Anopheles mosquitoes offer attractive targets to understand interactions with sporozoites, blood feeding behavior, homeostasis, and immunological evaluation of malaria vectors and parasite interactions. To date limited studies have been carried out to elucidate salivary proteins of An. stephensi salivary glands. The aim of the present study was to provide detailed analytical attributives of functional salivary gland proteins of urban malaria vector An. stephensi. A proteomic approach combining one-dimensional electrophoresis (1DE), ion trap liquid chromatography mass spectrometry (LC/MS/MS), and computational bioinformatic analysis was adopted to provide the first direct insight into identification and functional characterization of known salivary proteins and novel salivary proteins of An. stephensi. Computational studies by online servers, namely, MASCOT and OMSSA algorithms, identified a total of 36 known salivary proteins and 123 novel proteins analysed by LC/MS/MS. This first report describes a baseline proteomic catalogue of 159 salivary proteins belonging to various categories of signal transduction, regulation of blood coagulation cascade, and various immune and energy pathways of An. stephensi sialotranscriptome by mass spectrometry. Our results may serve as basis to provide a putative functional role of proteins in concept of blood feeding, biting behavior, and other aspects of vector-parasite host interactions for parasite development in anopheline mosquitoes.Salivary gland proteins of  An. stephensi is an important urban malaria vector of Indo-Pakistan subcontinent [ An. stephensi to both human and rodent malaria species, it turns out to be significant to use as a reference laboratory model to study salivary gland-parasite interactions [Malaria has been prevalent for a long time in tropical developing regions causing great morbidity and mortality . The worontinent . Due to ractions . Salivarractions . Plasmodium sporozoites invade the salivary glands and are injected with the saliva into vertebrate hosts during blood feeding [ Culex mosquitoes [The salivary gland proteins are thus relevant for malaria research since the feeding . In addi feeding  and bloo feeding , and som feeding  that helsquitoes . An. stephensi have been studied for molecular and genetic studies and for malaria transmission. Many products of salivary gland gene expression have been studied in An. stephensi with help of applications of transcriptomics and proteomics [ An. stephensi sialome studies were elucidated using transcriptomic studies that include full-length cDNA library sequence of An. stephensi [ An. stephensi salivary glands [ An. gambiae salivary gland [ Anopheles stephensi have been studied. Hence, to fully understand high biological actions of salivary gland proteins and to elucidate their role in different biosynthetic pathways, application of proteomics is very much needed. Mass spectrometry based proteomics data, when applied in conjunction with mosquito salivary gland genomic and transcriptomics databases, provides a comprehensive account that can be used to identify proteins as putative functional components of the salivary glands for novel malaria control strategies [Salivary gland tissues ofoteomics . Howeveroteomics . Howevertephensi , 5 and dy glands , 10. Thory gland  with larry gland , thus farategies . Plasmodium parasite. Transcriptomics studies combined with genetic variations across evolutionarily related mosquitoes for targeting specific RNA sequences are generally inconsistent to generate functional proteomic data sets [ An. stephensi salivary gland in order to establish functional phylogeny among different anophelines and other mosquitoes to validate their evolutionary functions.Unfortunately, to date, only limited studies exist to efficiently explore molecular interactions and role of salivary gland proteins of the mosquito and the sporozoites of theata sets , 9, 10.  An. stephensi. We have achieved this by analyzing mass spectrometry data using MASCOT and OMSSA algorithm. We report, herewith, the catalogue of 159 known and novel proteins obtained from LC-MS/MS data through a detailed bioinformatics analysis which should serve as a first preliminary step for putative functional identification of several salivary glands extracts (SGEs) proteins and proteomes at molecular levels that may provide novel targets for interrupting parasitic transmission life cycle. Our study thus opens up the possibilities of elucidating salivary gland-parasite interactions during blood meals and may provide relevant baseline information for characterizing proteomes of other mosquitoes for development of novel vector control strategies.Here we describe an in-gel proteomic approach using 1D and LC-MS/MS to characterize the proteome of the salivary gland extracts (SGEs) ofAnopheles stephensi mosquitoes reared in our insectary  were used in this study. 3-4-day-old sugar fed mosquitoes were used in the experiments and were maintained and reared under identical standard conditions at 27°C ± 2°C with 70% ± 10% relative humidity and a photoperiod of 12 : 12 (light/dark) hours. Adult mosquitoes were maintained on a 10% sucrose solution.Anopheles stephensi salivary glands were dissected on slide using fine needles under a stereomicroscope at 4x magnification using phosphate-buffered saline (PBS) and were pooled. After dissection the tissues were immediately placed in a PBS buffer (100 μL) with protease inhibitors  and stored at −80°C until use. An. stephensi were used to prepare salivary gland extracts (SGEs). Dissected salivary glands (100 pairs) in PBS were sonicated on ice with three pulses for 20 sec. Afterward the suspension was centrifuged for 10 min at 5000 rpm at 4°C to remove cell debris. The extracted supernatant was collected and stored at −80°C for further analysis. Protein estimation was carried out by Lowry's method [A total of 100 pairs of salivary glands of females method  and analμg of SGE sample was dissolved in sample buffer  containing β-mercaptoethanol (10% vol/vol) and heated at 95°C for 5 min. 30 μL sample was then loaded onto an acrylamide gel (3% stacking gel and 10% resolving gel) and subjected to electrophoresis on a Bio-Rad apparatus . Protein molecular weight markers  were also run on the gel. The gel was silver-stained according to the manufacturer's protocol (G-Biosciences). Stained gel was then sliced into different bands and these gel bands were individually subjected to digestion with proteomic grade trypsin .SGE samples were first fractionated on SDS-PAGE for separation. Briefly, 50–75 μL of ammonium bicarbonate (100 mM) for 10 min, dehydrated by addition of acetonitrile, swelled by rehydration in ammonium bicarbonate (100 mM), and shrunk again by addition of the same volume of acetonitrile. After removal of the liquid phase, the gel pieces were completely dried in a vacuum centrifuge. Gel slices were then swollen in a digestion buffer containing ammonium bicarbonate (50 mM), CaCl2 (5 mM), and trypsin solution (12.5 ng/μL) (ratio 1 : 100) in an ice cold bath. The supernatant was removed after 45 mins and replaced with 5–10 μL of the same buffer, but without trypsin, to keep the gel pieces wet during enzymatic cleavage . Peptides were extracted by one change of ammonium bicarbonate (20 mM) and three changes of 5% formic acid in acetonitrile (50%) (20 min for each change) at room temperature and dried down. This peptide mixture was then stored for analysis by LC/MS/MS.Proteins were reduced, alkylated with iodoacetamide, and digested with trypsin overnight at 37°C. Briefly, the excised gel slices were subjected to reduction and were dried in a vacuum centrifuge. DTT (10 mM) in ammonium bicarbonate (100 mM) was added to gel pieces and proteins were reduced for 1 hour at 56°C. After cooling to room temperature reduced proteins were alkylated with IAA (55 mM) in ammonium bicarbonate (100 mM) for 45 min at 25°C. After incubation in the dark with occasional vortexing the gel pieces were washed with 50–100 μL of each sample were injected. Peptides were first trapped and preconcentrated on a C-18 precolumn (Dionex) at 30 μL flow for 5 min and later eluted on the separation column with a flow of 220 nL/min . Solvents used for elution of peptides were solvent A: water, ACN (1.0%), and formic acid (0.1%) and solvent B: ACN and formic acid (0.1%). All samples were measured in “auto” MS/MS mode, positive ion mode on the Bruker nanospray source with a capillary voltage 1,500 Volts, dry gas flow 6.0 L/min, dry temperature: 130°C, 1 MS followed by 5 MS/MS of the most intense ions, total cycle time 4.4–8.8 sec, m/z 400–1,400 taken as precursor ions for MS/MS, active exclusion after 2 spectra for 0.5 min, and threshold for MS/MS set to 1,000.In-gel digested peptides were analyzed by nano LC-ESI-QTOF-MS/MS on a Bruker micrOTOF-Q II system. For LC-MS/MS analysis 15 MASCOT Server. LC/MS/MS data were analyzed using Bruker Daltonics ProteinScape database system. Raw data were converted into MGF format and database searches were performed on a MASCOT server  using fixed modification (none), variable modification carbamidomethyl (Cys), and methionine oxidation for each protein band sample (16 digested samples). Parameters used were trypsin as an enzyme, max missed cleavages: 1, peptide mass tolerance: ±0.05 Da or 10 ppm, fragment mass tolerance: ±0.05 Da, data format: MASCOT generic, instrument type: ESI-QUAD-TOF, and databases used SwissProt and NCBInr. Searches were made against Anopheles and other mosquito species. All searches were performed as decoy searches; a minimum score of 30 for at least one peptide was required for proteins to be reported. All the protein sequences were compared against the nonredundant database for homology searching through a BLASTP search (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi?PAGE=Proteins) and SwissProt. Along with that, protein sequences were also searched against data of An. stephensi peptides present in VectorBase (https://www.vectorbase.org/organisms/anopheles-stephensi/). All identifications were manually validated and the proteins were selected and validated on basis of MOWSE scores, peptides matches, and % sequence coverage.OMSSA Server. In order to evaluate other novel proteins which could not be detected with MASCOT, we have also used another search tool, that is, the open mass spectrometry search algorithm (ftp://ftp.ncbi.nih.gov/pub/lewisg/omssa/CURRENT/), under which probability score was used to determine specificity [ Anopheles gambiae present in the taxonomy list. Almost all the parameters were kept the same for OMSSA as used earlier for MASCOT. All searches were conducted by using database SwissProt and NCBInr like MASCOT and only those peptides were reported as significant for which E value was statistically significant (E < 0.05).cificity . Here mo An. stephensi by transcriptomics [ An. stephensi salivary glands for functional identification of such proteins.Role of salivary glands and their proteins is important in the mosquito because parasites mature to form infectious sporozoites in salivary glands. Various active protein molecules must be annotated/expressed in salivary glands of mosquito which may help in food ingestion and digestion and facilitate blood feeding, immune defenses, and haemostasis . In earliptomics , 10. Tra An. stephensi expressed by proteome analysis coupled with LC/MS/MS as an initial step towards the cataloging of the hundreds of proteins and peptides in the salivary proteome for future use in blood feeding experiments. The peak list/spectra obtained after LC/MS/MS were analyzed by both MASCOT (Matrix Science) and OMSSA algorithm and matched against databases of Anopheles, Aedes, and Culex mosquito species.Mass-spectrometry-based proteomics is now a powerful and reliable method that allows characterization of protein assemblies, and when this is combined with molecular, cellular, and bioinformatics techniques it provides a framework for translating complex molecules into simple molecules for in-depth analysis of expressed proteomes , 16. The An. gambiae has led us to large-scale EST projects to identify potential genes and transcriptomes expressed in different mosquito tissues following blood meal. These EST projects are no doubt descriptive in nature and generate hypothesis on the evolution and function of genes [Availability of genome sequence for mosquitoof genes . Still t An. stephensi of India. Total proteins of the salivary glands homogenate were first analyzed by in-gel approach on 10% SDS-PAGE. 16 gel bands of salivary gland homogenate sample were visualized after silver staining .In the present study, we employed a MS-based approach to categorize different putative functional proteins of salivary glands of an urban malaria vectorstaining . In-gel Different proteins are also assigned according to gel bands. In www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) [http://smart.embl-heidelberg.de/) [http://www.cbs.dtu.dk/services/SignalP/) which shows the indication of secretion [Among all identified proteins by LC/MS/MS, further conserved domains were searched by using NCBI domain programs (psb.cgi) , Interprerg.de/) . Signal ecretion .Among the known proteins, GE rich salivary gland protein was found with 56% sequence similarity with the highest score (609) and a total of 8 peptide matches. Further signal peptide for GE rich salivary gland protein was identified at amino acid positions 1 to 19 which depicts a secreted protein . Next to An. stephensi also revealed about 9 cDNA clusters similarities to An. gambiae SG1 proteins family [ An. stephensi that were related to different clusters of SG1 family [A sort of salivary gland proteins termed as SG1 family , 21 was s family . Valenzu1 family  and a si An. gambiae, Aedes aegypti, Anopheles funestus, and Culex quinquefasciatus ( Aedes aegypti FOF1-type ATP synthase beta subunit with 16 peptide matches and 22% sequence similarity. Another protein identified was similar to Histone H4 of Culex quinquefasciatus (21%). One novel protein was found to be similar to tetraspanin of An. gambiae . This protein was well studied in Drosophila which is a conserved membrane protein and it was known to be involved in intracellular signaling and cellular motility [13 novel hypothetical proteins were identified by MASCOT analysis that has features similar to proteins in other mosquito species likeasciatus . One novftp://ftp.ncbi.nih.gov/pub/lewisg/omssa/CURRENT/) by matching sequences of An. gambiae present in the database. We have identified and chosen only significant novel proteins matches on basis of E value. A total of 111 novel proteins from both SwissProt and NCBI nonredundant protein entries were identified by OMSSA algorithm. Information like molecular weight, % sequence coverage, and domain information of some putative functional significant proteins is depicted in All 16 digested samples were also analyzed by OMSSA algorithm after MS/MS analysis (Anopheles gambiae) that was analyzed by both b and y type ions. Signal peptide of this protein was identified at position 1 to 29 and 20% similarity was found. Many other proteins were also identified that have secreted function like defensin  and lysozyme c6 and protein O-fucosyltransferase 1 .During LC/MS/MS analysis, one of the peptides eluted with an amino acid sequence NWATSGETVDECLEEMAGSACEQAYFFTRCVMTR was matched to putative odorant-binding protein OBPjj9 .A total of 36 known proteins and 123 novel proteins were identified from both MASCOT and OMSSA algorithm. Putative functional annotation according to both biological approach and cellular approach was prepared among the identified proteins. These were identified by GO analysis , extracellular (13), cytoplasm (11), mitochondria (10), nucleus (8), intracellular (5), cytoskeleton (8), and so forth. We are unable to find location of a large number of proteins that were assigned under unknown category (65) (novel or known) .On the basis of biological approach, the majority of proteins were scrutinized marked for their role in signal transduction, metabolism, cytoskeleton protein, transcription and translational regulation, energy pathways, regulation of blood coagulation cascade and intracellular trafficking and transport, stress response, and so forth . 16 prot An. stephensi was found to be 23% similar to An. gambiae protein. After analyzing with SMART programme, interestingly we also found Knot1 domain which represents the antimicrobial peptides and has a role in defensive mechanism. In the salivary gland lysozyme c6 may help to check the bacterial growth in sugar meals of mosquitoes [ Anopheles stephensi such as proteins involved in signal transduction as STAT protein, SCRBQ2 protein, Anlar, STAT 1, calpain, TEP 2 protein, and others involved in transport such as tryptophan transporter, nicotinic acetylcholine receptor subunit β1, and ACP receptor putative cation proton antiporter.Various proteins that play an important role in immune responses were identified such as defensins, fibrinogen binding proteins (FBN9), majority of serine proteases, CLIPB , serine protease 14, immune factor (rel homology domain), and lysozyme c6. Such proteins may also be responsible for reduction in microbial load in ingested blood. Among them defensin protein insquitoes . Varioussquitoes . Several An. gambiae [ An. stephensi also showed identity with salivary apyrase protein.Long lists of enzymes were also identified that function as vasodilators, that is, peroxidases . Three p gambiae . Another gambiae –33. This gambiae . TranscrAmong different tables some proteins with sequence coverage below 5% were identified which were actually not native proteins; in fact they are degraded product of the putative proteins.http://string-db.org/) [ An. gambiae proteins  database of physical and functional interactions (db.org/) . String  Figures . Anopheles mosquitoes are believed to be important in the development of the plasmodium as these molecules are involved in the antihemostatic activity, which may assist during the blood feeding process and play a critical role in the transmission of malaria parasite. Our idea was to analyze the putative functional role of the previously known and other novel salivary proteins that may be essential for parasite development in the mosquito directly or indirectly. Here, we report our initial studies using proteomic approaches to characterize the salivary gland proteomic repertoire of an urban malaria vector An. stephensi and its identification by searching protein sequence databases. For that, two different algorithms were used to identify the proteins or peptides from databases , namely, MASCOT and OMSSA, and a total of 36 known proteins and 123 novel proteins were analyzed. These identified proteins were analyzed functionally  by using bioinformatics software so that such salivary proteins can be further employed to understand the concept of feeding, insecticide resistance mechanisms, signal transduction, immunological properties, and various aspects of vector-parasite host interactions.Mass spectrometric based proteomics techniques coupled with high throughput bioinformatic analysis are a powerful platform to understand comprehensive biology and interaction of functional proteins. Salivary gland proteins of theSuch proteins may be used for development of novel antimalarial control strategies for improving innate protection against malaria and help to elucidate the various aspects of salivary gland-malaria parasite interactions.Additional Table 1: Long list of known proteins identified by using in-gel digestion strategy using MASCOT algorithm.Additional Table 2: Long list of novel proteins identified by using in-gel digestion strategy using OMSSA algorithm. th line).Additional table 2 is the supporting information mentioned in Results & discussion section 3.1.2 OMSSA algorithm (11


Nunu, a spontaneously fermented yoghurt-like product, is produced and consumed in parts of West Africa. A total of 373 predominant lactic acid bacteria (LAB) previously isolated and identified from Nunu product were assessed in vitro for their technological properties . Following the determination of technological properties, Lactobacillus fermentum 22-16, Lactobacillus plantarum 8-2, Lactobacillus helveticus 22-7, and Leuconostoc mesenteroides 14-11 were used as single and combined starter cultures for Nunu fermentation. Starter culture fermented Nunu samples were assessed for amino acids profile and rate of acidification and were subsequently evaluated for consumer acceptability. For acidification properties, 82%, 59%, 34%, and 20% of strains belonging to Lactobacillus helveticus, L. plantarum, L. fermentum, and Leu. mesenteriodes, respectively, demonstrated fast acidification properties. High proteolytic activity (>100 to 150 μg/mL) was observed for 50% Leu. mesenteroides, 40% L. fermentum, 41% L. helveticus, 27% L. plantarum, and 10% Ent. faecium species. In starter culture fermented Nunu samples, all amino acids determined were detected in Nunu fermented with single starters of L. plantarum and L. helveticus and combined starter of L. fermntum and L. helveticus. Consumer sensory analysis showed varying degrees of acceptability for Nunu fermented with the different starter cultures. Nunu is a spontaneously fermented milk (yoghurt-like) product in Ghana and other parts of West Africa including Nigeria and Burkina Faso. Unlike other African fermented milk products where milk of goats, sheep, and camels is used, Nunu is solely prepared from cow milk. The traditional processing of Nunu involves collecting fresh cow milk into containers and then allowing it to ferment for a day or two days at ambient temperature. Nunu is yoghurt-like in taste (a sharp acid taste) and it can be taken alone or with Fura [ Nunu is largely home-based and the fermentation is spontaneous. Thus, starter cultures are not available, but old stocks of previous ferments and fermentation containers are used to initiate fermentation in new batches. The dependence on such undefined and diverse microbial consortium during Nunu fermentation may result in product of variable quality and stability.ith Fura , 2. Like Nunu fermentation. However, few investigations have been carried out on the microbiology of Ghanaian traditionally fermented milk products [ Nunu through controlled fermentation using starter culture. The culture should, however, be well-defined. Such starter cultures must be developed with a clear understanding of the ecology of the microbial species associated with the desirable traditional fermentation process, and their contributions to the products safety and quality are determined. The first stage in designing such starter culture(s) is to characterize and identify the technologically important microorganisms associated with the traditional fermentation of the product and then to test the use of the identified organisms in fermentation trials.Currently, there is no information on the use of starter cultures forproducts –4. The p Nunu in view of their application as starter cultures in Nunu production.The objective of the present study was therefore to evaluate the technological potential of lactic acid bacteria isolated from spontaneously fermented Nunu in Ghana using a combination of phenotypic and genotypic characteristics [ L. fermentum, 44 L. plantarum, 40 Ent. faecium, 41 L. mesenteroides, 35 L. helveticus, and 39 Ent. italicus.A total of 373 LAB isolated and identified from spontaneously fermentederistics  were assat time − pHzero time. The cultures were considered as fast, medium, or slow acidifying when a ΔpH of 0.4 U was achieved at 3, 3–5, and >5 h, respectively.Acidification properties of the LAB were measured by the change in pH with time . The strµL was added to 3 mL of OPA reagent. The mixture was held at room temperature (~20°C) for 2 minutes and the absorbance of each solution was measured by using a spectrophotometer  at 340 nm. The proteolytic activity of the bacterial cultures was expressed as the absorbance of OPA derivatives at 340 nm. A relative degree of proteolysis was determined as the difference between proteolytic activities in fermented milk to that of untreated milk.The proteolytic activity of the isolates during fermentation of milk was measured by assessing the free amino groups using the o-phthaldialdehyde (OPA) method . BrieflyStrains were grown overnight at 37°C in MRS broth. A loopful of fresh culture was placed on tributyrin agar [(1) Indicator Strains. The indicator strains included Bacillus cereus PA24, Staphylococcus aureus ATCC 19095, Escherichia coli O157:H7, Listeria monocytogenes Scott A, Salmonella typhi ATCC 13311, and Pseudomonas aeruginosa BFE 162. Bacillus cereus PA24, Escherichia coli O157:H7, and Pseudomonas aeruginosa BFE 162 were obtained from the Department of Applied Biology, DANIDA Microbiology Laboratory of the University for Development Studies, while Staphylococcus aureus ATCC 19095, Listeria monocytogenes Scott A, and Salmonella typhi ATCC 13311 were obtained from the Department of Nutrition and Food Science of the University of Ghana.(2) Preparation of Cell-Free Supernatant (CFS). Each LAB isolate was inoculated in 10 mL of MRS broth and incubated at 30°C for 48 hrs. After incubation, a cell-free supernatant was obtained by centrifuging the bacterial culture at 6000 ×g for 15 min followed by filtration of the supernatant through 0.20 µm pore size syringe filters .(3) Screening for Antimicrobial Activities. The agar-well diffusion method was employed in the screening of LAB for antimicrobial activities. Indicator lawns were prepared by inoculating 20 mL of BHI molten agar media with 100 μL (approximately 107 cfu/mL) of an overnight culture of each indicator organism and allowing them to solidify in a Petri dish. Wells were cut into the agar with a sterile 6 mm diameter cork-borer and sealed with two drops of sterile agar. Fifty microliters (50 μL) of the filtered cell-free supernatant of test strains was separately placed into the wells. The plates, prepared in duplicate, were kept at 4°C for 24 h [for 24 h  to allowThe screening of the isolates for EPSs production was carried out according to the method described by Guiraud . The isoμg/mL.The positive isolates were confirmed growing them on MRS sucrose broth and incubating them at 30°C for 24 h. A volume of 1.5 mL of the 24 h culture was centrifuged at 5000 g for 10 min (4°C) and 1 mL of the supernatant was put in a glass tube and an equal volume of ethanol 95% was added. An opaque link formed at the interface of the tube indicates the presence of EPSs. Samples were centrifuged 2500 ×g for 20 min and the pellets were dried at 100°C. The total carbohydrate content of the EPS was determined using the phenol-sulfuric acid procedure of Dubois et al. . Briefly Nunu. These include Lactobacillus fermentum 22-16 (LF-22-16), Lactobacillus plantarum 8-2 (LP-8-2), Lactobacillus helveticus 22-7 (LH-22-7), and Leuconostoc mesenteroides 14-11 (LM-14-11). The strains were selected based on their predominance in traditional spontaneous Nunu fermentation as well as their desirable technological properties including faster rates of acidification, high proteolytic and low lipolytic activities, ability to produce exopolysaccharides, and the possession of antimicrobial properties in milk.Four (4) strains of LAB were selected and used as starter cultures during the fermentation of milk to produce Nunu are shown in The LAB strains to be used as inocula were prepared by transferring a loopful of an overnight culture from MRS agar into 10 mL MRS broth and incubated at 35°C for 24 h. One hundred microliters of the 24 h old culture was transferred into 10 mL MRS broth and incubated at 35°C for 16 h (overnight). Subsequently, cells were harvested by centrifugation at 5000 g for 10 min (4°C); washed three times with 20 mL sterile diluent (Merck), pH 7.2 ± 0.2; and finally suspended in 10 mL of sterile diluent, and these served as the isolate inocula. Flasks containing 500 mL of fresh milk pasteurized at 60°C for 30 min in a water bath were inoculated in duplicate at 35°C for 24 h. Single and combined starter cultures used for the fermentation of milk to produce Nunu was determined using a digital pH meter . The pH meter was calibrated using standard buffer solutions and measurements were taken at ambient temperature of 30 ± 2°C. All measurements were carried out in triplicate and means and standard deviations were determined.The pH of fermenting(1) Preparation of Samples. Ten milliliters of Nunu samples was defatted with distilled petroleum ether  in a Soxhlet apparatus and stored in screw-capped plastic tubes at −20°C until they were required. Preparation of each sample was carried out in triplicate.(2) Analysis of Free-Amino Acids Profile. The free-amino acids profile analysis of the samples was assayed according to Ojinnaka and Ojimelukwe, [μm filter. Ten microliters of the filtrate was then placed in a sample vial for drying and redrying. The hydrolyzed dried samples were derivatized automatically on the Waters HPLC by allowing the samples to react, under basic situations with phenylisothiocyanate  to get phenylthiocarbamide (PTC) amino acid derivatives. The duration for this reaction was 45 minutes per sample, as calibrated on the instrument. A set of standard solutions of the amino acids were prepared from Pierce Reference standards H (5 μL) into autosampler cups and they were also derivatized. These standards  were used to generate a calibration file that was used to determine the amino acid contents of the samples. After the derivatization, a methanol solution (1.5 N), containing the PTC amino acids, was transferred to a narrow bore (Waters 600) HPLC system for separation. The separation and identification of amino acids were done in reverse phase C18 silica column and the analytes were detected at the wavelength of 254 nm. The elution of the whole amino acids in the samples took 12 minutes. The buffer system used for separation was 140 mM sodium acetate pH 6.40 as buffer A and 80% acetonitrile as buffer B. The program was run using a gradient of buffer A and buffer B concentration and ending with a 55% buffer B concentration at the end of the gradient. The intensity of the chromatographic peaks areas were automatically and digitally identified and quantified using Empowers 2 software data analysis system which was attached to the Waters 600 HPLC System. The calibration curve or file prepared from the average values of the retention times (in minutes) and areas (in Au) of the amino acids in 5 standards runs was used. Since a known amount of each amino acid in the standard was loaded into the HPLC, a response factor (Au/pmol) was calculated by Empowers 2 software that was interphased with the HPLC. This response factor was used to calculate the amount of each of the amino acid (in pmol) in the sample. The amount of each amino acid in the sample was finally calculated by the software by dividing the intensity of the peak area of each by the internal standard in the chromatogram and multiplying this by the total amount of internal standard added to the original sample.melukwe,  with minNunu products prepared by fermentation with different starter cultures were served to 35 volunteered untrained panelists (drawn from the Faculty of Applied Sciences of the University for Development Studies and the Navrongo Community) who are familiar with Nunu. The panel independently, in separate sensory evaluation booths, evaluated the various products for their sensory qualities including taste, colour/appearance, odour, texture, and overall acceptability, using a nine-point hedonic scale . All eleven Nunu products were presented to the panelists randomly placed side-by-side, with each panelist receiving 2 rounds of each product and water for rinsing. Spontaneously fermented Nunu (without added known starter culture) served as control sample. Before tasting the products, panelists were asked to evaluate the products' appearance using a 9-point hedonic scale ranging from “dislike extremely” to “like extremely.” After judging appearance, the panelists were then allowed to taste the samples and evaluate other sensory properties using a 9-point hedonic scale, once again ranging from “dislike extremely” to “like extremely.” The judges were made to wash their mouth with water after evaluating each product.P < 0.05) using the MINITAB statistical software package .All analyses were carried out in triplicate. Data obtained were subjected to one-way analysis of variance (ANOVA) and means were separated by Tukey's family error rate multiple comparison test  of Ent. italicus were fast acidifiers while no strain of Ent. faecium showed fast acidification. These results are in agreement with Sarantinopoulos et al. [ Enterococcus strains were poor acidifiers in milk and Ent. faecium and Enterococcus faecalis have been reported to degrade lactose in milk slowly [Nunu). It has been observed that the faster the decrease in pH to <4, the faster the growth inhibition of the fermenting medium against pathogens such as Salmonella spp. [Rapid acidification is a priority for development of starter cultures for fermented milk products. The acidification properties of predominant LAB isolated from spontaneously fermentednd Karam  indicateHaddadin , L. plans et al. , who ind percent % of Ent.lla spp. . L. fermentum, 27% of L. plantarum, 10% of Ent. faecium, 50% of L. mesenteroides, and 41% of L. helveticus showed good (>100–150 μg tyrosine/mL) proteolytic activities. The result is in contrast with those of Durlu-Ozkaya et al. [In general most of the strains tested showed proteolytic activities to varying degrees . Forty pa et al.  and Dagda et al.  who repoa et al.  reporteda et al. , 22. The L. fermentum screened for lipolytic activities, 32% were negative (showed no clear zones on the agar plates). Similarly, L. plantarum and L. helveticus had 9% and 8.6% strains not showing lipolytic activities, respectively , L. fermentum (30%), L. plantarum (27%) and L. mesenteroides (18%) demonstrated good (>100–150 μg/mL) exopolysaccharide production abilities. Most of the Ent. faecium (95%) and Ent. italicus (85%) strains were poor producers, producing below 50 μg/mL of exopolysaccharides. Exopolysaccharide production is a desirable feature of bacteria applied in dairy products because EPSs act as natural biothickener leading to higher consistency and viscosity of the product and reduced syneresis [ Streptococcus, Lactobacillus, Lactococcus, Leuconostoc, and Pediococcus [ bifidobacteria showing prebiotic potential [As shown in yneresis . Howeverlantarum % and L. yneresis . Most ofiococcus . EPS-proermentum %, L. plaiococcus , 32, rediococcus  and moduiococcus , and incotential , 36. Hen Nunu are shown in  Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Escherichia coli, and Proteus vulgaris. Raccah et al. [The antimicrobial properties of predominant LAB isolated fromh et al. , Smith ah et al. , and Cinh et al.  have demh et al.  indicateh et al.  reportedh et al. , 47. Nunu prepared with different single and combined starter cultures are shown in The rates of acidification ofThe rate of acid development is a critical factor in milk fermentation. The rapid acidification of the raw material prevents growth of undesirable microorganisms and is also essential for aroma, texture, and flavor of the end-product. Nunu prepared with various single and combined starter cultures. The results from this study show that the free amino acid profiles of Nunu produced with different starter cultures varied comparatively and were significantly different. All amino acids determined were detected in samples produced by fermenting milk with single starter cultures of L. plantarum (LP) and L. helveticus (LH) as well as the sample produced with a combined starter culture of L. fermentum and L. helveticus (LF + LH). Lysine, methionine, isoleucine, proline, glutamine, asparagine, alanine, and leucine were detected in all Nunu samples irrespective of the starter culture used. Except serine, all amino acids determined were detected in spontaneously fermented Nunu at varying concentrations. Many studies have shown that concentrations of most of the amino acids slightly increase due to fermentation. Muradyan et al. [ Nunu produced with LAB starter culture can serve as a good source of essential and nonessential amino acids required in metabolism. The amino acids indicated as essential nutrients for infant growth [ Nunu. Sulphur-containing amino acids (methionine and cysteine) are the most critical essential amino acids because they were easily lost from the body [ Nunu samples, but cysteine was not detected in Nunu fermented with L. mesenteroides (LM) starter culture and the combined starter culture of L. fermentum and L. mesenteroides (LF + LM).n et al.  reportedn et al. . It was n et al. . The rest growth , such asthe body  and ther Nunu fermented with the different starter cultures in relation to their sensory attributes such as taste, odour, colour, texture, and overall acceptability  and L. fermentum and L. plantarum (LF + LP) showed significantly higher overall acceptability.Consumer sensory analysis showed varying degrees of acceptability fortability . GeneralNunu) have demonstrated desirable technological properties and have subsequently been successfully used as starter cultures for Nunu fermentation. Lactobacillus fermentum, L. helveticus, and L. plantarum starter cultures  were able to produce yoghurt with desirable consumer sensory characteristics. Therefore, further development and application of these cultures in commercial Nunu production will improve safety and consumer acceptability of the product.Lactic acid bacteria isolated from Ghanaian traditional fermented milk product (


Curcuma longa L.). Under field conditions, the application of chitosan (crab shell) to turmeric plants by foliar spray method induces defense enzymes such as chitinases and chitosanases. Such an increase in enzyme activity was enhanced by spraying chitosan (0.1% w/v) on leaves of turmeric plants at regular intervals. Gel electrophoresis revealed new chitinase and chitosanase isoforms in leaves of turmeric plants treated with chitosan. Treated turmeric plants showed increased resistance towards rhizome rot disease caused by Pythium aphanidermatum, whereas control plants expressed severe rhizome rot disease. Increased activity of defense enzymes in leaves of chitosan treated turmeric plants may play a role in restricting the development of disease symptoms. The eliciting properties of chitosan make chitosan a potential antifungal agent for the control of rhizome rot disease of turmeric.Chitosan was evaluated for its potential to induce antifungal hydrolases in susceptible turmeric plant ( Pythium aphanidermatum is the most destructive disease of turmeric plants in India, which reduces its economic and commercial value [The rhizome rot disease caused byal value , 2. At pal value . Plant dal value , 5. Chital value . Chitosaal value , 7–9. Thal value , 9–11. T Curcuma longa (L.) cultivar Erode local (susceptible) were obtained from a farmer's field at Erode, Tamilnadu, India. The fungus P. aphanidermatum was obtained from ITCC (Indian Type Culture Collection), New Delhi, India, and was maintained on potato dextrose agar.Rhizomes of1 g chitosan  was dissolved in 40 mL of distilled water containing 9 mL of 1 M acetic acid. The pH was adjusted to 6.0 using sodium acetate. From this stock, 0.1% (w/v) chitosan solution was prepared and used for elicitor treatment.Rhizomes were thoroughly washed with running tap water thrice followed by glass distilled water; surface was sterilized by immersion in a sodium hypochlorite 0.001% (v/v) solution for 15 min followed by several rinses of sterile distilled water. For the field experiment, rhizomes were sown in plots and each plot had 30 plants, respectively, at a farmer's field, Erode district of Tamilnadu, India. For foliar spray, 30-day-old plants were treated with 0.1% chitosan (10 mL/plant) and the foliar spray was performed at a regular interval of 30 days up to 210 days. Water sprayed plants served as control. Each experiment was repeated three times.Leaf samples were collected at regular intervals and used for extraction. Turmeric leaves (1 g/2 mL) were homogenized with potassium phosphate buffer  and centrifuged. The clear supernatant was used as a source of protein/defense enzyme. Protein content was determined by the method of  using BSμmol of N-acetyl glucosamine per minute under assay condition.Chitinase was assayed by the method of Reissig et al.  using coμmol of reducing sugar as chitosamine per minute under assay condition.Chitosanase activity was determined by the method of measuring the reducing sugars released from chitosan. The reducing sugars were estimated by the Nelson and Somogyi , 15 methμg protein) were separated on 10% SDS-PAGE. For chitinase localization, 0.1% (w/v) glycol chitin was included in the separation gel. After electrophoresis, the gel was stained with calcofluor white M2R according to the procedure of Trudel and Asselin [SDS-PAGE was carried out according to Laemmli . Samples Asselin .Chitosanase localization was carried out according to the method of Grenier and Asselin  in which P. aphanidermatum (1 × 105 spores/mL). Disease severity was observed at different age levels.Control and chitosan treated plants (60-day-old) were challenged with 5 mL spores ofP < 0.01 and 0.05 levels. All statistical analysis was conducted using SPSS 16 software support.All the data were subjected to one-way analysis of variance to determine the significance of individual differences in An increase in protein content in leaves of chitosan (0.1% w/v) treated plants was evident compared to control (water treated) plants .Turmeric plants treated with chitosan showed higher chitinase activity than untreated plants . There wOn SDS-PAGE, different new isoforms of chitinase were observed in treated plants . In contTurmeric plants treated with chitosan showed an increase in chitosanase activity over a period of time and reached maximum on the 7th month. Nearly a threefold increase in chitosanase was observed in treated plants .Chitosanase separated on SDS-PAGE showed new polypeptides in chitosan treated turmeric plants. In control plants, chitosanase polypeptide with molecular mass 19 kDa was observed in all the months. Apart from this, 23 kDa chitosanase polypeptide was observed in the 5th month of control plants. In chitosan treated plants, apart from the constitutive chitosanase polypeptide (19 kDa), a new isoform was observed after the 4th, 5th, 6th, and 7th month with molecular mass ranging from 23–30 kDa . P. aphanidermatum showed a significant decrease in disease severity when compared to control (Chitosan treated plants challenged with control .β-1,3 glucanase isoforms [ Pythium aphanidermatum [ Apium graveolens, treated with chitosan showed a 20-fold increase in chitinase activity compared to that of chitosan-untreated plants and exhibited a delay in symptom expression caused by F. oxysporum [Inducing the plants own defense mechanisms by application of a biological inducer is thought to be a novel plant protection strategy. Chitosan can induce defense reactions in plants, including the induction of chitinase, chitosanase, and isoforms . Chitosaisoforms , 20. Chiisoforms , 22. In dermatum . Celery,xysporum . It has xysporum . Prapagdxysporum  reported P. aphanidermatum infection in turmeric plants. Increase in chitinase and chitosanase activity may play a role in enhanced resistance in turmeric plants against P. aphanidermatum infection.Chitosan played an important role in the growth suppression of


There was a graded association between Hcy tertiles and eGFR decline. Subjects in the 3rd tertile of Hcy levels had an accelerated rate of eGFR decline and an increased risk of incident CKD, as compared with those in the 1st tertile, after adjusting for age, gender, baseline diabetes, SBP, BMI, smoking, dyslipidemia, eGFR, folate and vitamin B12 levels. In conclusion, in this prospective cohort of Chinese hypertensive adults, elevated baseline plasma Hcy can serve as an independent biomarker to predict renal function decline and incident CKD.Hyper-homocysteinemia (HHcy) is associated with microalbuminuria and glomerular injury in general and diabetic populations. However, HHcy’s role in hypertensive patients was not studied. We investigated whether HHcy is an independent risk factor for renal function decline and development of chronic kidney disease (CKD) in hypertensive men and women. This was a community-based prospective cohort study of 2,387 hypertensive adults without CKD at baseline, with a mean follow-up of 4.4 years. Baseline and follow-up levels of plasma Hcy, folate, vitamin B12, blood pressure and other pertinent covariables were obtained. CKD was defined as an estimated glomerular filtration rate (eGFR) <60 ml/min/per 1.73 m Chronic kidney disease (CKD) is a serious clinical and public health challenge globally124579101112814Hcy is a sulfur-containing amino acid and an intermediate metabolite in methionine metabolism. Deficiency in Hcy metabolic enzymes and its co-factors, such as folate and other B vitamins, could lead to increased Hcy levels in both humans and mice18It is estimated that hypertensive disease affects 266 million adults in China, affecting nearly 27% of the adult population2121In this community-based prospective cohort study, we sought to investigate the independent effect of baseline Hcy, folate and vitamin B12 levels on renal function decline and incident CKD in Chinese hypertensive adults without baseline CKD.2 at baseline were analyzed. The mean blood pressure at baseline and at the end of the follow-up was 168 ± 98 and 152 ± 84 mmHg, respectively. Forty-four percent of participants received antihypertensive treatment. The percentage of participants achieving the target BP of <140/90 mmHg at the end of the follow-up was 21.2%. At baseline, men tended to be older, more likely to be a current smoker, and had higher DBP and lower serum total cholesterol, triglycerides and eGFR levels  and mean estimated glomerular filtration rate (eGFR) of 93.9 ml/min/per 1.73 mR levels . The medDuring 10,554 person-years (mean follow-up: 4.4 years), 69 (2.9%) participants developed CKD. As shown in When analyzed as a continuous variable, higher log-transformed Hcy was associated with higher incident CKD , even afFolate and vitamin B12 were not significantly associated with incident CKD in either unadjusted or adjusted models, respectively . When Hc2/year. As shown in The mean yearly change for eGFR in the cohort was −0.87 (Q1–Q3: −2.02 ~ 0.20) ml/min per 1.73 mHigher Hcy level was associated with a faster rate of eGFR decline in fully adjusted models . The hig2 as compared to those with eGFR>90 ml/min/per 1.73 m2. However, tests for interaction were not significant (p for interaction >0.05)  . In addin >0.05) . In addin >0.05) . HoweverIn this prospective study of 2,387 hypertensive adults, we identified a graded association between plasma Hcy levels and an increased risk of accelerated renal function decline and incident CKD. This association was independent of plasma folate and vitamin B12 levels and other traditional risk factors. Our findings suggest that elevated plasma Hcy levels, but not low folate and/or vitamin B12 levels, may predict progression of renal function decline and incident CKD in hypertensive adults.Increased plasma levels of Hcy, termed hyperhomocysteinemia, is diagnosed when plasma Hcy levels are greater than 15 μmol/L. HHcy is recognized as a potent risk factor and predictor for cardiovascular disease (CVD) in general populations25262729303132347Our study findings suggest that elevated Hcy levels might be a modifiable risk factor for CKD, and that preventive interventions should be considered for hypertensive adults with increased plasma Hcy levels. A number of strategies, including dietary modification of food intake components and supplementation with folic acid and vitamins B6 and B12, have been reported to be effective at lowering Hcy levels and reducing cardiovascular riskIt is to our surprise that plasma folate and vitamin B12 levels did not predict or correlate with the incident CKD and rapid decline of eGFR . These f13373839Our study has some limitations. As it was conducted in Chinese hypertensive adults, caution is needed when generalizing our findings to other populations. This study did not have the data on microalbuminuria, an important marker for the development and progression of CKD, which limits our ability to estimate the true incidence of CKD in this study.In conclusion, our study demonstrated that elevated plasma Hcy level is an independent predictor of rapid decline in renal function and incident CKD in a Chinese hypertensive population. Our findings, if further confirmed, may have important clinical and public health implications.2) at time of screening; (2) diagnosed secondary hypertension; (3) a history of stroke, myocardial infarction, heart failure, coronary revascularization, and/or congenital or acquired heart diseases; (4) cancer; and/or (5) liver disease and/or severe mental disorders.This community-based cohort study was approved by the Ethics Committees of the Institute of Biomedicine, Anhui Medical University. Investigation was performed in accordance with relevant guidelines and regulations. All participants were provided with written consent. The study was conducted in rural communities in Lianyungang, China. Eligible participants were those: (1) aged 45 to 75 years and (2) with hypertension (BP ≥ 140/90 mmHg or currently on antihypertensive therapy). Participants were excluded from the study if they had (1) confirmed CKD  were assessed at follow-up and participated in a subsequent scheduled visit after five years. Among the 2,518 participants, 131 were excluded from the analysis because of missing follow-up data. The final analysis included 2,387 participants.Data collection at baseline and at the follow-up visit were conducted by trained research staff according to a standard operating procedure.Resting blood pressures were measured using a mercury sphygmomanometer in a seated position after ≥10 minutes of rest. The mean of the three readings on the first baseline screening day was recorded and confirmed by three measurements on the second baseline screening day with an interval of at least one day. The mean of the three BP measurements on the second baseline screening day was used as the baseline BP for analyses.Questionnaires were administered to collect information on socio-demographic status, medical history and medications. Diabetes was defined as physician-diagnosed diabetes, or currently receiving hypoglycemic therapy, or fasting glucose level higher than 7.0 mmol/L.Anthropometric measurements, including height and weight, were taken using a standard operating procedure.Venous blood was drawn after overnight fasting during the baseline visit and the follow-up visit. Both plasma and serum samples were collected and stored at −80 °C. The samples were shipped by commercial cold chain transportation and measured in a central laboratory.Plasma Hcy, folate and vitamin B12 were measured by enzyme-cycling method using a commercial kit.Serum creatinine was measured by enzymatic method (sarcosine oxidase-PAP) using a commercial kit  according to the manufacturer’s protocol. The Beckman assay was calibrated to the Roche/Hitachi P module Creatinase Plus enzymatic assay , traceable to an isotope-dilution mass spectrometry assay at the National Institute of Standards and Technology41Fasting glucose, total cholesterol, triglycerides and HDL-C were measured using a Hitachi 7020 Automatic Analyzer .2. To avoid excluding participants with minor fluctuations in eGFR that may be due to “noise”, the definition also included a >1 ml/min/per 1.73 m2/year decrease in eGFRwas defined as follow-up eGFR <60 ml/min/per 1.73 m2/year. A negative eGFR change indicates a fall in eGFR.was calculated as the difference between the follow-up eGFR and baseline eGFR, which was divided by follow-up duration and expressed as ml/min/per 1.73 mt tests, signed rank tests, or chi-square test as appropriate. Multivariable logistic regression was used to assess the relations of Hcy, folate and vitamin B12 with incident CKD. Multivariate linear regression models were used to evaluate the associations between Hcy, folate and vitamin B12 with change in kidney function. In these analyses, Hcy, folate and vitamin B12 were modeled both as continuous variables and as categorical variables (categorized into tertiles). Hcy was natural log-transformed to normalize its skewed distributions. Because of gender differences in Hcy and folate distribution, gender-specific tertiles of Hcy and folate were used. The multivariate models were adjusted for age, gender, diabetes, SBP, BMI, smoking, cholesterol, HDL-C, triglycerides, eGFR, and previous use of ACEIs/ARBs. Effect modification was tested using a multivariate regression by stratifying the potential risk factors for renal outcomes. Adjusted splines of generalized additive models were created to evaluate the associations between baseline Hcy, folate and vitamin B12 and the outcomes. All tests were two-tailed and P < 0.05 was considered significant.All of the analyses were conducted using the statistical package R software, version 3.0.1How to cite this article: Xie, D. et al. Hyperhomocysteinemia predicts renal function decline: a prospective study in hypertensive adults. Sci. Rep.5, 16268; doi: 10.1038/srep16268 (2015).


However, present work evidenced that molecular docking of aloe emodin modulates the anti-inflammatory activity, as well as expression of COX-2 (cyclooxygenase-2) in rodent. The AEC  was synthesized using aloe emodin as starting material. The study was planned for evaluation of possible anti-inflammatory and antiarthritic activity in carrageenan rat induced paw oedema and complete Freund's adjuvant induced arthritis in rats. The AE  and AEC significantly (P < 0.001) reduced carrageenan induced paw edema at 50 and 75 mg/kg. Complete Freund's adjuvant induced arthritis model showed significant (P < 0.001) decrease in injected and noninjected paw volume, arthritic score. AE and AEC showed significant effect on various biochemical, antioxidant, and hematological parameters. Diclofenac sodium 10 mg/kg showed significant (P < 0.001) inhibition in inflammation and arthritis.Aloe emodin is isolated compound of aloe vera which is used traditionally as an anti-inflammatory agent. In vitro pharmacokinetic data suggest that glucuronosyl or sulfated forms of aloe emodin may provide some limitations in its absorption capacity. Aloe emodin was reported to have in vitro anti-inflammatory activity due to inhibition of inducible nitric oxide (iNO) and prostaglandin E Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by nonspecific inflammation of peripheral joints, destruction of articular tissues, and deformities in the joints. As the disease progresses, there are enhanced chances of bone damage and destruction of cartilage causing substantial disability . The conα, interleukin- (IL-) 1β, and IL-6, play key roles in the inflammation and joint damages during the development of RA [In particular, it was reported that the inflammatory cytokines, such as tumor necrosis factor- (TNF-) nt of RA . Epidemint of RA . Nonsternt of RA .In spite of their extensive usage, NSAIDs are associated with many adverse effects like myocardial infarction: Rofecoxib , gastric2, via its action on murine macrophages [The naturally occurring 1,8-dihydroxyanthraquinone derivatives  were obtained from various families such as Rhamnaceae , Liliaceae , Polygonaceae (rhubarbs), and Caesalpiniaceae (senna) . Aloe emrophages .Hence, taking into consideration reported activity of aloe emodin and molecular docking methodology, anthraquinone derivative was synthesized. The objective of forming this derivative was an attempt to reach an active anti-inflammatory agent with potent activity and selectivity toward COX-2. Molecular docking studies were carried out on these compounds to identify the structured feature required for effective bind to COX-2 enzyme. The most effectively bound ligand was taken as the active compound.Hence, the present study was planned to explore possible modulation of pro-anti-inflammatory potential by modifying the aloe emodin into its derivative for evaluation of anti-inflammatory potential by considering the docking results, because no such study has been carried out in the past.The aloe emodin was procured from aloe vera synthesis, Mumbai (India). Carrageenan was purchased from S. D. Fine lab. (India), Diclofenac was obtained from Hindustan Antibiotics Ltd. (India), and complete Freund's adjuvant was obtained from Sigma-Aldrich (USA).http://www.rcsb.org/pdb/, entry code 4COX) [ Gbinding = Gcomplex − (GΔligand + Gprotein), Gcomplex = G(ligand + protein), Gprotein = the energy of the protein after optimization (uncomplexed), Gligand = the energy of the ligand after optimization (uncomplexed), Gbinding is binding energy required for a ligand to bind a protein.ΔThe coordinates for the three-dimensional structure of COX-2 were obtained from Protein Data Bank (de 4COX) , 14. WatSynthesis of 4,5-Dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2 Carboxylic Acid from Aloe Emodin  See SchReaction. See Procedure. In this procedure the oxidizing medium was prepared by dissolving 2.5 g of sodium nitrite in 12 mL of sulphuric acid; the solution was heated to about 120°C. One gram of aloe emodin was added in parts to this mixture over a period of 30 min. The reaction mixture was kept at this temperature for 3 h. At the end of 3 h the reaction mixture was poured into 700 mL distilled water at 2°C to get orange brown precipitate . The precipitate so formed was filtered and dried to obtain crude 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2 carboxylic acid. This was then dissolved in sodium carbonate solution pH below 9.5 and extracted with organic solvent. The unreacted aloe emodin present gets extracted into the organic solvent (Dichloromethane). The compound is again regenerated from sodium bicarbonate solution using hydrochloric acid. The precipitate is then filtered, washed, dried, and recrystallized from methanol to obtain pure compound with yield of 95%.Rats. Albino Wistar rats of either sex weighing 150–250 g were used for present study. They were kept in polypropylene cages in an air-conditioned area at 22 ± 3°C in 10–14 h light dark cycle. They were provided with balanced feed and water ad libitum.The experimental protocol was approved by IAEC . Laboratory animal handling and experimental procedures were performed in accordance with CPCSEA guidelines .Carrageenan Induced Paw Edema in Rats. Anti-inflammatory activity of AE and AEC was tested using the carrageenan induced rat paw edema model [Vt is paw volume in mL, at time t, after carrageenan administration. Vc is paw volume in mL, before carrageenan administration. ConsiderEc is edema volume of control group. Et is edema volume of treated group.ma model . ExperimComplete Freund's Adjuvant Induced Arthritis in Rats. Adjuvant arthritis was induced as previously described by [ Mycobacterium tuberculosis per milliliter sterile paraffin oil into tibiotarsal joint of the left hind paw of female rats. The female Wistar rats weighing 180–230 g were divided into eight groups of six animals in each group as follows: group I: arthritic control/CFA (intraplantar injection of 0.1 mL CFA); group II: standard treated with Diclofenac sodium 10 mg/kg after intraplantar injection of 0.1 mL CFA, from 12th to 28th day; groups III–V: treated with AE 25, 50, and 75 mg/kg, respectively, after intraplantar injection of 0.1 mL CFA, from 12th to 28th day; groups VI–VIII: treated with AEC 25, 50, and 75 mg/kg, respectively, after intraplantar injection of 0.1 mL CFA, from 12th to 28th day.ribed by  as modifribed by . On day The following parameters were measured.(A) Paw Volume Evaluation (in mL). Paw volume was measured on days 0, 4, 8, 14, 21, and 28 by using Plethysmometer . Mean changes in injected and noninjected paw edema, with respect to initial paw volume, were calculated on respective day and % inhibition of paw edema with respect to untreated group was calculated using the following formula:i is % inhibition of paw edema and Δv treated is mean changes in paw volume of treated rat. Δv untreated is mean changes in paw volume of untreated rat.(B) Visual Arthritis Scoring System. The visual arthritis scoring system described by [ribed by , 20 was (C) Antioxidants. On the 28th day, animals were anaesthetized by ether and sacrificed by cervical dislocation. The liver of each animal was removed rapidly and washed with ice cold Tris buffer. Liver of each animal was cut into small pieces and homogenized with homogenizer, so that clear homogenate is formed. Homogenates were used for estimation of LPO (lipid hydroperoxide), GSH, SOD, and catalase. Reduced glutathione (GSH) in liver was estimated according to the method described by [μg of GSH/g of tissue. Superoxide dismutase (SOD) activity was estimated using the technique of [μmol H2O2 decomposed/min/g of tissue. The LPO (lipid hydroperoxide) end product malondialdehyde (MDA) measured in the homogenate was estimated by using method of [ribed by . The connique of . The SODnique of , 24. CATethod of . The con(D) Nitrite Content. The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide (NO), was determined with a colorimetric assay with Griess reagent (0.1% N-(1-Naphthyl)ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% phosphoric acid). The concentration of nitrite i was determined from a sodium nitrite standard curve.(E) Hematological Parameters. On the 28th day, blood was withdrawn through retroorbital plexus puncture from all groups by under light ether anesthesia and the hematological parameters like hemoglobin content, total WBC count, ESR, and RBC were analyzed using Culter CB-9000, Chariot.(F) Biochemical Parameter. On the 28th day, blood was withdrawn through retroorbital plexus of all groups and the biochemical parameters SGPT (serum glutamate pyruvic transaminase), SGOT , and ALP were analyzed using standard kits followed by fully automated analyzer.n = 6). Statistical significance was determined using one-way ANOVA followed by Dunnett test. ∗P < 0.05, ∗∗P < 0.01 when compared with control group.Values were expressed as mean ± SEM (G) on cyclooxygenase protein (4COX) using VLife MDS were as shown in The results of docking studies of anthraquinone derivatives  at doses of 50 and 75 mg/kg when compared with control group  as compared to arthritic control group. Group treated with AEC 25 showed significant decrease in paw volume on the 28th day (P < 0.05) as compared to arthritic control (P < 0.01) as compared to arthritic control. AE 50 treated groups showed significant decrease in the noninjected paw edema volume on the 14th day (P < 0.05) also on the 21st and 28th day (P < 0.01) as compared to arthritic control. AEC 25 treated groups showed significant decrease in the noninjected paw edema volume on the 28th day (P < 0.05) as compared to arthritic control (Treatment with Diclofenac sodium (10 mg/kg), AE (50 and 75 mg/kg), and AEC (50 and 75 mg/kg) showed significant decrease in injected paw edema volume on the 14th, 21st, and 28th day ( control . Groups  control .P < 0.01) as compared to arthritic control. Groups treated with AE 50, AE 75, AEC 50, and AEC 75 showed significant decrease in the arthritic index, on the 21st and 28th (P < 0.001) day, respectively, while AEC 25 treated groups showed significant decrease in the arthritic index on the 28th day (P < 0.05) as compared to arthritic control , while it showed significant increase in RBC and Hb count (P < 0.01), as compared to arthritic control. Groups treated with AE 50, AE 75, AEC 50, and AEC 75 showed significant decrease in WBC count, RF, and ESR (P < 0.01), while they showed significant increase in RBC and Hb count (P < 0.01), as compared to arthritic control. Group treated with AEC 25 showed significant decrease in WBC count and RF (P < 0.05) when compared with arthritic control , and erythrocyte sedimentation rate (ESR) (l Tables .P < 0.01) as compared to arthritic control and significant increase in GSH, CAT, and SOD (P < 0.01) as compared to arthritic control. Groups treated with AE 50, AE 75, AEC 50, and AEC 75 showed significant increase in GSH, CAT, and SOD (P < 0.01) as compared to arthritic control. Group treated with AEC 25 showed significant decrease in LPO and NO level (P < 0.05), while it showed significant increase in GSH, CAT, and SOD (P < 0.05) as compared to arthritic control  as compared to arthritic control. Groups treated with AE 50, AE 75, AEC 50, and AEC 75 showed significant decrease in SGPT, SGOT, and ALP levels (P < 0.01) as compared to arthritic control. Group treated with AEC 25 showed significant decrease in SGOT level (P < 0.05) as compared to arthritic control  is a symmetric polyarticular arthritis that primarily affects the small diarthrodial joints of the hands and feet. In addition to inflammation in the synovium, which is the joint lining, the aggressive front of tissue called pannus invades and destroys local articular structures. The synovium is normally a relatively a cellular structure with a delicate intimal lining. In RA, CD4+ T cells, B cells, and macrophages infiltrate the synovium and sometimes organize into discrete lymphoid aggregates with germinal centres. Hyperplasia of the intimal lining results from a marked increase in macrophage-like and fibroblast-like synoviocytes. Locally expressed degradative enzymes, including metalloproteinases, serine proteases, and aggrecanases, digest the extracellular matrix and destroy the articular structures .The structure of COX-2 was obtained from Protein Data Bank (PDB Code: 4COX) and used as target for docking studies. The docking study was carried out on VLife MDS docking software. The docking study was carried out on aloe emodin (AE) and carboxylic acid derivative of aloe emodin (AEC). The docking results of AEC were compared with that of AE based on various parameters such as Dock score, bond interactions, and binding free energy. The docking result showed that the AEC have favorable interactions with active site residues and also have favorable Dock score and binding free energy as compared with aloe emodin (AE), indicating that AEC may be having a better anti-inflammatory activity than aloe emodin.In the present study, AE and AEC exhibited significant anti-inflammatory and antiarthritic activity. The carrageenan is known for its classic biphasic effect; the first phase is mediated by release of histamine and serotonin during the first hour and release of kinins up to 2.5 h, while the second phase is mediated by release of prostaglandins from 2.5 to 6 h . It has α, IL-12, IL-6, and IFN-γ and several chemokines [In the present study, rats were selected to induce arthritis because rats develop a chronic swelling in multiple joints with influence of inflammatory cells, erosion of joint cartilage, and bone destruction. It has close similarities to human rheumatoid disease . The patemokines .γ, and TNF-α which have been implicated in immune arthritis [α is mainly involved in the perpetuation of the inflammatory cascades in autoimmune diseases, which affect connective tissues where the connective tissues become hypercontracted due to inflammation [Paw swelling is an index of measuring the antiarthritic activity of various drugs . The detrthritis , 37. IL-rthritis . TNF-α iammation .Prostaglandins greatly potentiate exudates by inducing relaxation of arteriolar smooth muscle cells, increasing the blood supply to the tissue . In the The appearance of secondary lesions, that is, CFA-noninjected paw swelling, is a manifestation of cell-mediated immunity. The appearance of secondary lesions is due to development of biochemical reactions into CFA-noninjected hind leg which results in swelling around tibiotarsal joint and paw. The suppression of such secondary lesions by a drug shows its immunosuppressive activity , 42. Theα/β globulin, and such a rise in the ESR indicates an active but obscure disease process [Anemia is commonly noted in patients with chronic arthritis . The two process . The acu process .Prominent immunologic abnormalities that may be important in pathogenesis of RA include immune complexes that are found in joint fluid cells and in vasculitis. Plasma cells produce antibodies  that contribute to these complexes. Serum RF measures the amount of antibody IgM titre present in the serum . RF is tIn arthritic condition there is a mild to moderate rise in WBC count due to release of IL-Ib inflammatory response. IL-Ib increases the production of both granulocyte and macrophages colony stimulating factor , 53. In The body has effective antioxidant mechanism to prevent and neutralize the free radical induced damage. This is accomplished by a set of endogenous antioxidant enzymes, such as SOD and CAT. When the balance between ROS (reactive oxygen species) production and antioxidant defense is lost, “oxidative stress” results, which through a series of events deregulates the cellular function leading to various pathological conditions .2O2, respectively, thereby protecting the cells against oxidative free radicals induced damage. H2O2 may be reduced by enzymes glutathione peroxide but, alternatively, may react again with superoxide anion to form free hydroxyl radicals, which have a greater toxicity and a longer half-life than superoxide anion. Although catalase is significantly increased in rheumatoid arthritis its concentration is very low to expect considerable protection against H2O2 [2O2 and suggests that these enzymes may play an important role in the rheumatic process and increased oxidative stress [Biological systems have evolved an array of enzymatic and nonenzymatic antioxidant defense mechanisms to combat the deleterious effects of oxidative free radicals (OFRs). SOD is a metalloprotein while CAT is a hemoprotein, localized in the peroxisomes or the microperoxisomes. Both superoxide dismutase (SOD) and catalase play an important role in the detoxification of superoxide anion and Hnst H2O2 , 56. In e stress .The GSH is a predominant low molecular weight thiol in the cytoplasm, which protects the tissue against in vivo toxicity of sulfhydryl—binding toxicants , 59. TheLipid peroxidation (LPO) is an important marker of oxidative stress and is analyzed by malonaldehyde. Increased ROS levels in RA may result in a prooxidation environment, which in turn could result in increased MDA levels.2O2 or by reducing the oxidative stress.As a result, LPO may have a role in the pathogenesis of the RA . In the Studies have revealed increased nitric oxide (NO) levels in the serum and synovial fluids of arthritic patients owing to the upregulation of inducible nitric oxide synthase (iNOS), indicating thereby a role of NO in arthritis. In experimental models of arthritis, selective inhibitors of iNOS have been observed to ameliorate the symptoms of joint inflammation . NO leveAssessment of liver injury is generally done by ascertaining the levels of biomarkers such as SGOT, SGPT, and ALP. Elevated levels of these enzymes in serum suggest injury to the architecture of hepatic cells resulting in leaching of these enzymes into the circulation. Liver impairment is a typical feature in adjuvant arthritis. Tissue damage in adjuvant induced arthritis was assessed based on enzyme levels in serum. The present study in which significant rise in the level of aminotransferase was observed in animals treated with Freund's complete adjuvant suggests that it might be released from the damaged cells of the liver . In the A growing body of evidence suggests a potential interplay between AE (Aloe emodin) and AEC . In fact, AEC have a protective role as compare to AE on inflammatory and arthritic abnormalities besides Increase specificity lowering effect, and their Anti-inflammatory potentials could be partly related to a mechanism by inhibiting COX-2 .


Purpose. To associate baseline patient characteristics and relapse across consecutive COG studies. Methods. We analyzed risk factors for LESFT patients in three randomized COG trials. We evaluated age at enrollment, primary site, gender, tumor size, and treatment (as randomized). We estimated event-free survival  and compared risk across groups (log-rank test). Characteristics were assessed by proportional hazards regression with the characteristic of interest as the only component. Confidence intervals (CI) for RR were derived. Factors related to outcome at level 0.05 were included in a multivariate regression model. Results. Between 12/1988 and 8/2005, 1444 patients were enrolled and data current to 2001, 2004, or 2008 were used. Patients were with a median age of 12 years (0–45), 55% male and 88% Caucasian. The 5-year EFS was 68.3% ± 1.3%. In univariate analysis age, treatment, and tumor location were identified for inclusion in the multivariate model, and all remained significant (p < 0.01). Since tumor size was not collected in the last study, the other two were reanalyzed. This model identified age, treatment, tumor location, and tumor size as significant predictors. Conclusion. Age > 18 years, pelvic tumor, size > 8 cms, and chemotherapy without ifosfamide/etoposide significantly predict worse outcome. AEWS0031 is NCT00006734, INT0091 and INT0054 designed before 1993 (unregistered). The 5-year event-free survival (EFS) for nonmetastatic Ewing sarcoma patients has improved to 60–70% –6, but 2The Children's Oncology Group (COG) and its predecessors conducted three studies 1988–2005) for newly diagnosed localized Ewing sarcoma (ES) or peripheral primitive neuroectodermal tumor (PNET) of bone [988–2005 We sought to pool the high-quality dataset represented by those COG trials , 2, 5 toWe analysed selected factors at study enrolment for 1444 patients treated in three COG studies: (1) INT-0091 ; and (3)Briefly, patients < 30 years of age with newly diagnosed, histologically confirmed primary ES or PNET were eligible for enrolment in INT-0091 . The dos2 (2 mg maximum dose), doxorubicin 75 mg/m2/dose , and cyclophosphamide 1200 mg/m2, followed by mesna uroprotection. Dactinomycin 1.25 mg/m2/d was substituted for doxorubicin when a total doxorubicin dose of 375 mg/m2 was reached in INT-0091. Courses of IE included ifosfamide 1800 mg/m2/d for 5 days, given with mesna uroprotection and etoposide 100 mg/m2/d over the same 5 days.The standard chemotherapy doses included the following: VCD—vincristine 2 mg/mThe studies prescribed local control following 12 weeks of therapy, which in the first two studies . AlthougWe defined EFS as the time from study entry until the occurrence of an analytic event or date of last contact, whichever came first. An analytic event was defined as disease progression, diagnosis of second malignant neoplasm (SMN), or patient death prior to the development of disease progression or SMN. A patient who had not experienced an event by the date of last follow-up was censored.Exploratory analysis was complicated by the fact that some data were not collected in all studies and even when intended to be collected some data were missing. The major sources and types of missing data were as follows: (1) INT-0091 excluded patients with extraosseous ES from enrollment; (2) in AEWS0031 tumor size was not collected; and (3) tumor size was not reported for 266 participants in the other two studies. We therefore excluded tumor size and soft-tissue ES from our first multivariate model. Participants whose data were truly missing  were also eliminated from the analysis.The distributions of EFS and overall survival were estimated by the method of Kaplan and Meier . Risk ofOnly potential prognostic factors measured at study enrollment were assessed in the dataset. In particular, the relationship between risks of an event and death and local control modalities were not analyzed. The potential prognostic factors considered included the following: (1) patient age at enrollment; (2) site of primary tumor; and (3) patient sex. Age at enrollment was categorized.All the factors noted above were explored further to assess their relative prognostic effects when considered jointly. To determine whether tumor size had an impact on outcome using COG-directed therapy, we performed a second multivariate analysis including only patients enrolled in INT-0091 and INT-0154, where tumor size in two perpendicular dimensions was requested but not required for eligibility. This allowed us to determine whether tumor size was an independent predictor of EFS using US treatment (in COG). For this analysis, tumor size was categorized.A total of 1444 patients were enrolled in INT-0091, INT-0154, and AEWS0031 between December 1988 and August 2005 . The INTp = 0.0071). This is likely driven by the absence of IE in the control arm of INT-0091, since INT-0154 and AEWS0031 have very similar EFS.The 5-year EFS for the 1444 patients enrolled in the three studies was 68.3%  ± 1.3% . The 5-yp = 0.061) and are just outside conventional statistical significance. Additionally, the RR of an event using the non-IE arm of INT-0091 relative to the standard timing arm of AEWS0031 is 1.5 (1.12–2). This confirms the inferiority of non-IE containing regimens as the risk of an event is 50% higher in those patients. The RRs are similar for patients in the standard timing IE treatments.As seen in Univariate analysis identified three variables for consideration in assessing the relative values of prognostic factors measured at study enrollment : (1) patp = 0.000)) compared to patients ≤ 9 years.p = 0.2587).Tumor location and assigned treatment also retain their role as significant predictors of EFS in the presence of one another. Patients with a pelvic tumor have a higher event risk RR 1.34 (1.07–1.67) than patients with nonpelvic tumors. Assigned treatment was also an important predictor of outcome and patients treated with non-IE containing treatment had an increased event risk RR 1.84 (1.33–2.53). In our multivariate analysis, risk of event was unrelated to patient sex. The estimates of the effects of age, tumor site, and treatment did not differ significantly between trials  and patients with soft-tissue tumors (not included in INT-0091). We excluded these factors from our first multivariate model rather than employ methods to impute the missing information and adjust We were able to confirm the importance of age ≥ 18 years as an independent predictor of worse EFS. Though older age has been consistently identified as predicting higher event rates in multiple series , 13, 14,Tumor location also predicted increased event risk; patients with pelvic primaries were particularly at high risk. This finding agrees with other studies of ES , 15, 24 Assigned chemotherapy treatment was an important predictor of EFS in our study. As expected from results of our previous trials , 2, the We were able to document the importance of tumor size in the context of tumor location in our second multivariate analysis (limited to INT-0091 and INT-0154), which revealed a twofold increased risk of an event for patients with tumors larger than 13 cm (RR 2.00 (1.44–2.79)). On average, patient risk appeared to increase with larger tumor size. The size of our population limited our sensitivity to these more subtle differences. Tumor size and/or volume have been identified in a number of studies as significant predictor of event risk , 17 and In conclusion, this is the largest series of US treated ES patients receiving a similar therapy backbone. We confirmed that primary tumor site (in the pelvis) and age ≥ 18 years are predictive of an increased event risk and should be considered at the time of treatment assignment. Patients ≥ 18 years and those with pelvic tumors might benefit from new treatment strategies. For example, chemoradiotherapy may allow resection for a larger number of pelvic tumors. Larger tumor size is also a predictor of worse EFS and should be included as a stratification factor in future US trials.