New adjuvants to enhance anticoagulant activity of Warfarin

New adjuvants of warfarin anticoagulant activity have been developed. These compounds, which are 1,4-methano-1,2,3,4-tetrahydroanthracene-9,10-diol derivatives, act synergistically with warfarin to potentiate its anticoagulant effect. None of the compounds tested is an effective oral anticoagulant in the absence of warfarin.     

Relationship among plasma adipokines, insulin and androgens level as well as biochemical glycemic and lipidemic markers with incidence of PCOS in women with normal BMI

Polycystic ovary syndrome (PCOS) is an endocrine disorder in women. Omentin-1 and vaspin are secretary adipokines that are produced by the visceral adipose tissue. These levels change in obese women with PCOS. The aim of this study is to investigate whether omentin and vaspin levels change in nonobese PCOS subjects. This study is a cross-sectional case control study in which 39 women with PCOS were picked out for this study. The inclusion criteria were based on the Rotterdam 2003 diagnostic criteria. The control group consisted of 39 women with normal pelvic sonographic reports having regular menstruation and showing no signs of infertility. The fasting plasma glucose (FPG), triglyceride (TG), Chol, and high-density lipoprotein cholesterol (HDL-C), insulin, testosterone, omentin and vaspin were measured by the enzymatic methods. The differences within these groups were calculated by the un-paired t-test and the Mann-Whitney test. The results from this study show a significant increase in the amount of insulin, testosterone, homeostasis model assessments for insulin resistance, TG and lower HDL in the patient group. No significant differences were seen in omentin, vaspin, FPG, Cho, low-density lipoprotein, very low-density lipoprotein cholesterol, blood urea nitrogen, Cr and homeostasis model assessments for B cell function levels between groups. Results show that PCOS is not a determinant of decreased omentin and vaspin plasma levels and those high androgen level and insulin resistances are warning signs of PCOS.     

Shotgun proteomic analysis of long-distance drought signaling in rice roots

Rice (Oryza sativa L. cv. IR64) was grown in split-root systems to analyze long-distance drought signaling within root systems. This in turn underpins how root systems in heterogeneous soils adapt to drought. The approach was to compare four root tissues: (1) fully watered; (2) fully droughted and split-root systems where (3) one-half was watered and (4) the other half was droughted. This was specifically aimed at identifying how droughted root tissues altered the proteome of adjacent wet roots by hormone signals and how wet roots reciprocally affected dry roots hydraulically. Quantitative label-free shotgun proteomic analysis of four different root tissues resulted in identification of 1487 nonredundant proteins, with nearly 900 proteins present in triplicate in each treatment. Drought caused surprising changes in expression, most notably in partially droughted roots where 38% of proteins were altered in level compared to adjacent watered roots. Specific functional groups changed consistently in drought. Pathogenesis-related proteins were generally up-regulated in response to drought and heat-shock proteins were totally absent in roots of fully watered plants. Proteins involved in transport and oxidation-reduction reactions were also highly dependent upon drought signals, with the former largely absent in roots receiving a drought signal while oxidation-reduction proteins were strongly present during drought. Finally, two functionally contrasting protein families were compared to validate our approach, showing that nine tubulins were strongly reduced in droughted roots while six chitinases were up-regulated, even when the signal arrived remotely from adjacent droughted roots.     

Differential regulation of aquaporins, small GTPases and V-ATPases proteins in rice leaves subjected to drought stress and recovery

Mechanisms of drought tolerance are complex, interacting, and polygenic. This paper describes patterns of gene expression at precise physiological stages of drought in 35-day-old seedlings of Oryza sativa cv. Nipponbare. Drought was imposed gradually for up to 15 days, causing abscisic acid levels to rise and growth to cease, and plants were then re-watered. Proteins were identified from leaf samples after moderate drought, extreme drought, and 3 and 6 days of re-watering. Label-free quantitative shotgun proteomics resulted in identification of 1548 non-redundant proteins. More proteins were down-regulated in early stages of drought but more were up-regulated as severe drought developed. After re-watering, there was notable down regulation, suggesting that stress-related proteins were being degraded. Proteins involved in signalling and transport became dominant as severe drought took hold but decreased again on re-watering. Most of the nine aquaporins identified were responsive to drought, with six decreasing rapidly in abundance as plants were re-watered. Nine G-proteins appeared in large amounts during severe drought and dramatically degraded once plants were re-watered. We speculate that water transport and drought signalling are critical elements of the overall response to drought in rice and might be the key to biotechnological approaches to drought tolerance.     

Halogenated Peptides as Internal Standards (H-PINS): INTRODUCTION OF AN MS-BASED INTERNAL STANDARD SET FOR LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY*

As the application for quantitative proteomics in the life sciences has grown in recent years, so has the need for more robust and generally applicable methods for quality control and calibration. The reliability of quantitative proteomics is tightly linked to the reproducibility and stability of the analytical platforms, which are typically multicomponent (e.g. sample preparation, multistep separations, and mass spectrometry) with individual components contributing unequally to the overall system reproducibility. Variations in quantitative accuracy are thus inevitable, and quality control and calibration become essential for the assessment of the quality of the analyses themselves. Toward this end, the use of internal standards cannot only assist in the detection and removal of outlier data acquired by an irreproducible system (quality control) but can also be used for detection of changes in instruments for their subsequent performance and calibration. Here we introduce a set of halogenated peptides as internal standards. The peptides are custom designed to have properties suitable for various quality control assessments, data calibration, and normalization processes. The unique isotope distribution of halogenated peptides makes their mass spectral detection easy and unambiguous when spiked into complex peptide mixtures. In addition, they were designed to elute sequentially over an entire aqueous to organic LC gradient and to have m/z values within the commonly scanned mass range (300–1800 Da). In a series of experiments in which these peptides were spiked into an enriched N-glycosite peptide fraction (i.e. from formerly N-glycosylated intact proteins in their deglycosylated form) isolated from human plasma, we show the utility and performance of these halogenated peptides for sample preparation and LC injection quality control as well as for retention time and mass calibration. Further use of the peptides for signal intensity normalization and retention time synchronization for selected reaction monitoring experiments is also demonstrated.As proteomics and systems biology converge, the need for the generation of high quality, large scale quantitative proteomics data sets has grown, and so-called label-free quantification has emerged as a very useful platform for their generation (1). Label-free quantitative experiments are usually designed to detect differentially abundant features in biologically relevant samples by comparing mass versus retention time feature maps generated by LC-MS. Although label-free proteomics experiments are time- and cost-effective, they require high levels of reproducibility at every step of the process (2). Too much variation resulting from sample preparation, LC performance (e.g. injection, gradient delivery, and flow rate), and MS performance (e.g. ionization efficiency, mass accuracy, and detector performance) could lead to an increase in the false discovery rate of detected peptides. Thus it is crucial to minimize such variation to adequately control the quality of the data. In addition, label-free experiments are often followed by directed MS/MS analyses in which selected peptides are specifically targeted for identification, a procedure that also requires high system reproducibility (3, 4). The total variation in the acquired data is the result of accumulating variation at each step. This variation, regardless of its source, be it from sample handling, injection irreproducibility, change in analyte volume, matrix and co-eluter interference (both suppression and enhancement), system instability, or finally variations in the ion source performance, can be accounted for if an appropriate internal standard (ISTD)¹ system is used.A more recent development in the field of quantitative proteomics is multireaction monitoring (MRM) also referred to as selected reaction monitoring (SRM). This MS-based technology is aimed at fast, sensitive, and reproducible screening of large sets of known targets and is ideal for building biological assays in which the presence and quantity of specific analytes is being determined in multiple samples. Certain inputs, such as transitional values (m/z values for the precursor ion and its fragment ions), collision energies, and chromatographic retention time are required to build a validated S/MRM assay. These values are either extracted from MS/MS data acquired from biological samples with the same type of instrument used for the S/MRM analyses or from a set of peptide standards (5). To maximize the number of S/MRM measurements in one LC-MS/MS run, the use of elution time constraints has proven to be highly beneficial (6). ISTDs could therefore play an integral role in building S/MRM assays if used to synchronize input values such as retention times between instruments or to monitor the retention time consistency in sequences of scheduled S/MRM experiments.ISTDs are usually designed to best fit the analytical system for which they are being used. Because the currency of quantitative proteomics is ionized peptide ions, peptides thus represent the best candidates for ISTDs for proteomics measurements. The use of peptides as ISTDs for proteomics applications, however, is not new. Both natural peptides and heavy isotope-labeled peptides (either chemically synthesized or produced by tryptic digestion of biologically expressed quantification concatamers (QconCATs)) have been used as internal standards by spiking (7, 8). Peptides from the biological analyte have also been used as pseudo-internal standards for normalization (9). But a limitation with all these methods that use native and heavy isotope-labeled peptides as ISTDs is signal detection. The MS-based signal detection for this type of peptide can be challenging when trying to confidently detect their signal in ion chromatograms acquired by mass spectral analysis of biological fluids or other samples of similar complexity where densely packed features cover the entire mass and time range (10). In addition, there is always a chance that a peptide with the same elemental composition as the internal standard might exist in the analyte and thus completely throw off the calibration curve (11). The same argument is valid for heavy isotope-labeled peptides because in many quantitative applications the analytical matrix is made of heavy isotope-labeled peptides (12–14). Obviously utilization of ISTDs in complex mixtures requires highly confident detection of corresponding signals, and for natural and heavy isotope-labeled peptides MS/MS analysis is the only way to accomplish that. But CID attempts on mass spectral features do not necessarily result in identification. First the MS features from ISTDs have to be picked for CID, and then the fragmentation should result in high quality MS/MS spectra that could be matched to the ISTD sequence with high confidence. This process is not always successful and consequently can result in an incomplete set of ISTD signals. The other limitation of MS/MS-based ISTDs is processing time. All MS/MS data have to be searched and curated before ISTD signals can be used.On the other hand, if ISTD signals could be easily detected at the MS level, then all the aforementioned limitations are lifted. For such a peptide to be an MS-based ISTD, it should really have unusual properties that make it easily detectable in a background of biological peptides.In this study we introduce the use of a set of halogenated peptides as internal standards (H-PINS) with unique isotopic distributions and mass defect that are easily detectable at the MS level by manual search and automated peak picking algorithms. The pattern of the isotopic distribution and mass defect are essential for detection of H-PINS at the MS level. Hence these peptides are best suited for high resolution and mass accuracy instruments. These peptides are similar to ordinary peptides in any other respect and can be treated similarly during purification and LC-MS analysis. We go on to illustrate their use for quality control (QC) at various steps of a proteomics experiment including sample preparation, LC-MS, and mass calibration and retention time synchronization between various analytical platforms.     

Quantitative shotgun proteomics reveals extensive changes to the proteome of the orbitofrontal cortex in rats that are hyperactive following withdrawal from a high sugar diet

In most Westernized societies, there has been an alarming increase in the consumption of sugar‐sweetened drinks. For many adults these drinks represent a substantial proportion of their total daily caloric intake. Here we investigated whether extended exposure to sugar changes behavior and protein expression in the orbitofrontal cortex (OFC). Male adult Sprague‐Dawley rats (n = 8 per group) were treated for 26 days with either water or a 10% sucrose solution. Locomotor behavior was measured on the first and last day of treatment, then 1 week after treatment. Following the 1‐week period free from treatment, sucrose treated rats were significantly more active than the control. Two hours following final behavioral testing, brains were rapidly removed and prepared for proteomic analysis of the OFC. Label free quantitative shotgun proteomic analyses of three rats from each group found 290 proteins were differentially expressed in the sucrose treated group when compared to the control group. Major changes in the proteome were seen in proteins related to energy metabolism, mitochondrial function and the cellular response to stress. This research does not seek to suggest that sugar will cause specific neurological disorders, however similar changes in proteins have been seen in neurological disorders such as Alzheimer's disease, Parkinson's disease and schizophrenia.     

Enhancement of osteogenic differentiation of adipose-derived stem cells by PRP modified nanofibrous scaffold

Recent developments in bone tissue engineering have paved the way for more efficient and cost-effective strategies. Additionally, utilization of autologous sources has been considered very desirable and is increasingly growing. Recently, activated platelet rich plasma (PRP) has been widely used in the field of bone tissue engineering, since it harbours a huge number of growth factors that can enhance osteogenesis and bone regeneration. In the present study, the osteogenic effects of PRP coated nanofibrous PES/PVA scaffolds on adipose-derived mesenchymal stem cells have been investigated. Common osteogenic markers were assayed by real time PCR. Alkaline phosphate activity, calcium deposition and Alizarin red staining assays were performed as well. The results revealed that the highest osteogenic differentiation occurred when cells were cultured on PRP coated PES/PVA scaffolds. Interestingly, direct application of PRP to culture media had no additive effects on osteogenesis of cells cultured on PRP coated PES/PVA scaffolds or those receiving typical osteogenic factors. The highest osteogenic effects were achieved by the simplest and most cost-effective method, i.e. merely by using PRP coated scaffolds. PRP coated PES/PVA scaffolds can maximally induce osteogenesis with no need for extrinsic factors. The major contribution of this paper to the current researches on bone regeneration is to suggest an easy, cost-effective approach to enhance osteogenesis via PRP coated scaffolds, with no additional external growth factors.     

Exosomes and microRNAs: New potential therapeutic candidates in Alzheimer disease therapy

Exosomes are biological nanocarriers which could be involved in a variety of basic physiological events. They exert their effects via targeting their cargos (i.e., DNAs, messenger RNAs, microRNAs [miRNAs], and proteins) to host cells, which led to change behaviors of recipient cells. One of the important aspects of exosomes is the roles of them in disease conditions. Increasing evidence indicated that exosomes are one of the main players in Alzheimer’s disease (AD) pathogenesis. Hence, it seems that these nanocarriers could be used as diagnostic and therapeutic biomarkers in AD treatment. Another important player in AD pathogenesis is miRNA. MiRNAs are short noncoding RNAs which exert their effects as epigenetic regulators. These molecules involved in different stages of AD. Therefore, miRNAs could be used as prognostic, diagnostic, and therapeutic biomarkers in AD. Here, we summarized various roles of exosomes and application of them in AD pathogenesis. Moreover, we highlighted the utilization of miRNAs as a therapeutic option in AD therapy.