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International audience; In vertebrates, intercellular communication is largely mediated by connexins (Cx), a family of structurally related transmembrane proteins that assemble to form hemichannels (HCs) at the plasma membrane. HCs are upregulated in different brain disorders and represent innovative therapeutic targets. Identifying modulators of Cx-based HCs is of great interest to better understand their function and define new treatments. In this study, we developed automated versions of two different cell-based assays to identify new pharmacological modulators of Cx43-HCs. As HCs remain mostly closed under physiological conditions in cell culture, depletion of extracellular Ca 2+ was used to increase the probability of opening of HCs. The first assay follows the incorporation of a fluorescent dye, Yo-Pro, by real-time imaging, while the second is based on the quenching of a fluorescent protein, YFP QL , by iodide after iodide uptake. These assays were then used to screen a collection of 2242 approved drugs and compounds under development. This study led to the identification of 11 candidate hits blocking Cx43-HC, active in the two assays, with 5 drugs active on HC but not on gap junction (GJ) activities. To our knowledge, this is the first screening on HC activity and our results suggest the potential of a new use of already approved drugs in central nervous system disorders with HC impairments.

Development of a mitochondria-targeting fluorescent probe with large Stokes shift and long-wavelength emission was benefit for accurate detection of hypoxic status, which was known as a major factor of the tumor physiology and influence important pathological processes. However, an efficient optical approach for simultaneously achieving such merits was still lacking. In this work, a turn-on fluorescence probe (HBT-NP) was designed to assess the hypoxic condition of tumor cells by detecting nitroreductase (NTR). Probe HBT-NP was constructed by conjugating 4-nitrobenzyl moiety as reaction site for NTR to 2-(benzo[d]thiazol-2-yl)-4-methylphenol derived fluorescent dye HBT-Py which demonstrated large Stokes shift (Δλ = 243 nm) and long wavelength emission (λem = 640 nm) due to intrinsic mechanism of ESIPT together with ICT process. Upon incubated with NTR, HBT-NP could successively undergo nitro reduction reaction and then release HBT-Py. The reaction mechanism was further confirmed by mass spectra and HPLC analysis, and the docking calculation also indicated that the binding mode and docking affinity of probe HBT-NP with NTR play an important role in catalytic reduction reaction process. As a result, HBT-NP displayed a wide linear range (0.1–1.5 μg/mL) and low detection limit (2.8 ng/mL) response to NTR, and could be used to evaluate hypoxic condition of cancer cells with precise mitochondria-targeting.

Filamenting temperature-sensitive mutant Z (FtsZ) is recognized as a promising target for new antibiotics development because of its high conservatism and pivotal role in the bacteria cell division. The aromatic heterocyclic scaffold of indole is known showing merit medical functions in antiviral and antimicrobial. In the present study, a series of 1-methylquinolinium derivatives, which were integrated with an indole fragment at its 2-position and a variety of amino groups (cyclic or linear, mono- or di-amine) at the 4-position were synthesized and their antibacterial activities were evaluated. The results of antibacterial study show that the representative compounds can effectively inhibit the growth of testing strains including MRSA and VRE, with MIC values of 1–4 μg/mL by bactericidal mode. The mode of action assays revealed that c2 can effectively disrupt the rate of GTP hydrolysis and dynamic polymerization of FtsZ, and thus inhibits bacterial cell division and then causes bacterial cell death. In addition, the result of resistance generation experiment reveals that c2 is not likely to induce resistance in S. aureus.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family of ligand-dependent transcription factors. PPARs are important regulators of glucose and fatty acid metabolism, apoptosis, angiogenesis, cell proliferation and differentiation, and immune response. Their possible role in the female reproductive tract was demonstrated. In the present study, cultured luminal epithelial (LE) and stromal (ST) cells of the porcine endometrium were used to examine (1) the effect of conceptus exposed medium (CEM) on mRNA and protein expression and DNA binding activity of PPARA, PPARD, and PPARG isoforms, and (2) the effect of PPARA, PPARD, and PPARG agonists on the expression of selected genes, apoptosis, and cell proliferation. The addition of CEM stimulated PPARA expression and DNA binding activity of this isoform in LE and ST cells (P 0.05). Increased expression of PPARD mRNA in the presence of CEM was detected in ST cells (P 0.05), while the concentration of PPARG transcripts decreased in response to CEM in both cell types (P 0.05). LE and ST cells of the pig endometrium possess PPARA, PPARD, and PPARG proteins, with clear nuclear staining visible predominately in ST cells. In LE cells, activation of PPARG with 15-deoxy-Δ12,14-prostaglandin(PG)J2 down-regulated the expression of genes encoding amino acid transporter 1 (SLC38A1), leukemia inhibitory factor (LIF) and enzymes involved in PG synthesis (P 0.05). In ST cells, activation of PPARD isoform with both agonists used (L-165,041 and cPGI2) and PPARG isoform with 15-deoxy-Δ12,14-PGJ2 increased vascular endothelial growth factor A (VEGFA) mRNA expression (P 0.05). Moreover, GW9578 (PPARA agonist) and 15-deoxy-Δ12,14-PGJ2 stimulated glucose transporter 1 (SLC2A1) gene expression in ST cells. 15-deoxy-Δ12,14-PGJ2 was also effective in up-regulation of the ratio of BAX/BCL2 mRNA expression and active caspase-3 concentration in ST cells (P 0.05). Finally, GW9578 stimulated LE and ST cell proliferation, while rosiglitazone (PPARG agonist) increased the number of viable ST but not LE cells. In conclusion, this study demonstrated that conceptus products differentially modulate PPARs expression and activity in the porcine endometrium. Activation of PPARs may in turn affect nutrient transport, PG synthesis, angiogenesis, apoptosis, or cell proliferation in this tissue. Therefore, PPAR isoforms seem to play an important role in development and function of the porcine uterus.

G-quadruplex (G4) DNA plays a vital role in myriad biological process and is linked to several human diseases, including Alzheimer’s disease. Probing G4s with fluorescent probes can provide a better understanding their mechanisms of action and of their roles in Nature. In this study we developed a quinolinium–vinylaniline molecular rotor probe, featuring a diethylaminosalicylaldehyde unit that could discriminate the hybrid-22AG G4 sequence selectively amongst other G4 sequences. This probe underwent a significant red-shift upon binding to the target G4 (broad 575 nm → sharp 630 nm) with enhanced fluorescence (up to 14-fold). We suspect that the vinylaniline unit of the molecular rotor, when bound to the hybrid-22 A G4, experienced restricted rotation, thereby undergoing enhanced intramolecular charge transfer. The presence of the diethylaminosalicylaldehyde moiety appeared to play a major role in the enhanced selectivity toward the 22AG G4.

Bipolar cancellation refers to a phenomenon when applying a second electric pulse reduces (“cancels”) cell membrane damage by a preceding electric pulse of the opposite polarity. Bipolar cancellation is a reason why bipolar nanosecond electric pulses (nsEP) cause weaker electroporation than just a single unipolar phase of the same pulse. This study was undertaken to explore the dependence of bipolar cancellation on nsEP parameters, with emphasis on the amplitude ratio of two opposite polarity phases of a bipolar pulse. Individual cells (CHO, U937, or adult mouse ventricular cardiomyocytes (VCM)) were exposed to either uni- or bipolar trapezoidal nsEP, or to nanosecond electric field oscillations (NEFO). The membrane injury was evaluated by time-lapse confocal imaging of the uptake of propidium (Pr) or YO-PRO-1 (YP) dyes and by phosphatidylserine (PS) externalization. Within studied limits, bipolar cancellation showed little or no dependence on the electric field intensity, pulse repetition rate, chosen endpoint, or cell type. However, cancellation could increase for larger pulse numbers and/or for longer pulses. The sole most critical parameter which determines bipolar cancellation was the phase ratio: maximum cancellation was observed with the 2(nd) phase of about 50% of the first one, whereas a larger 2(nd) phase could add a damaging effect of its own. “Swapping” the two phases, i.e., delivering the smaller phase before the larger one, reduced or eliminated cancellation. These findings are discussed in the context of hypothetical mechanisms of bipolar cancellation and electroporation by nsEP.

The antibacterial activity and the synergistic effect with β-lactam antibiotics of a new 1-methylquinolinium iodide derivative were investigated. The experimental results indicate that the compound possesses a strong antibacterial activity against a panel of bacteria including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and NDM-1 Escherichia coli with the MIC values from 0.75 μg/mL to 6 μg/mL. In addition, this compound combined with β-lactam antibiotics shows strong synergistic antimicrobial activities against antibiotic-resistant strains of S. aureus. The results of biochemical studies also reveal that this compound can effectively disrupt GTPase activity, polymerization of FtsZ, and cell division to cause cell death. The compound shows high potential for further development as a new generation of antibacterial agents to fight against the emergence of multidrug-resistant bacteria.

Decreasing the time gap between two identical electric pulses is expected to render bioeffects similar to those of a single pulse of equivalent total duration. In this study, we show that it is not necessarily true, and that the effects vary for different permeabilization markers. We exposed individual CHO or NG108 cells to one 300-ns pulse (3.7–11.6 kV/cm), or a pair of such pulses (0.4–1000 μs interval), or to a single 600-ns pulse of the same amplitude. Electropermeabilization was evaluated (a) by the uptake of YO-PRO-1 (YP) dye; (b) by the amplitude of elicited Ca(2+) transients, and (c) by the entry of Tl(+) ions. For YP uptake, applying a 600-ns pulse or a pair of 300-ns pulses doubled the effect of a single 300-ns pulse; this additive effect did not depend on the time interval between pulses or the electric field, indicating that already permeabilized cells are as susceptible to electropermeabilization as naïve cells. In contrast, Ca(2+) transients and Tl(+) uptake increased in a supra-additive fashion when two pulses were delivered instead of one. Paired pulses at 3.7 kV/cm with minimal separation (0.4 and 1 μs) elicited 50–100% larger Ca(2+) transients than either a single 600-ns pulse or paired pulses with longer separation (10–1000 μs). This paradoxically high efficiency of the closest spaced pulses was emphasized when Ca(2+) transients were elicited in a Ca(2+)-free solution (when the endoplasmic reticulum (ER) was the sole significant source of Ca(2+)), but was eliminated by Ca(2+) depletion from the ER and was not observed for Tl(+) entry through the electropermeabilized membrane. We conclude that closely spaced paired pulses specifically target ER, by either permeabilizing it to a greater extent than a single double-duration pulse thus causing more Ca(2+) leak, or by amplifying Ca(2+)-induced Ca(2+) release by an unknown mechanism.

Because the degree of labeling (DOL) of cell-bound antibodies, often required in quantitative fluorescence measurements, is largely unknown, we investigated the effect of labeling with two different fluorophores (AlexaFluor546, AlexaFluor647) in a systematic way using antibody stock solutions with different DOLs. Here, we show that the mean DOL of the cell-bound antibody fraction is lower than that of the stock using single molecule fluorescence measurements. The effect is so pronounced that the mean DOL levels off at approximately two fluorophores/IgG for some antibodies. We developed a method for comparing the average DOL of antibody stocks to that of the isolated, cell-bound fraction based on fluorescence anisotropy measurements confirming the aforementioned conclusions. We created a model in which individual antibody species with different DOLs, present in an antibody stock solution, were assumed to have distinct affinities and quantum yields. The model calculations confirmed that a calibration curve constructed from the anisotropy of antibody stocks can be used for determining the DOL of the bound fraction. The fluorescence intensity of the cell-bound antibody fractions and of the antibody stocks exhibited distinctly different dependence on the DOL. The behavior of the two dyes was systematically different in this respect. Fitting of the model to these data revealed that labeling with each dye affects quantum yield and antibody affinity differentially. These measurements also implied that fluorophores in multiply labeled antibodies exhibit self-quenching and lead to decreased antibody affinity, conclusions directly confirmed by steady-state intensity measurements and competitive binding assays. Although the fluorescence lifetime of antibodies labeled with multiple fluorophores decreased, the magnitude of this change was not sufficient to account for self-quenching indicating that both dynamic and static quenching processes occur involving H-aggregate formation. Our results reveal multiple effects of fluorophore conjugation, which must not be overlooked in quantitative cell biological measurements.

Fluorinated dendrimers have shown great promise in gene delivery due to their high transfection efficacy and low cytotoxicity, however, the structure-activity relationships of these polymers still remain unknown. Herein, we synthesized a library of fluorinated dendrimers with different dendrimer generations and fluorination degrees and investigated their behaviors in both DNA and siRNA delivery. The results show that fluorination significantly improves the transfection efficacy of G4-G7 polyamidoamine dendrimers in DNA and siRNA delivery. Fluorination on generation 5 dendrimer yields the most efficient polymers in gene delivery, and the transfection efficacy of fluorinated dendrimers depends on fluorination degree. All the fluorinated dendrimers cause minimal toxicity on the transfected cells at their optimal transfection conditions. This study provides a general and facile strategy to prepare high efficient and low cytotoxic gene carriers based on fluorinated polymers. Statement of Significance The structure-activity relationships of fluorinated dendrimers in gene delivery is still unknown and the behavior of fluorinated dendrimers in siRNA delivery has not yet been investigated. Herein, we synthesized a library of fluorinated PAMAM dendrimers with different dendrimer generations and fluorination degrees and investigated their behaviors in both DNA and siRNA delivery. The results clearly indicate that fluorination significantly improves the transfection efficacy of dendrimers in both DNA and siRNA delivery without causing additional toxicity. G5 PAMAM dendrimer is best scaffold to synthesize fluorinated dendrimers and the transfection efficacy of fluorinated dendrimers depends on fluorination degree. This systematic study provides a general and facile strategy to prepare high efficient and low cytotoxic gene carriers based on fluorinated polymers.

Chloride ion (Cl−) is one of the most abundant anions in our body. Increasing evidence suggests that Cl− plays fundamental roles in various cellular functions. We have previously reported that electroneutral cation-chloride cotransporters, such as Na+-K+-2Cl− cotransporter 1 (NKCC1) and K+-Cl− cotransporter 1 (KCC1), are involved in neurite outgrowth during neuronal differentiation. In the present study, we studied if there is correlation between intracellular Cl− concentrations ([Cl−]i) and the length of growing neurites. We measured [Cl−]i in the cell body and growing neurite tips using halide-sensitive fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), revealing that [Cl−]i in the tip of growing neurite was higher than that in cell body in a single cell. Importantly, there was a significant positive correlation between the length of growing neurite and [Cl−]i in neurite tip. Bumtanide (BMT), an inhibitor of NKCC1, significantly inhibited neurite outgrowth and decreased [Cl−]i in neurite tip. The results obtained in the present study and our previous studies together strongly suggest that high [Cl−]i in neurite tip region is crucial for efficient neurite outgrowth.

A benzofuroquinolinium derivative that exhibits excellent cell division inhibitory effect was discovered through cell-based screening approach. This compound possesses potent antimicrobial activity against both Gram-positive and Gram-negative bacteria including the drug-resistant strains. In addition, this compound is able to restore MRSA susceptibility to beta-lactam antibiotics. The biochemical results suggest that the compound inhibits bacterial cell division through the disruption of GTPase activity and the polymerization of FtsZ, which is probably the mechanism of antibacterial activity. Keywords: Bacterial resistance, Antibacterial activity, FtsZ inhibitor

Nanosecond electric pulses (nsEP's) are a well-studied phenomena in biophysics that cause substantial alterations to cellular membrane dynamics, internal biochemistry, and cytoskeletal structure, and induce apoptotic and necrotic cell death. While several studies have attempted to measure the effects of multiple nanosecond pulses, the effect of pulse repetition rate (PRR) has received little attention, especially at frequencies greater than 100 Hz. In this study, uptake of Propidium Iodide, FM 1–43, and YO-PRO-1 fluorescent dyes in CHO-K1 cells was monitored across a wide range of PRRs (5 Hz–500 KHz) using a laser-scanning confocal microscope in order to better understand how high frequency repetition rates impact induced biophysical changes. We show that frequency trends depend on the identity of the dye under study, which could implicate transmembrane protein channels in the uptake response due to their chemical selectivity. Finally, YO-PRO-1 fluorescence was monitored in the presence of Gadolinium (Gd3+), Ruthenium Red, and in calcium-free solution to elucidate a mechanism for its unique frequency trend.

The mitochondrial permeability transition pore (PTP) is now recognized as playing a key role in a wide variety of human diseases whose common pathology may be based in mitochondrial dysfunction. Recently, PTP assays have been adapted to high-throughput screening approaches to identify small molecules specifically inhibiting the PTP. Following extensive secondary chemistry, the most potent inhibitors of the PTP described to date have been developed. This review will provide an overview of each of these screening efforts, use of resulting compounds in animal models of PTP-based diseases, and problems that will require further study. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Sensitive electrophoretic method for determination of total sodium 2-mercaptoethanesulfonate (mesna) in human plasma, based on the stacking with high salt concentration in MEKC and in-capillary derivatization with 2-chloro-1-methyllepidinium tetrafluoroborate followed by UV detection was developed. In the method 0.03molL(-1)pH 7 phosphate buffer with the addition of 0.01molL(-1) SDS, and 10% ACN was used as a BGE. The limit of quantification (LOQ) of the method was 0.5μmolL(-1). Linearity in detector response was observed over the range of 0.5-10μmolL(-1) with the correlation coefficient 0.9971. The intra- and inter-day accuracy (three concentration levels, 5 days, n=3) of the method ranged from 97.2 to 110.0% and from 94.0 to 101.2%, respectively. The novel MEKC method with UV detection proved to be suitable for determination of total mesna in human plasma.

A novel molecule, TatBim-Alexa, consisting of the HIV1 Tat cell-penetrating peptide, the Bim apoptosis-inducing peptide, and Alexa Fluor 546 was synthesized for photoinducion of apoptosis. The Alexa Fluor 546 was used as a photosensitizer and covalently attached at the C-terminus of TatBim peptide by the thiol-maleimide reaction. Photo-dependent cytosolic internalization of TatBim-Alexa and photo-dependent apoptosis using TatBim-Alexa were demonstrated in several kinds of mammalian cells including human cancer cell lines.

We synthesized quinapyramine sulfate loaded-sodium alginate nanoparticles (QS-NPs) to reduce undesirable toxic effects of QS against the parasite Trypanosoma evansi, a causative agent of trypanosomosis. To determine the safety of the formulated nanoparticles, biocompatibility of QS-NPs was determined using Vero, Hela cell lines and horse erythrocytes in a dose-dependent manner. Our experiments unveiled a concentration-dependent safety/cytotoxicity (metabolic activity), genotoxicity (DNA damage, chromosomal aberrations), production of reactive oxygen species and hemolysis in QS-NPs treated cells. Annexin-V propidium iodide (PI) staining showed no massive apoptosis or necrosis. However, at very high doses (more than 300 times than the effective doses), we observed more toxicity in QS-NPs treated cells as compared to QS treated cells. QS-NPs were safe at effective trypanocidal doses and even at doses several times higher than the effective dose.

Trypanosomosis (Surra) is an economically important disease caused by Trypanosoma evansi which is an extracellular parasite present in the plasma, tissues and other body fluids of a wide range of hosts including domesticated animals. Currently, serological reports are based on detection of antibodies by ELISA using whole cell lysate (WCL) antigen, which has a limitation of persistence of anti-trypanosomal antibodies after successful treatment of the disease. Moreover, it has some ethical issues also like requirement of mice for in vivo maintenance of parasite for preparing the antigen. Therefore, in the present study, an attempt was made to evaluate the in vitro production of recombinant heat shock protein 70 (HSP70) for detection of antibodies in experimentally infected ponies. The amino acid sequence analysis of HSP70 revealed that N-terminal region of the protein was highly conserved while the C-terminal region was most divergent. The four different regions of HSP70 protein viz. HSP-1, HSP-2, HSP-3 and HSP-4 were cloned and expressed, among which HSP-1 (N-terminal region) & HSP-2 (C-terminal region) were truncated while HSP-3 & HSP-4 were complete C-terminal proteins. The recombinant fragments were probed with sequentially pooled experimental serum samples where antibodies were detected in these fragments from 10(th) day post infection till the termination of the experiment. Further, these recombinant fragments were also comparatively evaluated with WCL antigen in ELISA using experimental as well as field serum samples. It was observed that after successful treatment of infected ponies, there was a sharp fall in antibodies (within 90 days) when tested with recombinant HSP's fragments, while antibodies persisted even after 469 days when tested against WCL antigen. The sensitivity and specificity of all HSP70 fragments were also estimated from field serum samples with reference to WCL antigen ELISA. The HSP-1 showed minimum sensitivity (41.03%) among all the recombinant fragments. Among the C-terminal fragments, maximum sensitivity was observed with the HSP-2 (61.54%) while minimum was observed with HSP-4 (48.72%). The specificity increases for recombinant fragments from N-terminal to C-terminal region of protein and maximum specificity was observed with HSP-4 fragment (91.3%).

Tumour growth is closely related to the development of new blood vessels to supply oxygen and nutrients to cancer cells. Without the neovascular formation, tumour volumes cannot increase and undergo metastasis. Antiangiogenesis is one of the most promising approaches for antitumour therapy. The exploration of new antiangiogenic agents would be helpful in antitumour therapy. Quinoline is an aromatic nitrogen compound characterized by a double-ring structure which exhibits a benzene ring fused to pyridine at two adjacent carbon atoms. The high stability of quinoline makes it preferable in a variety of therapeutic and pharmaceutical applications, including antitumour treatment. This work is to examine the potential antiangiogenic activity of the synthetic compound 2-Formyl-8-hydroxy-quinolinium chloride. We found that 2-Formyl-8-hydroxy-quinolinium chloride could inhibit the growth of human umbilical vein endothelial cells in vitro. Using the diethylnitrosamine-induced hepatocarcinogenesis model, 2-Formyl-8-hydroxy-quinolinium chloride showed strong antiangiogenic activity. Furthermore, 2-Formyl-8-hydroxy-quinolinium chloride could inhibit the growth of large Hep3B xenografted tumour from the nude mice. We assume that 2-Formyl-8-hydroxy-quinolinium chloride could be a potential antiangiogenic and antitumour agent and it is worthwhile to further study its underlying working mechanism.

Layered silicate nanoparticles (LSN) are widely used in industrial applications and consumer products. They also have potential benefits in biomedical applications such as implantable devices and for drug delivery. To study how nanomaterials interact with cells and tissues, techniques to track and quantify their movement through different biological compartments are essential. While radiolabels can be very sensitive, particularly for in vivo studies, fluorescent labeling has been preferred in recent years because of the array of methods available to image and quantify fluorescent nanoparticles. However, labeling can be problematic, especially if it alters the physical properties of the nanomaterial. Herein is described a novel non-covalent labeling technique for LSN using readily available fluorescent dimeric cyanine dyes without the need to use excess amounts of dye to achieve labeling, or the need for removal of unbound dye. The approach utilizes the cationic binding properties of layered silicate clays and the multiple quaternary nitrogens associated with the dyes. Preparation of YOYO-1 labeled LSN with optimal dispersion in aqueous media is presented. The utilization of the labeled particles is then demonstrated in cell binding and uptake studies using flow cytometry and confocal microscopy. The labeled LSN are highly fluorescent, stable and exhibit identical physical properties with respect to the unlabeled nanoparticles. The general approach described here is applicable to other cyanine dyes and may be utilized more widely for labeling nanoparticles that comprise a crystalline plate structure with a high binding capacity.

MicroRNAs (miRNA) involve in regulating different physiological processes whose dysregulation is associated with a wide range of diseases including cancers, diabetes and cardiovascular problems. Herein, we report a direct, sensitive and highly selective detection assay for circulating microRNA (miRNA). This detection strategy employs magnetic nanoparticles as the reaction platform which can not only allow online pre-concentration and selective separation but also integrates ligation reaction with amplification to enhance the sensitivity of the detection assay. With the presence of the target miRNA, the locked nucleic acid (LNA)-modified molecular beacon (MB) opens up, exposing the binding sites at two ends. The 3’- and 5’-end of the MB responsible for the attachment onto the magnetic nanoparticles, and reporting probe for the attachment of the pair of amplification probes respectively. The ligase ligate RNA to DNA enhance the amplification efficiency. Upon labelled with intercalating fluorophores (YOYO-1) on the hybrids, the quantification of the target miRNA was determined by measuring the fluorescence intensity. A detection limit of 314 fM was achieved with trace amount of sample consumption (~20 μL). As a proof of concept, miRNA-149 was chosen as the target miRNA. This assay is capable of discriminating single-base and reliably quantifying circulating miRNA-149 in both healthy and cancer patient’s serums. The result obtained was comparable with that of quantitative reverse transcription polymerase chain reaction (qRT-PCR), suggesting that this direct and sensitive assay can be served as a promising, non-invasive tool for early diagnosis of breast cancer and colorectal cancer.

Exposure to intense, nanosecond-duration electric pulses (nsEP) opens small but long-lived pores in the plasma membrane. We quantified the cell uptake of two membrane integrity marker dyes, YO-PRO-1 (YP) and propidium (Pr), in order to test whether the pore size is affected by the number of nsEP. The fluorescence of the dyes was calibrated against their concentrations by confocal imaging of stained homogenates of the cells. The calibrations revealed a two-phase dependence of Pr emission on the concentration (with a slower rise at < 4 µM), and a linear dependence for YP. CHO cells were exposed to nsEP trains (1 to 100 pulses, 60 ns, 13.2 kV/cm, 10 Hz) with Pr and YP in the medium, and the uptake of the dyes was monitored by time-lapse imaging for 3 min. Even a single nsEP triggered a modest but detectable entry of both dyes, which increased linearly when more pulses were applied. The influx of Pr per pulse was constant and independent of the pulse number. The influx of YP per pulse was highest with 1- and 2-pulse exposures, decreasing to about twice the Pr level for trains from 5 to 100 pulses. The constant YP/Pr influx ratio for trains of 5 to 100 pulses suggests that increasing the number of pulses permeabilizes cells to a greater extent by increasing the pore number and not the pore diameter.

Transient receptor potential vanilloid 1 (TRPV1) has been shown to alter its ionic selectivity profile in a time- and agonist-dependent manner. One hallmark of this dynamic process is an increased permeability to large cations such as N-methyl-D-glucamine (NMDG). In this study, we mutated residues throughout the TRPV1 pore domain to identify loci that contribute to dynamic large cation permeability. Using resiniferatoxin (RTX) as the agonist, we identified multiple gain-of-function substitutions within the TRPV1 pore turret (N628P and S629A), pore helix (F638A), and selectivity filter (M644A) domains. In all of these mutants, maximum NMDG permeability was substantially greater than that recorded in wild type TRPV1, despite similar or even reduced sodium current density. Two additional mutants, located in the pore turret (G618W) and selectivity filter (M644I), resulted in significantly reduced maximum NMDG permeability. M644A and M644I also showed increased and decreased minimum NMDG permeability, respectively. The phenotypes of this panel of mutants were confirmed by imaging the RTX-evoked uptake of the large cationic fluorescent dye YO-PRO1. Whereas none of the mutations selectively altered capsaicin-induced changes in NMDG permeability, the loss-of-function phenotypes seen with RTX stimulation of G618W and M644I were recapitulated in the capsaicin-evoked YO-PRO1 uptake assay. Curiously, the M644A substitution resulted in a loss, rather than a gain, in capsaicin-evoked YO-PRO1 uptake. Modeling of our mutations onto the recently determined TRPV1 structure revealed several plausible mechanisms for the phenotypes observed. We conclude that side chain interactions at a few specific loci within the TRPV1 pore contribute to the dynamic process of ionic selectivity.

Successful glioma gene therapy lays on two important factors, the therapeutic genes and efficient delivery vehicles to cross the blood-brain barrier (BBB) and reach gliomas. In this work, a new gene vector was constructed based on dendrigraft poly-l-lysines (DGL) and polyethyleneglycol (PEG), conjugated with a cell-penetrating peptide, the nucleolar translocation signal (NoLS) sequence of the LIM Kinase 2 (LIMK2) protein (LIMK2 NoLS peptide, LNP), yielding DGL-PEG-LNP. Plasmid DNA encoding inhibitor of growth 4 (ING4) was applied as the therapeutic gene. DGL-PEG-LNP/DNA nanoparticles (NPs) were monodispersed, with a mean diameter of 90.6 ± 8.9 nm. The conjugation of LNP significantly enhanced the BBB-crossing efficiency, cellular uptake and gene expression within tumor cells. Mechanism studies suggested the involvement of energy, caveolae-mediated endocytosis and macropinocytosis in cellular uptake of LNP-modified NPs. MTT results showed that no apparent cytotoxicity was observed when cells were treated with synthesized vectors. Furthermore, LNP-modified NPs mediated strongest and most intensive apoptosis on the tumor site, and the longest median survival time of glioma-bearing mice. All the results demonstrated that LNP is a kind of efficient CPPs especially for BBB-crossing application, and DGL-PEG-LNP/DNA is a potential non-viral platform for glioma gene therapy via intravenous administration.

Quaternary ammonium salts substituted with a long alkyl chain exemplify a trustworthy group of medicinal compounds frequently employed as antifungal and antibacterial agents. A great asset of these surfactants underlying their widespread use is low local and system toxicity in humans. In this Letter, a series of novel quaternary 6-hydroxyquinolinium salts with varying length of N-alkyl chain (from C10 to C18) was synthesized and tested for in vitro activity against pathogenic bacterial and fungal strains. 6-Hydroxyquinolinium salt with C12 alkyl chain seems to be very interesting candidate due to a high antimicrobial efficacy and cytotoxic safety.

Intermolecular interaction of linear-type α-(1 → 6)-glucosyl megalosaccharide rich (L-IMS) and water-insoluble anionic ethyl red was firstly characterized in a comparison with inclusion complexation by cyclodextrins (CDs) to overcome the problem of poor solubility and bioavailability. Phase solubility studies indicated an enhancement of 3- and 9-fold over the solubility in water upon the presence of L-IMS and β-CD, respectively. (1)H NMR and circular dichrosim spectra revealed the dye forms consisted of 1:1 stoichiometric inclusion complex within the β-CD cavity, whereas they exhibited non-specific hydrophobic interaction, identified by solvent polarity changes, with L-IMS. The inclusion complex delivered by β-CD showed an uncompetitive inhibitory-type effect to azoreductase, particularly with high water content that did not promote dye liberation. Addition of the solid dye dispersed into coupled-enzyme reaction system supplied by L-IMS as the dye solubilizer provided usual degradation rate. The dye intermission in series exhibited successful removal with at least 5 cycles was economically feasible.

The objective of this study was to evaluate the effect of adding reduced glutathione (GSH) to a boar semen freezing extender supplemented with insulin-like growth factor I (IGF-I) or anti-IGF-I. Eight ejaculates from eight boars were extended to obtain insemination doses, which were supplemented with either recombinant human IGF-I (30 ng/mL) or anti-IGF-I (60 ng/mL) shortly after extension. After 24 h of liquid storage at 17 °C, the semen was frozen with or without GSH (5 mM) in the freezing extender for a total of six treatments. Osmotic resistance and acrosome integrity was greater in fresh semen (P 0.05) soon after adding IGF-I or the anti-IGF-I antibody. After 24 h of cooling, the supplementation with these compounds resulted in an increased (P 0.05) percentage of sperm with relatively greater mitochondrial activity and reduced the percentage of cells with relatively greater concentrations of superoxide. After thawing, there was a reduction (P 0.05) in the percentage and fluorescence intensity of sperm with greater quantities of superoxide and peroxide only in samples treated with GSH + IGF-I and GSH + anti-IGF-I. The addition of GSH (alone or in combination with IGF-I or anti-IGF-I), however, reduced the percentage of sperm with an intact acrosome (P 0.05). The same effect was not observed with IGF-I or anti-IGF-I alone. In conclusion, the addition of IGF-I or anti-IGF-I improved the quality of fresh or liquid-stored semen. Using GSH in the freezing extender improved the antioxidant potential of frozen semen only in combination with IGF-I or an anti-IGF-I antibody.

Aim Gastric cancer (GC) is the fourth most common cancer globally. Bufothionine is a major active constituent of Cinobufacini (Huachansu), which is extracted from the skin and parotid venom gland of the toad Bufo bufo gargarizans Cantor. It exhibits anti-cancer activities in vitro . However, whether bufothionine exerts anti-cancer activities against GC is unknown. This study was designed to evaluate the efficacy of bufothionine in vitro and in vivo . Material and methods MKN28 and AGS cells were chosen as cell models to study the anti-cancer effect of bufothionine. Cell viability was determined by CCK-8 assay, while the effect of bufothionine on cell membrane integrity was examined by LDH assay. Cell apoptosis was detected by Hoechst/PI staining and Annexin V-FITC/PI staining followed by flow cytometry analysis. The expression levels of proteins involved were examined using western blotting. I-Traq analysis was conducted to identify the differentially expressed genes in AGS cells following bufothionine treatment. The anti-growth effect of bufothionine was validated in vivo using a GC xenograft model. Key findings The results revealed that bufothionine prevented the growth, destroyed cell membrane and promoted apoptotic cell death of GC cells. iTRAQ analysis revealed thatPIM3 might be a molecular target responsible for the anti-cancer effects of bufothionine. It was also found that PIM3 knockdown significantly augmented the anti-growth and pro-apoptotic effects of bufothionine in GC cells. In contrast, ectopic PIM3 expression markedly dampened the anti-neoplastic activities of bufothionine. The expression of PIM3 was also suppressed by bufothionine treatment in xenograft tumor tissue. Significance Bufothionine exhibited anti-cancer activities in vitro and in vivo in GC via downregulating PIM3.

P2X7, an ATP-gated cation channel, is involved in immune cell activation, hyperalgesia and neuropathic pain. By regulating cytokine release in the brain, P2X7 has been linked to the pathophysiology of mood disorders and schizophrenia. We here assess the impact of 123 drugs that act in the central nervous system on human P2X7. Most prominently, the tricyclic antipsychotics prochlorperazine (PCP) and trifluoperazine (TFP) potently inhibited P2X7-mediated Ca2+ entry, dye permeation and ionic currents. In divalent cation-containing bath solutions or after prolonged incubation, ATP-evoked P2X7 currents were inhibited by 10 μM PCP. This effect was not related to dopamine receptor antagonism. Surprisingly, PCP co-applied with ATP enhanced inward currents in bath solutions with low divalent cation concentrations. Intracellular perfusion with PCP did not substitute for the extracellularly applied drug, indicating that its binding sites are accessible from the extracellular space. Since P2X7 current potentiation by PCP was voltage-dependent, at least one site may be located within the electrical field of the membrane. While the channel opening and closure kinetic was altered by PCP, the apparent affinity of ATP remained unchanged (potentiation) or changed slightly (inhibition). Measurements in human monocyte-derived macrophages confirmed the PCP-induced inhibition of ATP-evoked Ca2+ influx, Yo-Pro-1 permeability, and whole cell currents. Interestingly, neither heterologously expressed rat or mouse P2X7 nor native P2X7 in rat astrocyte cultures or in mouse bone marrow-derived macrophages were inhibited by perazines with a similar potency. We conclude that perazine-type neuroleptics are potent, but species-selective allosteric modulators of human but not murine P2X7 receptors.

Low throughput is an inherent problem associated with most single-molecule biophysical techniques. We have developed a versatile tool for high-throughput analysis of DNA and DNA-binding molecules by combining microfluidic and dense DNA arrays. We use an easy-to-process microfluidic flow channel system in which dense DNA arrays are prepared for simultaneous imaging of large amounts of DNA molecules with single-molecule resolution. The Y-shaped microfluidic design, where the two inlet channels can be controlled separately and precisely, enables the creation of a concentration gradient across the microfluidic channel as well as rapid and repeated addition and removal of substances from the measurement region. A DNA array stained with the fluorescent DNA-binding dye YOYO-1 in a gradient manner illustrates the method and serves as a proof of concept. We have applied the method to studies of the repair protein Rad51 and could directly probe the concentration-dependent DNA-binding behavior of human Rad51 (HsRad51). In the low-concentration regime used (100 nM HsRad51 and below), we detected binding to double-stranded DNA (dsDNA) without positive cooperativity.

Perineuronal net (PNN) is a specialized aggregate of the extracellular matrix, which is considered to be involved in regulation of structural plasticity of neuronal circuits. Here we examined the spatial and temporal differences in Wisteria floribunda agglutinin-labeled PNN intensity in single cells in the mouse hippocampus, where the neuronal circuits engaged in cognition and emotion are embedded in the dorsal and ventral parts, respectively. In young mice, the intensity of PNN was very low, and there were no significant dorsoventral differences in all hippocampal regions. Developmental increase in PNN intensity was larger in the dorsal part than in the ventral part. As a result, PNN intensity was higher in the dorsal part than in the ventral part in adult mice. Aging dissimilarly affects different regions of the dorsal hippocampus. Namely, PNN intensity in the dorsal part of old mice declined in the CA1 region, remained unchanged in the CA3 region, increased in the dentate gyrus. By contrast, there were no significant aging-related changes in PNN intensity in the ventral hippocampus. We also examined the intensity of parvalbumin (PV), an EF-hand calcium-binding protein, because it has been shown that PNNs are closely related to PV-containing GABAergic inhibitory neurons. Contrary to expectations, developmental and aging-related changes in PV intensity were not comparable to those seen in PNN intensity. The correlation coefficients between PNN and PV intensities in single cells showed gradual decline during development and aging in the CA1 and CA3 regions, while there were little correlations in the dentate gyrus regardless of age. In summary, PNNs are differentially expressed in the dorsal and ventral hippocampal circuits during development and aging, indicating their possible role for cognition and emotion control.

Two series of non-symmetrical bisquaternary pyridinium–quinolinium and pyridinium–isoquinolinium compounds were prepared as molecules potentially applicable in myasthenia gravis treatment. Their inhibitory ability towards human recombinant acetylcholinesterase and human plasmatic butyrylcholinesterase was determined and the results were compared to the known effective inhibitors such as ambenonium dichloride, edrophonium bromide and experimental compound BW284C51. Two compounds, 1-(10-(pyridinium-1-yl)decyl)quinolinium dibromide and 1-(12-(pyridinium-1-yl)dodecyl)quinolinium dibromide, showed very promising affinity for acetylcholinesterase with their IC50 values reaching nM inhibition of acetylcholinesterase. These most active compounds also showed satisfactory selectivity towards acetylcholinesterase and they seem to be very promising as leading structures for further modifications and optimization. Two of the most promising compounds were examined in the molecular modelling study in order to find the possible interactions between the ligand and tested enzyme.

The 3-dimensional quantitative structure-activity relationship (3D-QSAR) molecular modeling technique or comparative molecular field analysis (CoMFA) has been used to design analogs of the natural product cryptolepine (1). Twenty-three compounds with their in vitro biological activities (IC50 values) against Crytococcus neoformans were used to generate the training set database of compounds for the CoMFA studies. The cross-validated q(2), noncross-validated r(2), and partial least squares (PLS) analysis results were used to predict the biological activity of 11 newly designed test set compounds. The best CoMFA model produced a q(2) of 0.815 and an r(2) of 0.976 indicating high statistical significance as a predictive model. The steric and electrostatic contributions from the contour map were interpreted from the color-coded contour plots generated from the PLS model and the active structural components for potency against C. neoformans were determined and validated in the test set compounds. The 3-substituted benzylthio quinolinium salts (4) that make up the test set were synthesized and evaluated based on the predicted activity from the CoMFA model and the results produced a good correlation between the predicted and experimental activity (R=0.82). Thus, CoMFA has served as an effective tool to aid the design of new analogs and in this case, it has aided the identification of compounds equipotent with amphotericin B, the gold standard in antifungal drug design.

The molecular geometry, the normal mode frequencies and corresponding vibrational assignments, natural bond orbital analysis and the HOMO–LUMO analysis of 7-Amino-2,4-dimethylquinolinium formate in the ground state were performed by B3LYP levels of theory using the 6-31G(d) basis set. The optimised bond lengths and bond angles are in good agreement with the X-ray data. The vibrational spectra of the title compound which is calculated by DFT method, reproduces vibrational wave numbers and intensities with an accuracy which allows reliable vibrational assignments. The possibility of N–H⋯O hydrogen bonding was identified using NBO analysis. Natural bond orbital analysis confirms the presence of intramolecular charge transfer and the hydrogen bonding interaction.

Direct cellular entry of potentially useful polar compounds into cells is prevented by the hydrophobic barrier of the membrane. Toward circumventing this barrier, we used high throughput screening to identify a family of peptides that carry membrane-impermeant cargos across synthetic membranes. Here we characterize the plasma membrane translocation of these peptides with polar cargos under a variety of conditions. The spontaneous membrane-translocating peptides (SMTPs) delivered the zwitterionic, membrane-impermeant dye tetramethylrhodamine (TAMRA) into cells even when the conditions were not permissive for endocytosis. They also delivered the larger, anionic membrane-impermeant dye Alexa Fluor 546 but did not deliver a quantum dot nanoparticle. Under all conditions, the SMTP-cargo filled the cytoplasm with a diffuse, non-punctate fluorescence that was partially excluded from the nucleus. D-amino acid peptides behaved identically in vitro, ruling out proteolysis as an important factor in the diffuse cellular distribution. Thus, cytosolic delivery of SMTP-cargo conjugates is dominated by direct membrane translocation. This is in sharp contrast to Arg9-TAMRA, a representative highly cationic, cell-penetrating peptide, which entered cells only when endocytosis was permitted. Arg9-TAMRA triggered large scale endocytosis and did not appreciably escape the endosomal compartments in the 1-h timescales we studied. When injected into mice, SMTP-TAMRA conjugates were found in many tissues even after 2 h. Unconjugated TAMRA was rapidly cleared and did not become systemically distributed. SMTPs are a platform that could improve delivery of many polar compounds to cells, in the laboratory or in the clinic, including those that would otherwise be rejected as drugs because they are membrane-impermeant.

Piracetam is a cyclic derivative of γ-aminobutyric acid (GABA). It was the first nootropic drug approved for clinical use. However, mechanism of its action is still not clear. In present paper, I investigated effects of piracetam on neurotransmitter release, plasma membrane potential monitored by fluorescent dye DiSC3(5) and chloride transport monitored by fluorescent dye SPQ in rat brain synaptosomes. It was shown that piracetam (1 mM) induces slow weak plasma membrane depolarization. This effect was decreased on 43% and 58% by both AMPA/kainate receptor blockers NBQX (10 μM) and CNQX (100 μM), respectively, on 84% by GABA ionotropic receptor blocker picrotoxin (50 μM) and on 91% upon withdrawal of HCO(3-) ions from incubation medium. GABA (1 mM) and kainate (100 μM) were found not to produce changes of plasma membrane potential. Also, it was found that piracetam induces chloride efflux which seems to be the reason of depolarization. Thereby, piracetam induces depolarization of plasma membrane of isolated neuronal presynaptic endings by picrotoxin-sensitive way.

Previously, quinolinium-based tetraazacyclophanes, such as UCL 1684 and UCL 1848, have been shown to be extraordinarily sensitive to changes in chemical structure (especially to the size of the cyclophane system) with respect to activity as potent non-peptidic blockers of the small conductance Ca2+-activated K+ ion channels (SKCa). The present work has sought to optimize the structure of the linking chains in UCL 1848. We report the synthesis and SKCa channel-blocking activity of 29 analogues of UCL 1848 in which the central CH2 of UCL 1848 is replaced by other groups X or Y = O, S, CF2, C O, CHOH, C C, CH CH, CHMe to explore whether subtle changes in bond length or flexibility can improve potency still further. The possibility of improving potency by introducing ring substituents has also been explored by synthesizing and testing 25 analogues of UCL 1684 and UCL 1848 with substituents (NO2, NH2, CF3, F, Cl, CH3, OCH3, OCF3, OH) in the 5, 6 or 7 positions of the aminoquinolinium rings. As in our earlier work, each compound was assayed for inhibition of the afterhyperpolarization (AHP) in rat sympathetic neurons, an action mediated by the SK3 subtype of the SKCa channel. One of the new compounds (39, R7 = Cl, UCL 2053) is twice as potent as UCL 1848 and UCL 1684: seven are comparable in activity.

β-Adrenoceptor (β-AR)-mediated relaxation plays an important role in the regulation of vascular tone. β-AR-mediated vascular relaxation is reduced in various disease states and aging. We hypothesized that β-AR-mediated vasodilatation is impaired in DOCA-salt hypertension due to alterations in the cAMP pathway. β-AR-mediated relaxation was determined in small mesenteric arteries from DOCA-salt hypertensive and control uninephrectomized (Uni) rats. To exclude nitric oxide (NO) and cyclooxygenase (COX) pathways, relaxation responses were determined in the presence of l -NNA and indomethacin, NO synthase inhibitor and COX inhibitors, respectively. Isoprenaline (ISO)-induced relaxation was reduced in arteries from DOCA-salt compared to Uni rats. Protein kinase A (PKA) inhibitors (H89 or Rp-cAMPS) or adenylyl cyclase inhibitor (SQ22536) did not abolish the difference in ISO-induced relaxation between the groups. Forskolin (adenylyl cyclase activator)-induced relaxation was similar between the groups. The inhibition of IK Ca /SK Ca channels (TRAM-34 plus UCL1684) or BK Ca channels (iberiotoxin) reduced ISO-induced relaxation only in Uni rats and abolished the relaxation differences between the groups. The expression of SK Ca channel was decreased in DOCA-salt arteries. The expression of BK Ca channel α subunit was increased whereas the expression of BK Ca channel β subunit was decreased in DOCA-salt arteries. The expression of receptor for activated C kinase 1 (RACK1), which is a binding protein for BK Ca channel and negatively modulates its activity, was increased in DOCA-salt arteries. These results suggest that the impairment of β-AR-mediated relaxation in DOCA-salt mesenteric arteries may be attributable to altered IK Ca /SK Ca and/or BK Ca channels activities rather than cAMP/PKA pathway. Impaired β-AR-stimulated BK Ca channel activity may be due to the imbalance between its subunit expressions and RACK1 upregulation.

As a skin-resident member of the dendritic cell family, Langerhans cells (LCs) are generally regarded to function as professional antigen-presenting cells. Here we report a simple method to visualize the endocytotic activity of LCs in living animals. BALB/c mice received subcutaneous injection of FITC-conjugated dextran (DX) probes into the ear skin and were then examined under confocal microscopy. Large numbers of FITC + epidermal cells became detectable 12–24hours after injection as background fluorescence signals began to disappear. Most (>90%) of the FITC + epidermal cells expressed Langerin, and >95% of Langerin + epidermal cells exhibited significant FITC signals. To assess intracellular localization, Alexa Fluor 546-conjugated DX probes were locally injected into IAβ-enhanced green fluorescent protein (EGFP) knock-in mice and Langerin-EGFP-diphtheria toxin receptor mice—three dimensional rotation images showed close association of most of the internalized DX probes with major histocompatibility complex (MHC) class II molecules, but not with Langerin molecules. These observations support the current view that LCs constantly sample surrounding materials, including harmful and innocuous antigens, at the environmental interface. Our data also validate the potential utility of the newly developed imaging approach to monitor LC function in wild-type animals.

Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification cif pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.

Quindoline derivatives as telomeric quadruplex ligands have shown good biological activity for telomerase inhibition. In the present study, we used spectroscopic and calorimetric methods to investigate the interactions between a quindoline derivative (5-methyl-11-(2-morpholinoethylamino)-10-H-indolo-[3,2-b]quinolin-5-ium iodide, compound 1) and human telomeric G-quadruplex. The thermodynamic studies using isothermal titration calorimetry (ITC) indicated that their binding process was temperature-dependent and enthalpy-entropy co-driven. The significant negative heat capacity was obtained experimentally from the temperature dependence of enthalpy changes, which was consistent with that from theoretical calculation, and all suggesting significant hydrophobic contribution to the molecular recognition process. Based on the results from UV-vis, ITC, steady-state and time-resolved fluorescence, their binding mode was determined as two ligand molecules stacking on the quartets on both ends of the quadruplex. These results shed light on rational design and development of quindoline derivatives as G-quadruplex binding ligands.

In the present article, we summarize our studies of antrycide-resistance of Trypanosoma brucei evansi in four aspects in the last recent several years, the analysis of quinapyramine-sensitive situation of T. b. evansi in China, biological characteristics of T. b. evansi population in quinapyramine-resistance and biological materials of quinapyramine-resistance in T. b. evansi population. Firstly, the correlative assays of effective dosage of quinapyramine on T. b. evansi disease between in vivo and in vitro methods showed that their relationship was parabolic with positive correlation. On the other hand, the IC(50) and CD(100) values of 12 T. b. evansi isolates, AHB, GDB1, GDB2, HNB, JSB1, JSB2, YNB, ZJB, GDH, GXM, HBM and XJCA, collected from buffaloes, horses, mules and camels across nine provinces of China were examined using the two methods, respectively. Among them, the nine isolates, AHB, GDB1, GDB2, HNB, JSB1, JSB2, YNB, ZJB and GDH, became quinapyramine-sensitive T. b. evansi. Secondly, T. evansi populations could rapidly obtain antrycide-resistance when they were passed through immunosuppressed mice treated with low doses of the drug. But, the replication rate of trypanosomes with antrycide-resistance decreases as the level of drug-resistance increases. Thirdly, the analysis of the HK, G6PDH, ALAT and ASAT isoenzymes showed that they were not involved in the quinapyramine-resistance of T. b. evansi. But the protein bands of 15.79kDa and 19.76kDa might be involved in the antrycide-resistance of T. b. evansi population. At genetic level, the gene, TbTA1, could be amplified from the T. b. evansi isolate sensitive to quinapyramine-sensitivity but the T. b. evansi isolate with quinapyramine-resistance using not only the RT-PCR technique, but also PCR technique. We used the SSH (Suppression Subtractive Hybridization) to clone highly or low expressed cDNA fragments caused by production of antrycide-resistance in T. b. evansi. The 5 low and 9 high expressed new cDNA fragments were amplified. Among them, the 3 low expressed cDNA fragments had the same sequence of 65 amino acids and the 3 high expressed cDNA fragments were located in chromosome VI, like T. brucei. Lastly, more work needs to be done in order to elucidate the mechanism of quinapyramine-resistance of T. b. evansi.

Small conductance calcium (Ca(2+)) activated SK channels are critical regulators of neuronal excitability in hippocampus. Accordingly, these channels are thought to play a key role in controlling neuronal activity in acute models of epilepsy. In this study, we investigate the expression and function of SK channels in the pilocarpine model of mesial temporal lobe epilepsy. For this purpose, protein expression was assessed using western blotting assays and gene expression was analyzed using TaqMan-based probes and the quantitative real-time polymerase chain reaction (qPCR) comparative method delta-delta cycle threshold ( big up tri, open big up tri, openCT) in samples extracted from control and epileptic rats. In addition, the effect of SK channel antagonist UCL1684 and agonist NS309 on CA1 evoked population spikes was studied in hippocampal slices. Western blotting analysis showed a significant reduction in the expression of SK1 and SK2 channels at 10days following status epilepticus (SE), but levels recovered at 1month and at more than 2months after SE. In contrast, a significant down-regulation of SK3 channels was detected after 10days of SE. Analysis of gene expression by qPCR revealed a significant reduction of transcripts for SK2 (Kcnn1) and SK3 (Kcnn3) channels as early as 10days following pilocarpine-induced SE and during the chronic phase of the pilocarpine model. Moreover, bath application of UCL1684 (100nM for 15min) induced a significant increase of the population spike amplitude and number of spikes in the hippocampal CA1 area of slices obtained control and chronic epileptic rats. This effect was obliterated by co-administration of UCL1684 with SK channel agonist NS309 (1microM). Application of NS309 failed to modify population spikes in the CA1 area of slices taken from control and epileptic rats. These data indicate an abnormal expression of SK channels and a possible dysfunction of these channels in experimental MTLE.

Two rapid, sensitive and quantitative methods for the determination of the cysteine and cystine ratio in complex defined media feedstock using monolithic reversed-phase liquid chromatography (RPLC) and RPLC-MS are presented. Cysteine is pre-derivatised with purified 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) and separated from other derivatisation products on a narrow-bore 50mmx2mm I.D. monolithic C(18) column with UV detection at 355nm. For reversed-phase LC (RPLC) the separation is carried out isocratically using a mobile phase of 50mM trichloroacetic acid (TCA) adjusted to pH 2.5 with lithium hydroxide (LiOH) and acetonitrile (83:14) pumped at 1.5mL/min with an elevated column temperature. For RPLC-MS an ammonium acetate and acetonitrile gradient method was developed with a reduced flow rate of 0.3mL/min. The treatment of the samples consisted of dividing them into two aliquots, the first aliquot is analysed for cysteine and the second aliquot is analysed for cystine after its quantitative reduction to cysteine using tris(2-carboxyethyl)phosphine (TCEP). Both methods are linear, with R(2)>0.999 for 0.25-500microM for cysteine and 0.25-250microM for cystine using the LC-UV method, sensitive, with detection limit of 36nM for cysteine, and precise, with < or =1.1% RSD for both retention time and peak area (n=6). Samples (n=31) of an industry standard and supplied chemically defined media feedstock were analysed, finding cysteine ranging from 1.56 to 2.26microg/mL and cystine from 1062.02 to 1348.13microg/mL.

Nanosecond pulsed electric fields (nsPEFs) eliminates B16f10 melanoma in mice, but cell death mechanisms and kinetics of molecular events of cell death are not fully characterized. Treatment of B16f10 cells in vitro resulted in coordinate increases in active caspases with YO-PRO-1 uptake, calcium mobilization, decreases in mitochondria membrane potential with decreases in forward light scatter (cell size), increases in ADP/ATP ratio, degradation of actin cytoskeleton and membrane blebbing. However, there was no mitochondrial release of cytochrome c, AIF or Smac/DIABLO or generation of reactive oxygen species. Phosphatidylserine externalization was absent and propidium iodide uptake was delayed in small populations of cells. The results indicate that nsPEFs rapidly recruit apoptosis-like mechanisms through the plasma membrane, mimicking the extrinsic apoptosis pathway without mitochondrial amplification yet include activation of initiator and executioner caspases. nsPEFs provide a new cancer therapy that can bypass cancer-associated deregulation of mitochondria-mediated apoptosis in B16f10 melanoma.

Nijmegen breakage syndrome (NBS) is a genomic instability disease caused by hypomorphic mutations in the NBS1 gene encoding the Nbs1 (nibrin) protein. Nbs1 is a component of the Mre11/Rad50/Nbs1 (MRN) complex that acts as a sensor of double strand breaks (DSBs) in the DNA and is critical for proper activation of the broad cellular response to DSBs. Conditional disruption of the murine ortholog of the human NBS1, Nbs1, in the CNS of mice was previously reported to cause microcephaly, severe cerebellar atrophy and ataxia. Here we report that conditional targeted disruption of the murine NBS1 gene in the CNS results in mal-development, degeneration, disorganization and dysfunction of the murine visual system, especially in the optic nerve. Nbs1 deletion resulted in reduced diameters of Nbs1-CNS-Delta eye and optic nerve. MRI analysis revealed defective white matter development and organization. Nbs1 inactivation altered the morphology and organization of the glial cells. Interestingly, at the age of two-month-old the levels of the axonal guidance molecule semaphorin-3A and its receptor neuropilin-1 were up-regulated in the retina of the mutant mice, a typical injury response. Electroretinogram analysis revealed marked reduction in a- and b-waves, indicative of decreased retinal function. Our study points to a novel role for Nbs1 in the development, organization and function of the visual system.

A small library of carriers consisting of various combinations of the cell penetrating peptide TAT, the SV40 Large T protein nuclear localisation signal (NLS) and a cationic dendrimer of 7 lysine residues (DEN) was synthesised and each member was tested for its ability to deliver exogenous DNA to human HeLa cells. We found that the TAT peptide was essential, but not sufficient for efficient uptake of exogenous DNA. The addition of either NLS or DEN significantly enhanced uptake and expression with the most active carrier consisting of the TAT, NLS and DEN peptides. For those peptides that facilitated DNA uptake, the complexes were targeted to intracellular compartments that required incubation with a fusogenic agent such as chloroquine before gene expression was observed. However, our data suggest that chloroquine did not enhance expression solely by promoting endosomal release since a fusogenic peptide derived from the influenza virus haemagglutinin protein did not improve gene expression. Chloroquine was found to protect DNA from degradation and enhance transcription of DNA bound to the respective carriers. Our results demonstrate that multi-component peptide-based gene carriers can be designed to deliver exogenous DNA. The actions of lysosomotropic agents such as chloroquine reveal the multifactorial properties required for carriers used in non-viral gene delivery.

Choline kinase (ChoK) is reported to involve in cell signaling pathways and cell growth by regulating the intermediate, phosphocholine (PCho), which is the first step to biosynthesis a membrane phospholipid, phosphatidylcholine. The PCho levels are overexpressed due to elevated activation of the protein under carcinogenesis conditions. ChoK has thus evolved as a novel target for various cancers and a range of compounds has been reported in this course as potent ChoK inhibitors. However, not much information is known about the binding site of the inhibitors. Therefore, we ventured to unravel the possible binding site of 39 bis -quinolinium inhibitors from which the structural requirement for better protein–ligand complex was delved. Molecular docking and 3D-QSAR studies namely comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed on the series. The knowledge of the active site was obtained from the site id search and molcad surface calculations of Sybyl, which was further considered for docking studies. In 3D-QSAR, the best predictions were obtained from the model where 29 compounds were considered in the training set and remaining 10 in the test set. The best CoMFA statistics were obtained with r 2 of 0.99 and q 2 of 0.81 while, CoMSIA was resulted with r 2 of 0.98 and q 2 of 0.77. A comparative analysis was done with the resulted 3D-QSAR maps and the docked poses by overlaying the maps on the active site residues. Since, there is no reported ligand co-crystallized structure of ChoK the present study provides valuable clues on the binding conformation of the ligand and its interactions with the active site.

Acridine-iminodibenzyl chimeric compounds were previously introduced as a class of cholesterol-redistributing substances with antiprion effects. Here, we show that administration of the lead compound quinpramine to mice with experimental autoimmune encephalitis, an animal model of multiple sclerosis (MS), significantly ameliorates disease in preventive and therapeutic paradigms. Quinpramine treatment decreased the number of inflammatory CNS lesions, antigen-specific T-cell proliferation, and pro-inflammatory cytokines IFNgamma and IL-17. Quinpramine is thus an immunoregulatory drug that is a candidate pharmaceutical for MS.

ATP-sensitive P2X7 receptors are localized on cells of immunological origin including peripheral macrophages and glial cells in the CNS. Activation of P2X7 receptors leads to rapid changes in intracellular calcium concentrations, release of the pro-inflammatory cytokine IL-1beta, and following prolonged agonist exposure, the formation of cytolytic pores in plasma membranes. Data from gene knockout studies and recently described selective antagonists indicate a role for P2X7 receptor activation in inflammation and pain. While several species selective P2X7 antagonists exist, A-804598 represents a structurally novel, competitive, and selective antagonist that has equivalent high affinity at rat (IC50 = 10 nM), mouse (IC50 = 9 nM) and human (IC50 = 11 nM) P2X7 receptors. A-804598 also potently blocked agonist stimulated release of IL-1beta and Yo-Pro uptake from differentiated THP-1 cells that natively express human P2X7 receptors. A-804598 was tritiated ([3H]A-804598; 8.1Ci/mmol) and utilized to study recombinant rat P2X7 receptors expressed in 1321N1 cells. [3H]A-804598 labeled a single class of high affinity binding sites (Kd=2.4 nM and apparent Bmax=0.56 pmol/mg). No specific binding was observed in untransfected 1321N1 cells. The pharmacological profile for P2X antagonists to inhibit [3H]A-804598 binding correlated with their ability to block functional activation of P2X7 receptors (r=0.95, P