Biomolecular detection techniques are tending to develop in terms of miniaturization, automation, rapidity, sensitivity and low cost, and these techniques are urgently needed as “point of care tests” or “rapid tests” in clinical diagnosis, environmental monitoring and food safety. The rapid developments of microfluidics and nanomaterials, especially their interdisciplinary investigations, offer great opportunities to satisfy these immediate requirements. In this study, graphene oxide (GO) based nanobiosensors were integrated into microfluidic devices for gene-specific detection, combining their unique advantages in specificity, sensitivity, rapidity or miniaturization. The functional structure and fabrication technology of the microfluidic chip was investigated to meet the requirements for performing rapid and label-free detection of a specific gene using the GO based nanobiosensor, aiming at miniaturization, automation, low consumption of reagents and high throughout. In this paper a novel method is proposed for simple and rapid fabrication of microfluidic chips. Furthermore, the assay conditions of gene detection using the nanobiosensor were optimized in the microfluidic device, aiming at efficient specificity, sensitivity and rapidity for gene detection. Finally, the fabricated integrated microfluidic device was used to perform qualitative and quantitative analysis for detection of the target genes. Thus, this study demonstrates that the integrated microfluidic device can offer unique abilities such as rapid detection, portability, simplicity and low-cost detection of biomolecules in clinical diagnosis, environmental monitoring and industrial applications.

Biomolecular detection techniques are tending to develop in terms of miniaturization, automation, rapidity, sensitivity and low cost, and these techniques are urgently needed as “point of care tests” or “rapid tests” in clinical diagnosis, environmental monitoring and food safety. The rapid developments of microfluidics and nanomaterials, especially their interdisciplinary investigations, offer great opportunities to satisfy these immediate requirements. In this study, graphene oxide (GO) based nanobiosensors were integrated into microfluidic devices for gene-specific detection, combining their unique advantages in specificity, sensitivity, rapidity or miniaturization. The functional structure and fabrication technology of the microfluidic chip was investigated to meet the requirements for performing rapid and label-free detection of a specific gene using the GO based nanobiosensor, aiming at miniaturization, automation, low consumption of reagents and high throughout. In this paper a novel method is proposed for simple and rapid fabrication of microfluidic chips. Furthermore, the assay conditions of gene detection using the nanobiosensor were optimized in the microfluidic device, aiming at efficient specificity, sensitivity and rapidity for gene detection. Finally, the fabricated integrated microfluidic device was used to perform qualitative and quantitative analysis for detection of the target genes. Thus, this study demonstrates that the integrated microfluidic device can offer unique abilities such as rapid detection, portability, simplicity and low-cost detection of biomolecules in clinical diagnosis, environmental monitoring and industrial applications.

Pneumatic micro-valve controlled microfluidic chip provides precise fluidic control for cell manipulation. In this paper, a multi-functional microfluidic chip was designed for three separate experiments: 1. Different cell lines were dispensed and cultured; 2. Three transfected SH-SY5Y cells were introduced and treated with methyl-phenyl-pyridinium (MPP+) as drug delivery mode; 3. Specific protection and interaction were observed among cell co-culture after nerve damage. The outcomes revealed the potential and practicability of our entire multi-functional pneumatic chip system on different cell biology applications.

•Develop a new workflow for comparative proteomics across multiple samples.•Validation of the 18O-reference based comparison strategy.•Investigation of a quantitation and identification-decoupled strategy.Comparisons across large numbers of samples are frequently necessary in quantitative proteomics. Many quantitative methods used in proteomics are based on stable isotope labeling, but most of these are only useful for comparing two samples. For up to eight samples, the iTRAQ labeling technique can be used. For greater numbers of samples, the label-free method has been used, but this method was criticized for low reproducibility and accuracy. An ingenious strategy has been introduced, comparing each sample against a 18O-labeled reference sample that was created by pooling equal amounts of all samples. However, it is necessary to use proportion-known protein mixtures to investigate and evaluate this new strategy. Another problem for comparative proteomics of multiple samples is the poor coincidence and reproducibility in protein identification results across samples. In present study, a method combining 18O-reference strategy and a quantitation and identification-decoupled strategy was investigated with proportion-known protein mixtures. The results obviously demonstrated that the 18O-reference strategy had greater accuracy and reliability than other previously used comparison methods based on transferring comparison or label-free strategies. By the decoupling strategy, the quantification data acquired by LC-MS and the identification data acquired by LC-MS/MS are matched and correlated to identify differential expressed proteins, according to retention time and accurate mass. This strategy made protein identification possible for all samples using a single pooled sample, and therefore gave a good reproducibility in protein identification across multiple samples, and allowed for optimizing peptide identification separately so as to identify more proteins.Download high-res image (221KB)Download full-size image

Mitochondria are not only the main source of energy in cells but also produce reactive oxygen species (ROS), which result in oxidative stress when in space. This oxidative stress is responsible for energy imbalances and cellular damage. In this study, a rat tail suspension model was used in individual experiments for 7 and 21 days to explore the effect of simulated microgravity (SM) on metabolic proteins in the hippocampus, a vital brain region involved in learning, memory, and navigation. A comparative 18O-labeled quantitative proteomic strategy was used to observe the differential expression of metabolic proteins. Forty-two and sixty-seven mitochondrial metabolic proteins were differentially expressed after 21 and 7 days of SM, respectively. Mitochondrial Complex I, III, and IV, isocitrate dehydrogenase and malate dehydrogenase were down-regulated. Moreover, DJ-1 and peroxiredoxin 6, which defend against oxidative damage, were up-regulated in the hippocampus. Western blot analysis of proteins DJ-1 and COX 5A confirmed the mass spectrometry results. Despite these changes in mitochondrial protein expression, no obvious cell apoptosis was observed after 21 days of SM. The results of this study indicate that the oxidative stress induced by SM has profound effects on metabolic proteins.

The study of the composition of microsome proteins/complexes/interactions in adipocytes provides useful information for researchers related to energy metabolism disorders. The native gel coupled with LC-ESI-QTOF approach was employed here for separating protein complexes. We found a series of proteins functionally clustered in biological processes of protein metabolism, cellular carbohydrate catabolism, response to stimulus and wounding, macromolecular complex subunit organization, positive regulation of molecular function, regulation of programmed cell death and biomolecule transport. According to clustering of proteins’ electrophoresis profiles across native gel fractions and bioinformatics data retrieval, protein complexes/interactions involved in protein metabolism, cellular carbohydrate catabolism, macromolecular complex subunit organization and biomolecule transport were identified. Besides, the results also revealed some functional linkages, which may provide useful information for discovering previously unknown interactions. The interaction between SSAO and ALDH2 was verified by co-immunoprecipitation. The native gel combining mass spectrometry approach appeared to be a useful tool for investigating microsome proteins and complexes to complement the traditional electrophoresis approaches. The native gel strategy together with our findings should facilitate future studies of the composition of rat adipocyte microsome protein complexes under different conditions.

Norland Optical Adhesive 81 (NOA81) is an excellent material for a microfluidic chip, but this chip has been rarely used in the field of gene amplification due to the inhibition effect on the polymerase chain reaction (PCR). In this study, a NOA81 PCR chip (NP-chip) was developed with a simple, fast fabrication method. To overcome this inhibition effect, a simple bovine serum albumin (BSA) coating method was introduced and the BSA coated pH and concentration were optimized for a better gene amplification effect. The PCR results of HLA-DRB1 indicated that the BSA coating method could greatly improve the PCR efficiency on the NP-chip. Moreover, two kinds of PCRs were performed on the NP-chip to validate the effectiveness of the BSA coating method. (i) Gene mutation of anti EB variable region gene (AEB-HC6-6) was observed under the conditions of cobalt radiation. (ii) Short Tandem Repeats (STRs) PCR was performed on the NP-chip as the multiple-primers PCR. The results showed that the NP-chip with BSA coating was able to successfully realize single and multiple primer gene amplification. With further improvement of the precise temperature control, this kind of NP-chip would be widely applied in gene amplification and promote the development of the miniature gene amplification device in point of care testing (PCOT), forensic detection, etc.

Microfluidic chip has been applied in various biological fields owing to its low-consumption of reagents, high throughput, fluidic controllability and integrity. The well-designed microscale intermediary is also ideal for the study of cell biology. Particularly, microfluidic chip is helpful for better understanding cell-cell interactions. A general survey of recent publications would help to generalize the designs of the co-culture chips with different features. With ingenious and combinational utilization, the chips facilitate the implementation of some special coculture models that are highly concerned in a different spatial and temporal way.

Selective capture of microorganisms is an effective and rapid technique for microbe detection. In this article, we obtained aptamers targeting live bacterial cells E. coli as recognition and capture elements by using a whole-cell based aptamer selection, and integrated the isolated aptamers into a microfluidic device for selective capture of microorganisms. Eight rounds of selection were performed; the specificity and affinity of aptamers to the target bacterial cells were also characterized using flow cytometric analysis. A total of 40 aptamer sequences were obtained, and the apparent dissociation constant Kd of the representative aptamer displaying the highest binding affinity was estimated to be 24.8 ± 2.7 nM. After sequencing of the pool showing the highest binding affinity to E. coli, a DNA sequence of high affinity to the bacteria was integrated into the microfluidic device as recognition and capture elements for selective capture of microorganisms. The results demonstrated that the aptamer-based microfluidic device was able to capture E. coli selectively to distinguish different species of bacteria. This investigation is envisaged to provide a new avenue for the development of lab-on-chip devices for selective capture of microorganisms, which could be beneficial for portable, rapid and economic microbial detection in food safety, environmental monitoring and medical diagnosis.

Despite extensive efforts to study the inflammatory process in the central nervous system of Parkinson’s disease (PD) patients, little is known about the role of peripheral blood mononuclear cells (PBMCs) in PD. In the present study, we used an in vitro co-culture system to study the role of the human monocyte cell line THP-1 in medium conditioned by the neuroblastoma cell line SH-SY5Y damaged with the endogenous neurotoxin 1-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline (Salsolinol, Sal) in co-culture with the human glioma cell line U87. For this purpose, SH-SY5Y and U87 co-cultures were treated with Sal, and this conditioned medium containing mediators, including the potential effector CCL2, was isolated and applied to THP-1 cells. This treatment resulted in approximately 19 % cell proliferation as well as activation of mTOR and induction of phosphorylated 4E-BP1, S6K1, PI3K, and AKT proteins. Treatment with rapamycin, an mTOR inhibitor, attenuated the proliferation of THP-1 cells. U87 glial cells were essential for this as medium conditioned without them had no effect on THP-1 cells. These results suggest a positive effect of THP-1 cells on Sal-induced neurotoxicity in a cellular model of PD and this is likely mediated by the enhancement of cell proliferation through activation of the mTOR signaling pathway. Hence, PBMCs and their mTOR signaling pathway could be of therapeutic benefit in treating the endogenous neurotoxin-induced neuroinflammation in PD.

Recent studies provide the evidence that indirect effects of radiation could lead to neuronal cells death but underlying mechanism is not completely understood. On the other hand astroglial cells are known to protect neuronal cells against stress conditions in vivo and invitro. Yet, the fate of neuronal cells and the neuroprotective effect of coculture system (with glial cells) in response to indirect radiation exposure remain rarely discussed. Here, we purpose that the indirect effect of radiation may induce DNA damage by cell cycle arrest and receptor mediated apoptotic cascade which lead to apoptotic death of neuronal SH-SY5Y cells. We also hypothesized that coculture (with glial U87) may relieved the neuronal SH-SY5Y cells from toxicity of indirect effects radiation by reducing DNA damage and expression of apoptotic proteins in vitro. In the present study irradiated cell conditioned medium (ICCM) was used as source of indirect effect of radiation. Neuronal SH-SY5Y cells were exposed to ICCM with and without coculture with (glial U87) in transwell coculture system respectively. Various endpoints such as, cell survival number assay, Annexin V/PI assay, cell cycle analysis by flow cytometer, mRNA level of Fas receptor by q RT-PCR, expression of key apoptotic proteins by western blot and estimation of neurotrophic factors by ELISA method were analyzed into neuronal SH-SY5Y cells with and without co culture after ICCM exposure respectively. We found that ICCM induced DNA damage in neuronal SH-SY5Y cells by significant increase in cell cycle arrest at S-phase (***P < 0.001) which was further supported by over expression of P53 protein (**P < 0.01). While coculture (with glial U87), significantly reduced the ICCM induced cell cycle arrest and expression of P53 (###P < 0.001) neuronal SH-SY5Y cells. Further investigation of the underlying apoptotic mechanism revealed that in coculture system; ICCM induced elevated level of FAS mRNA level was significantly reduced (###P < 0.001) in neuronal SH-SY5Y cells which was followed by significant reduction in expression of key apoptotic protein i.e., FADD (###P < 0.001), caspase-8 (###P < 0.001), and cleaved caspase-3 (###P < 0.001) as compare to neuronal SH-SY5Y cells which received ICCM without coculture. Intriguingly, concentration of neurotrophic factors such as, GDNF and BDNF were significantly increased (###P < 0.001) in neuronal SH-SY5Y after coculture (with glial U87) cells. Hence, these findings infer that the receptor mediated pathway could be the one way through which indirect effects of radiation cause neurotoxicity. However, in co-cultures system (with glial U87) neuronal SH-SY5Y depicts remarkable resistance against ICCM induced neurotoxicity.

Based on the successful graft of P(N-isopropylacrylamide-4-vinylphenylboronic acid) [P(NIPAAm-co-VPBA)] on glass substrates by surface-initiated atom transfer radical polymerization, a microfludic chip with the performance of thermoresponsive boronate affinity was fabricated and was used for capture–release of the cis-diol biomolecule adenosine. The copolymers modified successfully on the surface of glass substrate were characterized by Fourier-transform infrared spectroscopy and X-ray photoelectron spectroscopy. The results demonstrated that, by simply adjusting the temperature from 4 to 55 °C, the microchip grafted with P(NIPAAm-co-VPBA) was specific to the cis-diol-containing molecules, and can selectively and quickly realize the capture and release of the cis-diol biomolecule adenosine from the mixture of adenosine and deoxyadenosine, which was validated by high performance liquid chromatography/electronic spray ion–ion trap mass spectrometry (HPLC/ESI–ion trap MS). Moreover, less than 10 min was taken from injection to the collection of the sample at the end of the channel. With further development, the P(NIPAAm-co-VPBA)-grafted microchip with high selectivity and repeatability may be chosen as a promising tool for the enrichment of the cis-diol-containing biomolecules, such as glycoproteins, carbohydrates, glycosylated peptides, nucleosides, etc.

The enzyme-linked immunesorbent assay (ELISA) is most frequently used for the determination of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). However, this technique is time-consuming and requires a substantial sample volume. We have developed (a) a new kind of colloidal gold-based immunochromatographic strip, and (b) a colloidal gold-based immunochromatographic microchip for simultaneous, rapid, and visual detection of IL-6 and TNF-α. The lowest limit of detection is 62.5 ng mL−1 for IL-6, and 30 ng mL−1 for TNF-α. Both the strip method and the microchip method are highly specific, user-friendly, and can be read out without the need for instrumentation.

We report a switchable chromatographic material for the selective capture-release of proteins with simpler adjustment of the temperature instead of the conventional need to change the mobile phase. This novel strategy greatly increased the number of LC/MS/MS-identified proteins from human serum, as well as their scores and coverage ratios.

The CP4 EPSPS gene is widely used in herbicide-tolerant crops/plants all over the world. In this study, a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated to quantify the amount of CP4 EPSPS expression in Nicotiana tabacum leaves. The quantification of protein was used to measure the unique peptides of the CP4 EPSPS protein. Two peptides unique to CP4 EPSPS were synthesized and labelled in H218O to give 18O stable isotope labelled peptides which served as internal standards. The validated method resulted in good specificity and linearity. The intra- and inter-day precisions and accuracy for all samples were satisfactory. The results demonstrated that the novel method was sensitive and selective to quantify CP4 EPSPS in the crude extract without time-consuming pre-separation or purification procedures.

The main purpose of this work was to develop an assay as a means of quality control in milk and dairy products. As specific and high abundant proteins in milk and diary products, casein and β-lactoglobulin were chosen as the targets, and an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for determination of the total concentration of casein and β-lactoglobulin. The detection limit of the assay was 79.07 ng/mL, and a working range of the calibration curve was from 140.6 to 36,000 ng/mL. The intra-assay and inter-assay coefficients of variations were 4.7 % and 6.5 %, respectively. The recoveries ranged from 94.1 % to 106.6 %. The developed ELISA assay was used to detect the total concentration of casein and β-lactoglobulin in liquid milk and milk powder. The results indicated that the developed assay was very specific, and the addition of nonprotein nitrogen (such as melamine, cyanuric acid, and urea) and vegetable protein did not affect the detection. The good performance of the assay makes it suitable for use in the quality control of milk and dairy products.

Reactive oxygen species (ROS) are produced due to oxidative stress which has wide range of affiliation with different diseases including cancer, heart failure, diabetes and neurodegenerative diseases like Alzheimer’s disease, Parkinson’s disease, ischemic and hemorrhagic diseases. This study shows the involvement of BNIP3 in the amplification of metabolic pathways related to cellular quality control and cellular self defence mechanism in the form of autophagy. We used conventional methods to induce autophagy by treating the cells with H2O2. MTT assay was performed to observe the cellular viability in stressed condition. MDC staining was carried out for detection of autophagosomes formation which confirmed the autophagy. Furthermore, expression of BNIP3 was validated by western blot analysis with LC3 antibody. From these results it is clear that BNIP3 plays a key role in defence mechanism by removing the misfolded proteins through autophagy. These results enhance the practical application of BNIP3 in neuroblastoma cells and are helpful in reducing the chances of neurodegenerative diseases. Although, the exact mode of action is still unknown but these findings unveil a molecular mechanism for the role of autophagy in cell death and provide insight into complex relationship between ROS and non-apoptotic programmed cell death.

Recent studies have evaluated the role of direct radiation exposure in neurodegenerative disorders; however, association among indirect effects of radiation and neurodegenerative diseases remains rarely discussed. The objective of this study was to estimate the relative risk of neurodegeneration due to direct and indirect effects of radiation. 60Co gamma ray was used as source of direct radiation whereas irradiated cell conditioned medium (ICCM) was used to mimic the indirect effect of radiation. To determine the potency of ICCM to inhibit neuronal cells survival colony forming assay was performed. The role of ICCM to induce apoptosis in neuronal SH-SY5Y cells was estimated by TUNEL assay and Annexin V/PI assay. Level of oxidative stress and the concentration of inflammatory cytokines after exposing to direct radiation and ICCM were evaluated by ELISA method. Expression of key apoptotic protein following direct and indirect radiation exposure was investigated by western blot technique. Experimental data manifest that ICCM account loss of cell survival and increase apoptotic induction in neuronal SH-SY5Y cells that was dependent on time and dose. Moreover, ICCM stimulate significant release of inflammatory cytokines i.e., tumor necrosis factor TNF-alpha (P < 0.01), Interleukin-1 (IL-1, P < 0.001), and Interleukin-6 (IL-6, P < 0.001) in neuronal SH-SY5Y cells and elevate the level of oxidative stress (MDA, P < 0.01). Up-regulation of key apoptotic protein expression i.e., Bax, Bid, cytochrome C, caspase-8 and caspase-3 confirms the toxicity of ICCM to neuronal cells. This study provides the evidence that indirect effect of radiation can be as much damaging to neuronal cells as direct radiation exposure can be. Hence, more focused research on estimation risks of indirect effect of radiation to CNS at molecular level may help to reduce the uncertainty about cure and cause of several neurodegenerative disorders.

Autophagy is a catabolic pathway that regulates homeostasis in cells. It is an exceptional pathway of membrane trafficking. Autophagy is characterized by the formation of double-membrane vesicles; autophagosomes that are responsible for delivering damaged organelle and extra proteins to lysosome for recycling. A series of actions including environmental and genetic factors are responsible for induction of autophagy. In the past few decades, the research on autophagy has been immensely expanded because it is a vital process in maintaining cellular balance as well as deeply connected with pathogenesis of a number of diseases. The aim of this review is to present an overview of modern work on autophagy and highlight some essential genetic role in the induction of autophagy. There is an emerging need to identify, quantify, and manipulate the pathway of autophagy, due to its close relationship with a variety of developmental pathways and functions especially in cancer, diabetes, neurodegenerative disorders, and infectious diseases.

The post-digestion of 18O labeling is widely used in comparative proteomics for the relative quantification of proteins and peptides. Application and precise quantitative analysis are hindered, since 18O labeling is pH sensitive and has back-exchange. Herein, in a study of peptides derived from BSA (bovine serum albumin), we demonstrated through a detailed evaluation that removal of soluble trypsin by ultrafiltration prevented back-exchange and effectively enhanced the stability of 18O-labeled peptides under off-gel separation even without trypsin inhibitor. After ultrafiltration, the 18O labeling efficiency was 95.8 ± 2.3% under off-gel separation. In addition, these peptides had a relative high, approximately 80% recovery and less than 5% 16O/18O ratio variation through ultrafiltration, indicating no apparent effect on quantification. Hence, the useful and economical method presented here effectively inhibited back-exchange in off-gel separation and might enable further applications towards large-scale biomarker discovery.

Ionizing radiation (IR) is of clinical importance for glioblastoma therapy; however, the recurrence of glioma characterized by radiation resistance remains a therapeutic challenge. Research on irradiation-induced transcription in glioblastomas can contribute to the understanding of radioresistance mechanisms. In this study, by using the total mRNA sequencing (RNA-seq) analysis, we assayed the global gene expression in a human glioma cell line U251 MG at various time points after exposure to a growth arrest dose of γ-rays. We identified 1656 genes with obvious changes at the transcriptional level in response to irradiation, and these genes were dynamically enriched in various biological processes or pathways, including cell cycle arrest, DNA replication, DNA repair and apoptosis. Interestingly, the results showed that cell death was not induced even many proapoptotic molecules, including death receptor 5 (DR5) and caspases were activated after radiation. The RNA-seq data analysis further revealed that both proapoptosis and antiapoptosis genes were affected by irradiation. Namely, most proapoptosis genes were early continually responsive, whereas antiapoptosis genes were responsive at later stages. Moreover, HMGB1, HMGB2 and TOP2A involved in the positive regulation of DNA fragmentation during apoptosis showed early continual downregulation due to irradiation. Furthermore, targeting of the TRAIL/DR5 pathway after irradiation led to significant apoptotic cell death, accompanied by the recovered gene expression of HMGB1, HMGB2 and TOP2A. Taken together, these results revealed that inactivation of proapoptotic signaling molecules in the nucleus and late activation of antiapoptotic genes may contribute to the radioresistance of gliomas. Overall, this study provided novel insights into not only the underlying mechanisms of radioresistance in glioblastomas but also the screening of multiple targets for radiotherapy.

Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline), the endogenous dopamine-derived catechol isoquinolines whose structure is similar with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), may be a possible candidate of dopaminergic neurotoxins to elicit Parkinson’s disease (PD). Catechol isoquinolines can selectively target dopaminergic neurons, leading to dopaminergic neuronal death. However, the formation and nosogenesis of these toxins remains unclear. Salsolinol synthase is a novel enzyme which condensate dopamine and acetaldehyde to salsolinol. It is the first key enzyme in the metabolic pathway of catechol isoquinolines which directly affects salsolinol and its derivative metabolism in vivo. It is also one kind of Pictet-Spenglerase, which has been little studied and need more characterization. PC12 cells and rat brains were performed to illustrate the existence of salsolinol synthase in our study. The results indicate that salsolinol synthase is a low molecular weight protein, showing enhanced activity with increase in dopamine concentration. It is suggested that salsolinol synthase is sensitive to strong acid and stable to high-temperature. In this research, existence of salsolinol synthase was confirmed in vivo, and also provided some new evidences to elucidate the endogenous catechol isoquinoline neurotoxin substances involved in the pathogenesis of PD.

A novel and simple coating method was developed by coating bovine serum albumin (BSA) onto the inner surface of a fused-silica capillary, to avoid the adsorption of analytes during CE. The advantage presented here was that the coating process is more simple, fast, stable, and reproducible. The coated capillary avoided the adsorption of analytes onto the inner surface of a fused-silica capillary and might be a promising candidate for separation of complex biological samples with further development. Meanwhile, the efficiencies of the coated capillary were evaluated by EOF, chromatographic peak shape, and theoretical plate number (N m−1) of RNase A. The optimal coating conditions were obtained from the results. The pH value of coating buffer PB was 4.2, the standing time was 12 h at 4 °C, and the coating concentration of BSA was 1.5 mg mL−1. The stability of the coating on the inner wall of the capillary and the reproducibility of the coated capillaries were good. The theoretical plate number values of RNase A were over 1.3 × 105 (N m−1) in the coated capillary. After successive electrophoresis for 48 h using the coated capillary, the RSD values of EOF and the theoretical plate number were 4.14 % and 9.14 %, respectively. In addition, the RSD values of EOF and the theoretical plate number (N m−1) in the coated capillaries were 13.19 % and 8.96 %, respectively. Finally, the coated capillary was successfully applied to separate the mixture of four basic proteins (RNase A, lysozyme, trypsin and myoglobin).

We have recently reported the new concept of temperature-responsive capillary electrochromatography (CEC) using a poly(N-isopropylacrylamide)-grafted capillary column. In this study, temperature-responsive CEC separation of six clinical drugs including thymopentin and octreotide acetate (two peptides), homatropinum and oxazepam, benazepril hydrochloridec and amlodipine acetate was conducted to evaluate the separation efficiency of the CEC column. For comparison purposes, a bare capillary column was used for the control experiments. It was found that the resolutions and migration times of thymopentin and octreotide acetate, homatropinum and oxazepam on the modified column were much larger than those on the bare column. Enhanced resolving power mainly resulted from the interactions between analytes and PNIPAAm-grafted capillary surfaces. Therefore, the modification with temperature-responsive PNIPAAm was evaluated to achieve a better separation of the four analytes. The resolutions of benazepril hydrochloridec and amlodipine acetate on the modified column were smaller than those on the bare column because peak broadening and tailing were also obtained, although the differences in migration time between them were much larger. Furthermore, significant changes in the resolution and migration time were only observed on the modified column around the lower critical solution temperature of PNIPAAm, demonstrating its temperature-responsive property.

A new thermoresponsive boronate affinity chromatographic material, P(N-isopropylacrylamide-4-vinylphenylboronic acid) P(NIPAAm-co-VPBA) grafted silica featuring a thermally switchable property, was prepared and successfully employed for the specific capture–release of cis-diol biomolecules with simpler adjustment to the temperature instead of the conventional need to change the pH of the mobile phase.

In this study, a simple, fast, and reliable method to fabricate a micro free-flow electrophoresis (μFFE) device on glass is presented. The two-dimensional depth channel in the chip was easily achieved by using a photocurable monomer (NOA 81) that served as the bonding material. In such a geometrical structure (two-dimensional depth channel), the effect of fluid behavior on the separation efficiency of micro free-flow zone electrophoresis (μFFZE) was simulated. The results of numerical simulation indicate that the pressure at the inlets may play an important role in the separation performance. Under the optimum separation conditions, four FITC-labeled amino acids were well separated, indicating the validity of the performance of the chip. Since the chip was fabricated by organic polymer bonding, it was easily recyclable through a simple re-fabrication process. The reproducibility of results from these recycling re-fabrication chips was investigated. The RSD of the resolution between FITC-L-glycine and FITC-L-phenylalanine was 5.3%. Furthermore, three FITC-labeled proteins were successfully separated with the resolution of 2.2 and 5.46, respectively, by using the coating of neutral liposome.

Cry1Ab is one of the most common Bacillus thuringiensis (Bt) proteins in genetically modified crops, which exhibits strong resistance against insect pests. In the present study, a sensitive and precise liquid chromatography stable isotope dilution multiple reaction monitoring tandem mass spectrometry (LC-SID-MRM-MS) assay was developed and validated to quantify the amount of Cry1Ab expression in transgenic maize leaves. The measurement of protein was converted to measurement of unique peptides to Cry1Ab protein. Two peptides unique to Cry1Ab were synthesized and labeled in H218O to generate 18O stable isotope peptides as internal standards. The validated method obtained superior specificity and good linearity. And the inter- and intra-day precision and accuracy for all samples were satisfactory. The results demonstrated Cry1Ab protein was 31.7 ± 4.1 μg g−1 dry weight in Bt-176 transgenic maize leaves. It proved that the novel LC-SID-MRM-MS method was sensitive and selective to quantify Cry1Ab in the crude extract without time-consuming pre-separation or purification procedures.

Salsolinol (Sal) and N-methylsalsolinol (NMSal) are endogenous neurotoxins in the brain, which are the key causative agents of the death of dopaminergic neurons. Due to their strong polarity, they have weak retention and are difficult to quantify using the C18 column. In the present study, a sensitive and selective LC/MS method for the determination of Sal and NMSal was developed and validated using a novel pentafluorophenylpropyl column performed on high performance liquid chromatography electrospray ionisation triple quadrupole. The calibration curve was linear over the range of 0.098–25 nM and 3.91–250 nM for Sal and NMSal, respectively. The lower limit of quantitation is 0.098 nM and 3.91 nM for Sal and NMSal. The inter-day and intra-day precision and accuracy for all samples were acceptable. Furthermore, the assay has also been applied for the determination of Sal and NMSal in rat brain.

Semicarbazide-sensitive amine oxidase (SSAO) is present in various mammalian tissues and in blood plasma. Elevation of SSAO activity is linked to vascular disorders associated with pathological conditions such as diabetic complications, heart failure and vascular dementia. In the present paper, a high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) method is developed to determine the SSAO activity. Methylamine is used as physiological substrate for the enzyme activity assay of SSAO. Formaldehyde, the enzymatic reaction product, was derivatized by dopamine, and separated by a silica-based pentafluorophenyl column. The calibration curve was linear over the range of 0.03–4.00 μM of formaldehyde concentration, with 0.03 μM the lower limit of quantification (LOQ). The inter-day and intra-day precisions ranged from 2.2% to 7.9% and 4.4% to 9.2% respectively for each quality control sample of formaldehyde at 0.06, 0.50, and 2.00 μM. The accuracy ranged from 96.0% to 111.5%. The limit of detection (LOD) for serum SSAO activity was 1 nmol h−1 mg−1 protein. The method was successfully applied for the determination of SSAO activity in both mouse serum and rat tissues. SSAO activity of the serum in diabetes mice was significantly increased compared with the control and was inhibited by 2-bromoethylamine in vivo. The levels of SSAO activity in rat lung, spleen, aorta, kidney and brain tissues were significantly decreased by semicarbazide in vitro.

An efficient chemoenzymatic route for the synthesis of enantiopure (R)-salsolinol through the use of Candida antarctica lipase A (CALA) was developed. Different parameters, including the acyl agent, the solvent, the temperature, and the amount of enzyme and substrates used, were investigated in order to establish the optimal reaction conditions for the enzymatic kinetic resolution of secondary amines. The combination of CALA with phenyl allyl carbonates in toluene allowed the isolation of amines with high conversion (50%) and ee (98%) values.An efficient chemoenzymatic route for the synthesis of (R)-salsolinol using phenyl allylcarbonates as an acyl donor. This approach allowed the isolation of amines with excellent enantioselectivity (ee 98%) and high conversion (50%).(R)-1-Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolineC10H13NO2ee = 98% from HPLC[α]D25=+30.2 (c 1, MeOH)Source of chirality: kinetic resolutionAbsolute configuration: (R)(R)-1-Methyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolineC12H17NO2ee = 98% from HPLC[α]D25=+59.2 (c 1, EtOH)Source of chirality: kinetic resolutionAbsolute configuration: (R)

For the most common neurodegenerative disorders, there is no simple, obvious, and effective biomarker for disease identification. Plasma biomarkers of neurodegenerative disorders should play an important role in early detection and diagnosis of diseases. The catechol isoquinoline compounds are considered as possible neurotoxins involved in the pathogenesis of Parkinson’s disease (PD). These catechol isoquinolines and enzymes involved in their biosynthesis and metabolism may be endogenous factors in the pathogenesis of PD. As we know, there exists a salsolinol N-methyltransferase (SNMT) in human brain and lymphocyte. In normal postmortem human brain regions, frontal cortex, caudate, putamen, and thalamus, the SNMT activity was significantly higher than the control, and was well relevant to PD. In parkinsonian lymphocytes, the SNMT activity was much higher than in control patients. In addition, there was significant positive correlation between the SNMT activity in the lymphocyte and the level of N-methylsalsolinol in cerebrospinal fluid from the same untreated PD patients. Mass spectrometry–based biomarker discovery, undoubtedly, is still in its early stage of infancy. However, in light of the potential of this technology to provide deep coverage of the body fluid proteomes, it will certainly consolidate its role in development molecular medicine into clinical practice.

Parkinson’s disease (PD) arises from the loss of dopaminergic neurons in the substantia nigra. 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) is well known to cause Parkinsonism in humans with neurotoxicity specific for dopaminergic neurons. The experience with MPTP supports the hypothesis that endogenous or xenobiotic neurotoxins are involved in the pathogenesis of PD in humans. In our study, 1-acetyl-6, 7-dihydroxy-1, 2, 3, 4-tetrahydro-isoquinoline (ADTIQ), a novel compound, was found in frozen human brain tissues. The formation of ADTIQ was demonstrated using dopamine and methylglyoxal under physiological conditions. Methylglyoxal is a by-product of glycolysis. ADTIQ and its precursors, dopamine and methylglyoxal, were detected in different regions of frozen human brains such as the substantia nigra, caudate nucleus, putamen, frontal cortex, and the cerebellum. A significant difference in ADTIQ levels between control and Parkinson’s patients was found; for instance, the ADTIQ level in putamen of PD patients was 0.76 ± 0.27 nmol/g compared to 0.10 ± 0.01 nmol/g in control. Our results might indicate that ADTIQ is possibly related to Parkinson’s disease.

High performance liquid chromatography diode array detector electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) was utilized to separate and identify flavone glycosides and aglycones in Cephalotaxus sinensis, a natural plant with an anti-hyperglycemic effect. The HPLC-DAD-ESI-MS system could provide a great amount of information on the structure of flavone aglycones and glycosides in C. sinensis without time-consuming pre-separation or purification procedures. A total of seven flavone glycosides and two flavone aglycones were unambiguously identified based on their retention times, ultraviolet spectra, molecular weights and MS/MS spectra. It's interesting that five 5-O-flavone glycosides were found in C. sinensis since 5-O-flavone glycosides are rare and difficult to form in the natural plant. Among them, quercitrin and apigenin-5-O-β-D-glucopyranoside were isolated from this genus for the first time. A novel flavone glycoside, chryoseriol-5-O-[α-L-rhamnopyranosyl-(1→4)-β-D-6-O-acetylglucopyranoside], was isolated for the first time and identified by the ultraviolet spectrum, infrared spectrum, mass spectrum and nuclear magnetic resonance spectrum. In addition, the proposed fragment pathway of the novel flavone glycoside was concluded in the negative ionization mode by HPLC-ESI-MS/MS.

In previous study, we demonstrated the hypoglycemic effect of aqueous extract of Belamcanda chinensis leaves in rats. Here, we separated the aqueous extract of B. chinensis leaves and investigated the spectrum-effect relationships between HPLC chromatograms and hypoglycemic activities of different isolates from B. chinensis leaf extract. Sequential solvent extraction with petroleum ether, chloroform, acetic ester and n-butanol provided several isolates showing similar hypoglycemic activities, making it difficult to discriminate the active fractions. Stepwise elution through HP20 macroporous resin by water, 40% and 95% ethanol provided isolates with distinct hypoglycemic activities, representing a simple, rapid and efficient preparative separation method. Combination of HPLC chromatogram and pharmacological effect targeted a hypoglycemic activity-related region in HPLC chromatogram. Each peak in this region was analyzed by UV spectrum scan. Most of them were flavonoids in which tectoridin and swertisin were known flavonoids with anti-diabetic activities. In together, this work provides a general model of combination of HPLC chromatography and pharmacological effect to study the spectrum-effect relationships of aqueous extract from B. chinensis leaves, which can be used to find principle components of B. chinensis on pharmacological activity.

Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; Sal) is structurally similar to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which is supposed to have a role in the development of Parkinson-like syndrome in both human and non-human subjects. In the human brain, the amount of (R)-enantiomer of Sal is much higher than (S)-enantiomer, suggesting that a putative enzyme may participate in the synthesis of (R)-salsolinol, called (R)-salsolinol synthase. In this study, the (R)-salsolinol synthase activity in the condensation of dopamine and acetaldehyde was investigated in the crude extracts from the brains of Sprague Dawley rats. Identification of the enzymatic reaction products and enzyme activity detection were achieved by HPLC-electrochemical detection. The discovery of this enzyme activity in rat’s brain indicates the natural existence of (R)-salsolinol synthase in the brains of humans and rats, and it is distributed in most brain regions of rat with higher activity in soluble proteins extracted from striatum and substantia nigra.

Multiple system atrophy (MSA) is a progressive neurodegenerative disease characterized by glial cytoplasmic inclusions containing insoluble α-synuclein. Since Ca2+ plays an important role in cell degeneration, [Ca2+]i in α-synuclein-overexpressed human glioma cells was analyzed by Fura-2 fluorometry. Overexpression of α-synuclein increased the basal level of [Ca2+]i, and a higher Ca2+ response to hydrogen peroxide was further observed. The effect that α-synuclein overexpression caused U251 cells to be more vulnerable to hydrogen peroxide was eliminated by Ca2+ chelator BAPTA-AM or transient receptor potential channels blocker SKF 96365 but not by L-type Ca2+ channel blocker nimodipine. These findings suggest that the dysregulation of cellular Ca2+ homeostasis caused by α-synuclein under oxidative stress may contribute to the glial cell death in MSA.

In this study, a new strategy for screening of enzyme inhibitors based on the interaction between enzyme and substrate was established. The bioactive proteoliposome reconstituted by monoamine oxidase (MAO) and liposome was added in the running buffers of capillary electrophoresis (CE) as pseudostationary phase to simulate the interaction between MAO and its substrate kynuramine (Kyn). The results showed that the relative migration time ratio (RMTR) of Kyn decreased obviously with increase of the proteoliposome, indicating strong interaction between proteoliposome and Kyn. After adding MAO inhibitors into the running buffers containing proteoliposome, the decline of the RMTR value of Kyn became slower. This occurred because MAO was inhibited to some degree by the inhibitors, weakening the interaction between MAO and Kyn. However, adding compounds which do not inhibit MAO activity into the running buffers did not affect the RMTR of Kyn obviously. The results obtained demonstrate that the method developed could characterize the inhibition of MAO inhibitors. Compared with traditional inhibitor screening methods, it does not need incubation outside the column to complete the enzyme catalytic reaction, so analysis is faster and less sample is consumed. With further development, the method might be chosen as a useful tool for screening of membrane protein inhibitors and identification of proteins associated with various diseases.

An offline two-dimensional RP/RPLC system was developed for the separation of components in Dracaena cochinchinensis (Lour.) S.C.Chen xylem containing a resin that is an important raw material in Chinese traditional medicine. In this separation method, a CN-column was used in the first dimension and a C18-column in the second dimension. Fractions collected from the CN-column were resolved into many additional components, which indicated that the two-dimensional RP/RPLC system based on the CN- and C18-columns is orthogonal. The method provides higher peak capacity and better resolution. It is suitable for the analysis of complex samples such as those found in traditional Chinese medicines.

The post-digestion 18O labeling method decouples protein digestion and peptide labeling. This method allows labeling conditions to be optimized separately and increases labeling efficiency. A common method for protein denaturation in proteomics is the use of urea. Though some previous studies have used urea-based protein denaturation before post-digestion 18O labeling, the optimal 18O labeling conditions in this case have not been yet reported. Present study investigated the effects of urea concentration and pH on the labeling efficiency and obtained an optimized protocol. It was demonstrated that urea inhibited 18O incorporation depending on concentration. However, a urea concentration between 1 and 2 M had minimal effects on labeling. It was also demonstrated that the use of FA to quench the digestion reaction severely affected the labeling efficiency. This study revealed the reason why previous studies gave different optimal pH for labeling. They neglect the effects of different digestion conditions on the labeling conditions. Excellent labeling quality was obtained at the optimized conditions using urea 1–2 M and pH 4.5, 98.4 ± 1.9% for a standard protein mixture and 97.2 ± 6.2% for a complex biological sample. For a 1:1 mixture analysis of the 16O- and 18O-labeled peptides from the same protein sample, the average abundance ratios reached 1.05 ± 0.31, demonstrating a good quantitation quality at the optimized conditions. This work will benefit other researchers who pair urea-based protein denaturation with a post-digestion 18O labeling method.

In the present study, a new enzymatic microreactor is developed by immobilizing trypsin on the matrix-HILIC (hydrophilic interaction chromatography). The influences of different types of buffer solutions (borate, Tris·HCl, and phosphate), their concentrations and pH values on the immobilizing rate and the activity of immobilized trypsin were investigated. The optimal condition for off-line immobilization of trypsin on matix-HILIC was Tris·HCl buffer (20 mmol/L, pH 8.0), and then, under the optimal condition, an enzymatic microreactor was prepared by trypsin dynamically immobilized on HILIC matrix which was packed in a Peeksil column. It can be found that the microreactor possessed high hydrolytic activity according to the hydrolysis results of N-α-benzoyl-DL-arginine P-nitroanilide (BAPNA), a substrate of trypsin. Moreover, bovine serum albumin (BSA), cytochrome C and myoglobin as standard proteins were chosen to characterize the enzymolysis of the microreactor demonstrating the high enzymolysis efficiency by this enzymatic microreactor. Besides, the deactivated trypsin can be easily eluted from the matrix-HILIC to re-immobilize trysin, which is highly cost-effective in the experiment. All the results show that the new enzymatic microreactor associated with HPLC-MS techniques can realize online enzymolysis of proteins, and the time to analyze and identify the proteins is greatly shortened.

Peak compression technique based on the difference of the solute migration velocity in two different mobile phases was described theoretically and confirmed using benzaldehyde and 4- hydroxyquinoline (4-HQ) as model compounds. After peak compression, the peak compression factors (the ratio of peak width at half-height under non-compression and that under compression condition) of benzaldehyde and 4-HQ were 0.19 and 0.13, respectively. By this application of the peak compression technique to the mixture, both enhanced peak height and good separation were obtained in one run cycle. This peak compression technique was introduced to determine benzaldehyde from semicarbazide-sensitive amine oxidase-catalyzed enzymetic reaction in order to illustrate the applicability of this technique to the real sample. As a result, the peak was compressed effectively, and 4.94-fold, 19.3-fold and 5.74-fold enhancement in peak height, plate number and signal to noise ratio were also achieved, respectively.

Monolayer of 3-(triethoxysilyl) propyl isocyanate was prepared on the slide by self-assembled technique. X-ray photoelectron spectroscopy (XPS) was employed to analyze the elementary composition of the film. Contact angle of distilled water was measured to characterize the surface state. It was shown that 3-(triethoxysilyl) propyl isocyanate had been successfully assembled on the slide. The increase of contact angle to 80° demonstrated that the hydrophobicity of the surface of chip was increased significantly. Moreover, further self-assembly of bovine serum albumin (BSA) on 3-(triethoxysilyl) propyl isocyanate was also carried out with the advantages such as simple and convenient preparation. Therefore, the potential of broader applications in the modification of micro-channel in the μ-TAS system, the immobilization of protein or peptide and the surface modification of materials are all expectative.

The selective loss of dopaminergic neurons in Parkinson’s disease is suspected to correlate with the increase of cellular iron, which may be involved in the pathogenesis of PD by promotion of oxidative stress. This research investigated dopamine-induced oxidative stress toxicity contributed by iron and the production of dopamine-derived neurotoxins in dopaminergic SH-SY5Y cells.After the SH-SY5Y cells were pre-incubated with dopamine and Fe2+ for 24 h, the cell viability, hydroxyl radical, melondialdehyde, cell apoptosis, and catechol isoquinolines were measured by lactate dehydrogenase assay, salicylic acid trapping method, thiobarbuteric acid assay, Hoechst 33258 staining and HPLC-electrochemical detection (HPLC-ECD), respectively.(1) Optimal dopamine (150 μmol/L) and Fe2+ (40 or 80 μmol/L) significantly increased the concentrations of hydroxy radicals and melondialdehyde in SH-SY5Y cells. (2) Induction with dopamine alone or dopamine and Fe2+ (dopamine/Fe2+) caused cell apoptosis. (3) Compared with untreated cells, the catechol isoquinolines, salsolinol and N-methyl-salsolinol in dopamine/Fe2+-induced cells were detected in increasing amounts.Due to dopamine/Fe2+-induced oxidative stress similar to the state in the parkinsonian substantia nigra neurons, dopamine and Fe2+ impaired SH-SY5Y cells could be used as the cell oxidative stress model of Parkinson’s disease. The catechol isoquinolines detected in cells may be involved in the pathogenesis of Parkinson’s disease as potential neurotoxins.帕金森氏病(Pakinson’s disease, PD)中多巴胺能神经元选择性缺失与胞内铁水平升高有密切关系, 提示铁可能通过参与氧化应激在PD发病机制中起重要作用。本研究使用一定浓度的Fe2+和多巴胺诱导人多巴胺能成神经细胞瘤SH-SY5Y细胞产生氧化应激状态, 并且检测胞内是否有多巴胺衍生类的神经内毒素物质产生。多巴胺添加不同浓度的Fe2+诱导SH-SY5Y细胞, 24 h后用乳酸脱氢酶法、 水杨酸捕获法、 硫代巴比妥酸法、 Hoechst33258染色法和带有电化学检测器的高效液相色谱仪分别检测细胞存活率、 羟自由基生成量、 丙二醛含量、 细胞凋亡和儿茶酚异喹啉物质的生成情况。(1) 150 μmol/L 多巴胺添加40或80 μmol/L Fe2+后, 胞内羟自由基和丙二醛含量较对照组显著增加; (2) 单独多巴胺以及多巴胺加40或80 μmol/L Fe2+诱导后细胞发生凋亡; (3)在诱导后的胞内检测到Salsolinol和N-methylsalsolinol的含量高于对照组。一定浓度的Fe2+和多巴胺诱导SH-SY5Y细胞可模拟帕金森氏病人黑质区多巴胺能神经元所受到的氧化应激状态, 胞内检测到的儿茶酚异喹啉物质, 如去甲猪毛菜碱和N-methyl-salsolinol, 可能作为一类潜在的神经毒性物质与帕金森氏病的发病有关。

Formaldehyde, a well-known environmental toxic hazard, has been found to produce endogenously via semicarbazide-sensitive amine oxidase-catalyzed oxidative deamination of methylamine. In diabetes, the activity of SSAO has been found to increase with a subsequent increase in endogenous formaldehyde production. It has been postulated that SSAO-induced production of formaldehyde may be involved in the alteration of protein structure, which may subsequently cause protein deposition associated with chronic pathological disorders. Formaldehyde has also been found to react (cross-link) with amino group of the N-terminal amino acid residue and with the side-chains of arginine, cysteine, histidine and lysine residues. Therefore, formaldehyde may be responsible, at least in part, for protein cross-linkage, oxidative stress and cytotoxicity. The cross-linking of formaldehyde with bovine serum albumin was studied using LC-MS and Mascot database. The peptides sequence for control BSA (untreated) digested with trypsin was matched in the online database search query by exporting the MS/MS data to online MASCOT database. In this way, a total of twenty-seven peptides were matched in the database search query. These twenty-seven peptides were then searched manually in all of the tryptic BSA samples treated with different concentrations of FA that were incubated in different time intervals. Six formaldehyde-treated BSA peptides (FKDLGEEHFK, HLVDEPQNLIK, KVPQVSTPTLVEVSR, RPCFSALTPDETYVPK, LVNELTEFAK, DAFLGSFLYEYSR) were found to be the possible markers for formaldehyde-protein/peptides adducts.

Wistar rats were treated with caffeine or 2-bromoethylamine, the effect of caffeine on the activity of semicarbazide-sensitive amine oxidase (SSAO) in rat serum and tissues was studied using various LC–MS methods. Caffeine was found to present in all tissues after administration for 10 days and accumulated for 25 days. The level of caffeine was high in brain and liver, and the SSAO activity in all tissues was found to be inhibited by caffeine. As the concentration of caffeine increased, the SSAO activity decreased. The inhibition ratio was correlated to the levels of caffeine present. We presume that caffeine may treat with SSAO activity associated diseases.Highlights► Caffeine distribute and accumulate in rat adipose, brain and serum. ► Caffeine inhibit the SSAO activity in all rat tissues. ► Efficiency of caffeine on adipose and aorta was high.

PurposeThe radioprotective effects of Dragon's blood (DB) and its extracts (DBE) were investigated using the chromosomal aberrant test, micronucleus and oxidative stress assay for anti-clastogenic and anti-oxidative activity.Materials and methodsAdult BALB/C mice were exposed to the whole body irradiation with 4 Gy 60Co γ-rays. DB and DBE were administered orally once a day from 5 days prior to irradiation treatment to 1 day after irradiation. The mice were sacrificed on 24 h after irradiation. The cells of bone marrow were measured by counting different types of chromosomal aberrations and the frequency of micronuclei. Oxidative stress response was carried out by analysis of serum from blood.ResultsDB and DBE significantly decreased the number of bone marrow cells with chromosome aberrations after irradiation with respect to irradiated alone group. The administration of DB and DBE also significantly reduced the frequencies of micronucleated polychromatic erythrocytes (MPCE) and micronucleated normochromatic erythrocytes (MNCE). In addition, DB and DBE markedly increased the activity of antioxidant enzymes and the level of antioxidant molecular. Malondialdehyde (MDA) and nitric oxide (NO) levels in serum were significantly reduced by DB and DBE treatment.ConclusionsOur data suggested that DB and DBE have potential radioprotective properties in mouse bone marrow after 60Co γ-ray exposure, which support their candidature as a potential radioprotective agent.

•Peripheral blood cells, bone marrow.•Apoptosis, Bax, Bcl-2, caspase-3.•ERK1/2, but not Akt.PurposeDragon's blood (DB), a Chinese traditional herb, was shown to have certain protective effects on radiation-induced bone marrow injury due to the presence of several phenolic compounds. The 50% ethanol extracts (DBE) were separated from DB by the methods of alcohol extracting-water precipitating. The protective effects of DBE on hematopoiesis were studied, particularly on megakaryocytes.Materials and methodsIn this study, we investigated the in vivo radioprotective effects of DBE on hematopoiesis and pathological changes using an irradiated-mouse model. Moreover, the protective effects and potential molecular mechanisms of DBE on megakaryocytopoiesis in vitro were explored in GM-CSF depletion-induced Mo7e cell model.ResultsDBE significantly promoted the recovery of peripheral blood cells in irradiated mice. Histology bone marrow confirmed the protective effect of DBE, as shown by an increased number of hematopoietic cells and a reduction of apoptosis. In a megakaryocytic apoptotic model, DBE (50 µg/mL) markedly alleviated GM-CSF withdrawal-induced apoptosis and cell-cycle arrest of Mo7e cells. DBE (50 µg/mL) also significantly decreased the ratio of Bax to Bcl-2 expression, inhibited the active caspase-3 expression. In addition, DBE could induce ERK1/2 phosphorylation in GM-CSF-depleted Mo7e cell, but not Akt.ConclusionsOur data demonstrated that DBE could effectively accelerate the recovery of peripheral blood cells, especially platelet. DBE attenuated cell apoptosis and cell cycle arrest through the decrease of Bax/Bcl-2 ratio and the reduction of active caspase-3 expression. The effect of DBE on Mo7e cells survival and proliferation is likely associated with the activation of ERK, but not Akt.

•α-Synuclein overexpression increases the oxidative stress in U251 cells.•α-Synuclein overexpression alters cell surface and mitochondrial morphologies.•α-Synuclein overexpression induces aggregation of cytoplasmic vesicles.•H2O2 increases the diameter of the cytoplasmic vesicles.α-Synuclein is the major component of Lewy bodies, Lewy neurites, and glial cytoplasmic inclusions. It plays an important role in neurodegenerative diseases such as Parkinson’s disease, multiple system atrophy, and other synucleinopathies. However, the pathogenesis and neurodegenerative effects of α-synuclein remain unknown. In this study, we established an α-synuclein and an α-synuclein-EGFP overexpressing U251 cell line. α-Synuclein overexpression increases oxidative stress and alters the cell surface and mitochondrial morphologies. We provide fluorescent-protein tagging, immunofluorescence and ultrastructural evidence showing that α-synuclein accumulations are associated with clusters of cytoplasmic vesicles and the diameter of these vesicles increases by H2O2 in a time- and dose-dependent manner.

Ethnopharmacological relevanceDragon׳s blood, a traditional Chinese herb, has been used to “panacea of blood activating” and its major biological activity appears to be from phenolic compounds. In this study, our research aims to examine the effects of Dragon׳s blood (DB) and its extracts (DBE) on radiation-induced myelosuppressive mice.Materials and methodsAdult BALB/C mice were exposed to the whole body irradiation with 4 Gy 60Co γ-rays. DB and DBE were respectively administered orally for 5 constitutive days prior to irradiation treatment. The radioprotective effects and relevant mechanisms of DB and DBE in radiation-induced bone marrow injury were investigated by ex vivo examination.ResultsWe found that the administration of DB and DBE significantly increased the numbers of peripheral blood cells and colony forming unit of bone marrow-derived stem/progenitor cells. Interestingly, compared with the irradiation group, the administration of DB and DBE significantly decreased the levels of the inflammatory cytokines such as IL-6, TNF-α and IFN-γ and oxidative stress injury such as SOD, CAT, GSH, MDA in serum of mice. Furthermore, DBE markedly improved the morphology of bone marrow histopathology.ConclusionsOur data suggest that DB and DBE effectively attenuate radiation-induced damage in bone marrow, which is likely associated with the anti-oxidative and anti-inflammatory properties of DB and DBE.Download high-res image (106KB)Download full-size image

Belamcanda chinensis (Iridaceae) belongs to the family of iridaceae and its rhizoma has been widely used for the treatment of throat ailment. Here we report a new pharmacological activity of B. chinensis leaf extract (BCL), that is, the hypoglycemic effect in normal and STZ-induced diabetic rats. Animals either healthy or STZ-induced diabetic show significantly lowered fasting blood glucose levels after treatment with BCL. The serum insulin concentration in normal rats is also enhanced. Additionally, the increase in blood glucose levels after administration of various carbohydrates in normal rats is significantly decreased and the oral glucose tolerance (OGTT) of STZ-induced diabetic rats is largely improved by BCL treatment. However, co-administration of BCL with Nifedipine, a Ca2+ ion channel blocker, or Nicorandil, an ATP-sensitive K+ ion channel opener thoroughly abolishes the hypoglycemic effect of BCL. HPLC analysis and compound identification showed that several isoflavone glycosides with antidiabetic activities were contained in BCL while pharmacological experiment showed that the polysaccharide fraction of BCL had no significant hypoglycemic effect on normal rats. Therefore, the isoflavone glycosides but not polysaccharides might be the active fraction of BCL in diabetes treatment.

Dragon's blood is a bright red resin obtained from Dracaena cochinchinensis (Lour.) S.C.Chen (Yunnan, China). As a traditional Chinese medicinal herb, it has great traditional medicinal value and is used for wound healing and to stop bleeding. Its main biological activity comes from phenolic compounds. In this study, phenolic compounds were made into dropping pills and their protective effects were examined by establishing focal cerebral ischemia rats model used method of Middle Cerebral Artery Occlusion (MCAO), and by investigating indexes of neurological scores, infarct volume, cerebral index, cerebral water content and oxidation stress. Compared to model group, high, middle and low groups of Dragon's blood dropping pills could improve the neurological function significantly (p < 0.01) and reduce cerebral infarct volume of focal cerebral ischemia rats remarkably (p < 0.05–0.01). Meanwhile, each group could alleviate cerebral water content and cerebral index (p < 0.05–0.01) and regulate oxidative stress of focal cerebral ischemia rats obviously (p < 0.05–0.01). Activities of middle group corresponded with that treated with positive control drug. The results obtained here showed that Dragon's blood dropping pills had protective effects on focal cerebral ischemia rats.Download high-res image (178KB)Download full-size image

Selective capture of microorganisms is an effective and rapid technique for microbe detection. In this article, we obtained aptamers targeting live bacterial cells E. coli as recognition and capture elements by using a whole-cell based aptamer selection, and integrated the isolated aptamers into a microfluidic device for selective capture of microorganisms. Eight rounds of selection were performed; the specificity and affinity of aptamers to the target bacterial cells were also characterized using flow cytometric analysis. A total of 40 aptamer sequences were obtained, and the apparent dissociation constant Kd of the representative aptamer displaying the highest binding affinity was estimated to be 24.8 ± 2.7 nM. After sequencing of the pool showing the highest binding affinity to E. coli, a DNA sequence of high affinity to the bacteria was integrated into the microfluidic device as recognition and capture elements for selective capture of microorganisms. The results demonstrated that the aptamer-based microfluidic device was able to capture E. coli selectively to distinguish different species of bacteria. This investigation is envisaged to provide a new avenue for the development of lab-on-chip devices for selective capture of microorganisms, which could be beneficial for portable, rapid and economic microbial detection in food safety, environmental monitoring and medical diagnosis.

The post-digestion of 18O labeling is widely used in comparative proteomics for the relative quantification of proteins and peptides. Application and precise quantitative analysis are hindered, since 18O labeling is pH sensitive and has back-exchange. Herein, in a study of peptides derived from BSA (bovine serum albumin), we demonstrated through a detailed evaluation that removal of soluble trypsin by ultrafiltration prevented back-exchange and effectively enhanced the stability of 18O-labeled peptides under off-gel separation even without trypsin inhibitor. After ultrafiltration, the 18O labeling efficiency was 95.8 ± 2.3% under off-gel separation. In addition, these peptides had a relative high, approximately 80% recovery and less than 5% 16O/18O ratio variation through ultrafiltration, indicating no apparent effect on quantification. Hence, the useful and economical method presented here effectively inhibited back-exchange in off-gel separation and might enable further applications towards large-scale biomarker discovery.

A new thermoresponsive boronate affinity chromatographic material, P(N-isopropylacrylamide-4-vinylphenylboronic acid) P(NIPAAm-co-VPBA) grafted silica featuring a thermally switchable property, was prepared and successfully employed for the specific capture–release of cis-diol biomolecules with simpler adjustment to the temperature instead of the conventional need to change the pH of the mobile phase.

Semicarbazide-sensitive amine oxidase (SSAO) is present in various mammalian tissues and in blood plasma. Elevation of SSAO activity is linked to vascular disorders associated with pathological conditions such as diabetic complications, heart failure and vascular dementia. In the present paper, a high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) method is developed to determine the SSAO activity. Methylamine is used as physiological substrate for the enzyme activity assay of SSAO. Formaldehyde, the enzymatic reaction product, was derivatized by dopamine, and separated by a silica-based pentafluorophenyl column. The calibration curve was linear over the range of 0.03–4.00 μM of formaldehyde concentration, with 0.03 μM the lower limit of quantification (LOQ). The inter-day and intra-day precisions ranged from 2.2% to 7.9% and 4.4% to 9.2% respectively for each quality control sample of formaldehyde at 0.06, 0.50, and 2.00 μM. The accuracy ranged from 96.0% to 111.5%. The limit of detection (LOD) for serum SSAO activity was 1 nmol h−1 mg−1 protein. The method was successfully applied for the determination of SSAO activity in both mouse serum and rat tissues. SSAO activity of the serum in diabetes mice was significantly increased compared with the control and was inhibited by 2-bromoethylamine in vivo. The levels of SSAO activity in rat lung, spleen, aorta, kidney and brain tissues were significantly decreased by semicarbazide in vitro.

Salsolinol (Sal) and N-methylsalsolinol (NMSal) are endogenous neurotoxins in the brain, which are the key causative agents of the death of dopaminergic neurons. Due to their strong polarity, they have weak retention and are difficult to quantify using the C18 column. In the present study, a sensitive and selective LC/MS method for the determination of Sal and NMSal was developed and validated using a novel pentafluorophenylpropyl column performed on high performance liquid chromatography electrospray ionisation triple quadrupole. The calibration curve was linear over the range of 0.098–25 nM and 3.91–250 nM for Sal and NMSal, respectively. The lower limit of quantitation is 0.098 nM and 3.91 nM for Sal and NMSal. The inter-day and intra-day precision and accuracy for all samples were acceptable. Furthermore, the assay has also been applied for the determination of Sal and NMSal in rat brain.

The CP4 EPSPS gene is widely used in herbicide-tolerant crops/plants all over the world. In this study, a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated to quantify the amount of CP4 EPSPS expression in Nicotiana tabacum leaves. The quantification of protein was used to measure the unique peptides of the CP4 EPSPS protein. Two peptides unique to CP4 EPSPS were synthesized and labelled in H218O to give 18O stable isotope labelled peptides which served as internal standards. The validated method resulted in good specificity and linearity. The intra- and inter-day precisions and accuracy for all samples were satisfactory. The results demonstrated that the novel method was sensitive and selective to quantify CP4 EPSPS in the crude extract without time-consuming pre-separation or purification procedures.