers and low schizotypy individuals do not exceed within-group variations. These observations may support the hypothesis of nicotine self-medication in schizophrenia patients.For the eyes closed condition, γ₁ rhythm PS was also observed to be higher than for the eyes open condition. The Euclidean distance-based classifier showed, on average, better than 80% correct classification between the eyes open and eyes closed subject conditions while using a subset of the available electrode pairs (i.e., electrode masking). This discrimination could be useful, for instance, for automated vigilance monitoring.     

Photoreceptor Spectral Sensitivity in the Bumblebee,Bombus impatiens (Hymenoptera: Apidae)

The bumblebee Bombus impatiens is increasingly used as a model in comparative studies of colour vision, or in behavioural studies relying on perceptual discrimination of colour. However, full spectral sensitivity data on the photoreceptor inputs underlying colour vision are not available for B. impatiens. Since most known bee species are trichromatic, with photoreceptor spectral sensitivity peaks in the UV, blue and green regions of the spectrum, data from a related species, where spectral sensitivity measurements have been made, are often applied to B impatiens. Nevertheless, species differences in spectral tuning of equivalent photoreceptor classes may result in peaks that differ by several nm, which may have small but significant effects on colour discrimination ability. We therefore used intracellular recording to measure photoreceptor spectral sensitivity in B. impatiens. Spectral peaks were estimated at 347, 424 and 539 nm for UV, blue and green receptors, respectively, suggesting that this species is a UV-blue-green trichromat. Photoreceptor spectral sensitivity peaks are similar to previous measurements from Bombus terrestris, although there is a significant difference in the peak sensitivity of the blue receptor, which is shifted in the short wave direction by 12–13 nm in B. impatiens compared to B. terrestris.     

Physiological responses of Pogonatum cirratum subsp. fuscatum living in different habitats to simulated nitrogen deposition

The effects of nitrogen (N) deposition on Pogonatum cirratum (Sw.) Bird. subsp. fuscatum (Mitt.) Hyvoenen, a species of moss widely distributed in South China, were investigated. Plots subjected to a gradient of N treatments were established in 3 habitats with different illumination and humidity conditions. N was sprayed onto plots with NH₄NO₃ solutions, at doses equivalent to 20, 40 and 60 kg N ha^?1, in 4 applications during May 2006 and January 2007. The results suggest that high N deposition affects both carbon (C) and N metabolism of P. cirratum subsp. fuscatum, as well as the interaction between them. On the other hand, the sensitivity of the moss varied according to the humidity and light conditions. Moss in a habitat with high humidity and moderate light was more tolerant to an N deposition increase than moss in a habitat with low humidity and low light, as the latter was extremely sensitive to an increase in N deposition.     

Translational discrimination against bacteriophage T7 gene 0·3 messenger RNA

Based on genetic manipulation of T7 late messenger RNA levels in vivo, we previously hypothesized that wild-type T7 infection of Escherichia coli develops in mRNA excess and that there is translational discrimination against T7 gene 0·3 mRNA (Strome & Young, 1978). The results presented here support our hypothesis. The discrimination against 0·3 mRNA translation observed in vivo can be mimicked in a cell-free system by increasing the concentration of T7 RNA beyond the level needed to saturate the translational machinery or by translating T7 RNA with a low concentration of ribosomes. This discrimination can be overcome by adding ribosomes to the cell-free system (increasing the ribosome to mRNA ratio) or by slowing the rate of polypeptide chain elongation. In addition 0·3 mRNA activity as well as a substantial fraction of T7 late mRNA activity is found to be shifted off of polysomes late in T7 infection. Our results are indicative of a low initiation rate constant for 0·3 mRNA compared to T7 late mRNAs.     

Evaluating the diagnostic test accuracy of molecular xenomonitoring methods for characterising the community burden of Onchocerciasis

BackgroundMolecular xenomonitoring (MX), the detection of parasite nucleic acid in the vector population, is recommended for onchocerciasis surveillance in elimination settings. However, the sensitivity of MX for detecting onchocerciasis-positive communities has not previously been evaluated. MX may have additional applications for control programmes but its utility is restricted by a limited understanding of the relationship between MX results and human prevalence.MethodsWe conducted a systematic review of studies reporting the prevalence of Onchocerca volvulus DNA in wild-caught Simulium spp. flies (MX rate) and corresponding prevalence of microfilaria (mf) in humans. We evaluated the sensitivity of MX for detecting onchocerciasis-positive communities and describe the characteristics of studies with reduced sensitivity. We conducted a linear regression to evaluate the relationship between mf prevalence and MX rate.ResultsWe identified 15 relevant studies, with 13 studies comprising 34 study communities included in the quantitative analyses. Most communities were at advanced stages towards elimination and had no or extremely low human prevalence. MX detected positive flies in every study area with >1% mf prevalence, with the exception of one study conducted in the Venezuelan Amazonian focus. We identified a significant relationship between the two measurements, with mf prevalence accounting for half of the variation in MX rate (R² 0.50, p<0.001).ConclusionMX is sensitive to communities with ongoing onchocerciasis transmission. It has potential to predict human mf prevalence, but further data is required to understand this relationship, particularly from MX surveys conducted earlier in control programmes before transmission has been interrupted.     

Modified Proofreading PCR for Detection of Point Mutations,Insertions and Deletions Using a ddNTP-Blocked Primer

The development of simple, accurate, rapid and cost-effective technologies for mutation detection is crucial to the early diagnosis and prevention of numerous genetic diseases, pharmacogenetics, and drug resistance. Proofreading PCR (PR-PCR) was developed for mutation detection in 1998 but is rarely applied due to its low efficiency in allele discrimination. Here we developed a modified PR-PCR method using a ddNTP-blocked primer and a mixture of DNA polymerases with and without the 3''-5'' proofreading function. The ddNTP-blocked primer exhibited the best blocking efficiency to avoid nonspecific primer extension while the mixture of a tiny amount of high-fidelity DNA polymerase with a routine amount of Taq DNA polymerase provided the best discrimination and amplification effects. The modified PR-PCR method is quite capable of detecting various mutation types, including point mutations and insertions/deletions (indels), and allows discrimination amplification when the mismatch is located within the last eight nucleotides from the 3''-end of the ddNTP-blocked primer. The modified PR-PCR has a sensitivity of 1-5 × 10² copies and a selectivity of 5 × 10^-5 mutant among 10⁷ copies of wild-type DNA. It showed a 100% accuracy rate in the detection of P72R germ-line mutation in the TP53 gene among 60 clinical blood samples, and a high potential to detect rifampin-resistant mutations at low frequency in Mycobacterium tuberculosis using an adaptor and a fusion-blocked primer. These results suggest that the modified PR-PCR technique is effective in detection of various mutations or polymorphisms as a simple, sensitive and promising approach.     

Application of comet assay for the assessment of DNA damage caused by chemical genotoxins in the dairy yeast Kluyveromyces lactis

Kluyveromyces lactis, also known as dairy yeast, has numerous applications in scientific research and practice. It has been approved as a GRAS (Generally Recognized As Safe) organism, a probiotic, a biotechnological producer of important enzymes at industrial scale and a bioremediator of waste water from the dairy industry. Despite these important practical applications the sensitivity of this organism to genotoxic substances has not yet been assessed. In order to evaluate the response of K. lactis cells to genotoxic agents we have applied several compounds with well-known cyto- and genotoxic activity. The method of comet assay (CA) widely used for the assessment of DNA damages is presented here with new special modifications appropriate for K. lactis cells. The comparison of the response of K. lactis to genotoxins with that of Saccharomyces cerevisiae showed that both yeasts, although considered close relatives, exhibit species-specific sensitivity toward the genotoxins examined.     

Transfection improvements of fish cell lines by using deacylated polyethylenimine of selected molecular weights

A new tool for DNA transfer to fish cell lines such as epithelioma papulosum cyprini (EPC) and rainbow trout gonad (RTG2), has been optimized by testing commercially available polyethylenimine (PEI) polymers as transfectant reagents. Deacylated 25 kDa PEI polymers were selected amongst the most active and then low toxicity deacylated PEIs fractions around 15 kDa were obtained by gel filtration chromatography to increase 3–4-fold their initial in vitro transfection efficiency. The EPC and plasmids coding for reporter genes were first used to optimize variable values for best expression by transfection with deacylated low toxicity PEI while both EPC/RTG2 and a plasmid coding for the glycoprotein G gene of the fish pathogen, viral haemorrhagic septicemia virus (VHSV) were then used to demonstrate some of their practical applications. Due to its relatively low price, defined chemical composition and availability, low toxicity deacylated PEI might be used for numerous applications for all those studying fish cell immunology in vitro as well as in vivo.