Differential Protein and Morphological Responses of Mosses to Heavy Metal Exposure: Insights from SDS-PAGE Analysis and Microscopic Examination

Antimicrobial efficacy of endodontic irrigation solutions against planktonic microorganisms and dual-species biofilm

Developing new plant-derived medicines for treating malignant tumors involves the selection of plants with antitumor activity. Only a few plants show such activity, and testing them on animals with transplanted tumors is laborious and restricts the scope for routine analysis. Therefore, for many years efforts have been devoted to selecting preparations from their effects on tumor cell suspensions in vitro. Here I consider methods of selecting preparations based on the optical differentiat ion of living and dead cells in aseitic fluid f rom mouse Ehrlich carcinoma with Schrek straining [1]. The preparation is mixed in a 1:1 ratio with fresh ascitic fluid and the mixture is kept in a thermostat at 37~ for 3 h. During that time, it acts on the cells, some of which die. Then an eosin solution is added to the mixture. The dead cells are stained, while the live ones remain colorless. In existing methods, the ratio of the number of dead cells to the total number is determined by counting under the microscope. This has low accuracy and restricted use in routine analysis because of visual fatigue on prolonged microscope use. My method defines the proportion of cells killed by the extract without the use of a microscope by means of a colorimeter. After the mixture of ascitic fluid (1 ml) and plant extract (1 ml) has been kept in the thermostat for 3 h and the dead cells have been stained with eosin (2 ml of 0.05% aqueous solution), the material is passed through a centrifuge, and the deposited cells are washed three times with distilled water. To the washed material, one adds 3 ml of 0.2 M potassium hydroxide solution and heats the mixture on a water bath for 15 min. The cells dissolve completely and the eosin f rom the stained ones becomes dark green. The color strength under otherwise identical conditions is directly related to the number of stained (i.e., dead) ceils. The colorimeter is used with a graph to determine the proportion of cells killed by the preparation. The calibration curve is constructed from two standard specimens: one where all the cells have been killed (for example, by heating or with mercury chloride) and the other where the cells have been exposed only to physiological saline. The standard specimens are prepared in the same way as the others and assayed. The extinction is plotted as a function of the proportion of dead cells with those two points, through which one draws a straight line, which is used in interpreting the colorimeter readings. When the samples and standards are prepared, all the solutions must be handled under identical conditions: in each series, one should use carefully mixed ascitic fluid, with each centrifugate washed with the same amount of water and so on. If the color provided by a standard specimen differs so much from that for the samples that the colorimeter scale does not allow one to compare them, one can dilute the solutions with 0.2 M potassium hydroxide, e.g., by a factor of two, and then take the proportion of dead cells as 50%, which corresponds to the dilution. I also examined how color in the specimens affects the result. Even highly colored aqueous extracts f rom dried plants do not influence the result substantially. Plant juices sometimes may be adsorbed by the ceils in ascitic fluid and thus strengthen the color in the solutions prepared for colorimetry. When tests are made on highly colored plant juices, one should use two solutions for each sample for colorimetry, not one. One of them is prepared as described above, and the other with the difference that after incubation in the thermostat one adds not eosin but 2 ml of water. The color in the first solution is due to the eosin and the plant juice residues. The color in the second is due only to the residues, whose concentration is as in the first. One takes the difference in the extinctions, and then determines the proportion of cells killed by the extract or juice.

On the Hydrogen-Ion Concentration of Hay Infusions, With Special Reference to its Influence on the Protozoan Sequence

Streptococcus suis serotype 2 (SS2) is a zoonotic pathogen that can cause meningitis both in pigs and in human beings. However, the pathogenesis of central nervous system (CNS) infection caused by SS2 have not yet been elucidated. To find the key molecules in cerebrospinal fluid (CSF) needed for the pathogenesis, a SS2 meningoencephalitic pig model and a SS2 non-meningoencephalitic pig model were established in this study. CSF was collected from infected piglets, and protein profiling was performed with label-free proteomics technology. A total of 813 differential proteins, including 52 up-regulated proteins and 761 down-regulated proteins, were found in the CSF of meningoencephalitic pigs compared with both non-meningoencephalitic pigs and healthy pigs. These 813 differential proteins were clustered into three main categories, namely, cellular component, biological process, and molecular function by gene ontology (GO) analysis. The most enriched subclasses of differential proteins in each category were exosome (44.3%), energy pathway (25.0%) and catalytic activity (11.3%), respectively. The most enriched subclasses of upregulated proteins were extracellular (62.1%), protein metabolism (34.5%) and cysteine-type peptidase activity (6.9%), and of downregulated proteins were exosomes (45.0%), energy pathway (24.0%) and catalytic activity (9.4%). Then, the differential proteins were further investigated by using the KEGG database and were found to participate in 16 KEGGs. The most enriched KEGG was citrate cycle (56.6%), and some of these differential proteins are associated with brain diseases such as Huntington's disease (18.6%), Parkinson's disease (23.8%) and Alzheimer's disease (17.6%). Sixteen of the 813 differential proteins, chosen randomly as examples, were further confirmed by enzyme-linked immunosorbent assay (ELISA) to support the proteomic data. To our knowledge, this is the first study to analyze the differential protein profiling of CSF between SS2 meningoencephalitic piglets and non-meningoencephalitic piglets by employing proteomic technology. The discovery and bioinformatics analysis of these differential proteins provides reference data not only for research on pathogenesis of SS2 CNS infection but also for diagnosis and drug therapy research.

Abstract. Sasmita R, Mabrur, Rahmy USA, Badruzsaufari. 2019. Genetic diversity and phylogenetic relationship among Anabantoidei fish (Anabantiformes) in South Kalimantan, Indonesia based on SDS-PAGE analysis. Biodiversitas 20: 2519-2527. Protein profile is a molecular marker for diversity and phylogenetic analysis of germplasm, including Anabantoidei fish which is abundant in freshwater ecosystem of South Kalimantan. The aim of this research was to analyze genetic diversity and construct phylogenetic relationship among Anabantoidei fish based on SDS PAGE analysis. Protein was extracted from muscle tissue of six species of Anabantoidei fish and precipitated using Ammonium sulfate salt. Soluble protein content was determined using Bradford assay and then separated based on SDS-PAGE method. Genetic diversity and phylogenetic relationship were constructed using PAST software based on UPGMA method. The results showed that the soluble protein of fish muscle can be precipitated optimally in different ammonium sulfate concentration. Based on SDS-PAGE analysis, 59 different protein bands have been separated from gels with molecular weight ranging from 28.15 to 181.61 kDa. On the protein level, the Anabantoidei fish showed high genetic polymorphism (greater than 90%) with 3, 2 and 5 monomorphic bands on non-precipitated, AS-precipitated and combination between non- and AS-precipitated proteins, respectively. The phylogenetic reconstruction also exhibited that the Anabantoidei fish has the unique phylogenetic trees, especially for the combined protein datasets. This information would be useful for freshwater fish conservation and breeding programs.

Approximately two million traumatic brain injury (TBI) incidents occur annually in the United States, yet there are no specific therapeutic treatments. The absence of brain injury diagnostic endpoints was identified as a significant roadblock to TBI therapeutic development. To this end, our laboratory has studied mechanisms of cellular injury for biomarker discovery and possible therapeutic strategies. In this study, pooled naïve and injured cortical samples (48 h postinjury; rat controlled cortical impact model) were processed and analyzed using a differential neuroproteomics platform. Protein separation was performed using combined cation/anion exchange chromatography-PAGE. Differential proteins were then trypsinized and analyzed with reversed-phase LC-MSMS for protein identification and quantitative confirmation. The results included 59 differential protein components of which 21 decreased and 38 increased in abundance after TBI. Proteins with decreased abundance included collapsin response mediator protein 2 (CRMP-2), glyceraldehyde-3-phosphate dehydrogenase, microtubule-associated proteins MAP2A/2B, and hexokinase. Conversely C-reactive protein, transferrin, and breakdown products of CRMP-2, synaptotagmin, and alphaII-spectrin were found to be elevated after TBI. Differential changes in the above mentioned proteins were confirmed by quantitative immunoblotting. Results from this work provide insight into mechanisms of traumatic brain injury and yield putative biochemical markers to potentially facilitate patient management by monitoring the severity, progression, and treatment of injury.

This study examined how aqueous extracts from Archidium ohioense, Hyophila involuta and Pelekium gratum influenced the seed germination of Capsicum chinense, Capsicum annuum and Capsicum frutescens. This was with a view to determining the suitability and effectiveness of the aqueous extracts in stimulating seed germination. The Capsicum seedlings were raised in Petri-dishes layered with Whatman No. 1 filter paper dampened with 5.0 ml of the respective moss extract, which was prepared by soaking 250 g of each of the moss species separately in 2.0 litres of distilled water for 72 hours. The Petri-dishes for the control were dampened with 5.0 ml distilled water. All the prepared Petri-dishes were incubated at ambient temperature in the laboratory for fourteen (14) days. The effects of the extracts were observed and recorded noting the germination percentage, plumule length and radical length. Data generated were analysed using Analysis of Variance (ANOVA) to identify significant differences (P < 0.05) among the treatments. Mean comparisons were performed using Least Significant Difference (LSD) Test. The findings revealed that the aqueous extracts from the selected moss species had a beneficial impact on the seed germination of the three species of Capsicum. Aqueous extract of H. involuta had the most stimulatory effect on all the three species of Capsicum, followed by A. ohioense, P. gratum and the control respectively. The highest germination percentage (76%) and plumule length (25.56 mm) were recorded in C. frutescens while the highest radical length (30.48 mm) was recorded in C. chinense. There was a significant difference in the results obtained for percentage germination, radical length and plumule length (P < 0.05) compared to that of the control. It was concluded that the three tested moss aqueous extracts stimulated the seed germination of the three Capsicum species and can be considered suitable for promoting seed germination in cultivating Capsicum.

Angiosperms and bryophytes, though distantly related plant groups, have many similar ecological and economic implications, including medicinal value. Therefore, this study aimed to analyse the phenolic and flavonoids composition and antioxidant and antimicrobial activities of Helianthus annuus L. (Angiosperms-Asteraceae) and another plant, a moss (Bryophyta), Hyophila involuta (Hook.) Jaeg., aerial parts (leaves), prepared in four different extracts (methanol, chloroform, distilled water, and petroleum ether). Phytochemical screening was conducted using standard methods of precipitation and colouration reactions. The Folin-Ciocalteu method was employed to determine the total phenol content, while the Aluminium Chloride Colorimetric method was used for flavonoid content determination. The antioxidant activity was measured through two methods: DPPH and NOSA scavenging activity. The phytochemical screening detected the presence and absence of fixed oils and fats, flavonoids, saponins, terpenoids, tannins, polyphenols, carbohydrates, and glycosides in both plants. The antibacterial and antifungal activity of both plants' methanolic extracts was examined against bacterial and fungal pathogens, i.e., Escherichia coli and Bacillus subtilis and fungal strains, i.e., Fusarium oxysporum and Aspergillus niger. The results were compared to a regular antibiotic disc and negative control that served as a methanol solvent. The methanolic extract of H. annuus has higher total phenol and flavonoid content, as well as antioxidant and antimicrobial activity, than H. involuta. Based on these data, it can be concluded that, while H. annuus is more effective than H. involuta, both distantly related plant species have similar phytochemical profiles and should be included equally in future herbal compositions.

Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an approximately 58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of approximately 3.6 microg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed alpha-chitin binding activity with Kd approximately 0.64 microm and Bmax approximately 4.68 micromol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an approximately 8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity.

Spatially targeted proteomics analyzes the proteome of specific cell types and functional regions within tissue. While spatial context is often essential to understanding biological processes, interpreting sub-region-specific protein profiles can pose a challenge due to the high-dimensional nature of the data. Here, we develop a multivariate approach for rapid exploration of differential protein profiles acquired from distinct tissue regions and apply it to analyze a published spatially targeted proteomics data set collected from Staphylococcus aureus-infected murine kidney, 4 and 10 days postinfection. The data analysis process rapidly filters high-dimensional proteomic data to reveal relevant differentiating species among hundreds to thousands of measured molecules. We employ principal component analysis (PCA) for dimensionality reduction of protein profiles measured by microliquid extraction surface analysis mass spectrometry. Subsequently, k-means clustering of the PCA-processed data groups samples by chemical similarity. Cluster center interpretation revealed a subset of proteins that differentiate between spatial regions of infection over two time points. These proteins appear involved in tricarboxylic acid metabolomic pathways, calcium-dependent processes, and cytoskeletal organization. Gene ontology analysis further uncovered relationships to tissue damage/repair and calcium-related defense mechanisms. Applying our analysis in infectious disease highlighted differential proteomic changes across abscess regions over time, reflecting the dynamic nature of host-pathogen interactions.

The hydroxamate siderophore receptor FhuA is a TonB-dependent outer membrane protein of Escherichia coli composed of a C-terminal 22-stranded beta-barrel occluded by an N-terminal globular cork domain. During siderophore transport into the periplasm, the FhuA cork domain has been proposed to undergo conformational changes that allow transport through the barrel lumen; alternatively, the cork may be completely displaced from the barrel. To probe such changes, site-directed cysteine mutants in the cork domain (L109C and Q112C) and in the barrel domain (S356C and M383C) were created within the putative siderophore transport pathway. Molecular modeling predicted that the double cysteine mutants L109C/S356C and Q112C/M383C would form disulfide bonds, thereby tethering the cork and barrel domains. The double cysteine FhuA mutants were denatured under nonreducing conditions and fluorescently labeled with thiol-specific Oregon Green maleimide. Subsequent SDS-PAGE analysis revealed two distinct species: FhuA containing a disulfide bond and FhuA with free sulfhydryl groups. To address the role of the putative siderophore transport pathway and to evaluate possible rearrangements of the cork domain during ferricrocin transport, disulfide bond formation was enhanced by an oxidative catalyst. Cells containing double cysteine FhuA mutants that were subjected to oxidation during ferricrocin transport exhibited disulfide bond formation to near completion. After disulfide tethering of the cork to the barrel, ferricrocin transport was equivalent to transport by untreated cells. These results demonstrate that blocking the putative siderophore transport pathway does not abrogate ferricrocin uptake. We propose that, during siderophore transport through FhuA, the cork domain remains within the barrel rather than being displaced.

Background: Current preoperative differentiation of premalignant and malignant pancreatic cysts from benign pancreatic cysts based on imaging and cyst fluid analysis is inadequate. This inability to accurately differentiate has resulted in unnecessary surgical resections. This is the first study applying proteomic technology to identify novel proteins or protein patterns in pancreatic cyst fluid which may more accurately predict pancreatic cyst pathology preoperatively and help triage patient care. Methods: Pancreatic cyst fluid was collected by endoscopic ultrasound guided FNA from patients with pathologically confirmed pancreatic mucinous and non-mucinous cysts, centrifuged and stored immediately at −80°C. Fluid from representative pancreatic cysts was prepared for proteomic analysis using centrifugation, acetone, ethanol and TCA precipitation, followed by solubilization into highly chaotropic nonionic detergents and buffer for 2-D electrophoresis and 2 dimensional liquid chromatography (PF2D: 1-D chromatofocussing, 2D-reverse phase). Spots from 2D gels were robotically excised, digested and mass spectrometry was used to identify unique differential proteins using liquid chromatography tandem mass spectrometry (LC-MS/MS) and MALTI-TOF. Proteins were identified using Mascot search engine and Sequest search algorithms. Results: 122 pancreatic cyst fluid samples were collected, stored and processed according to the protocol. Initial protein quantification for ranged from 1 to 5 mg/ml. Pancreatic cyst fluid from 5 samples (2 mucinous and 3 serous cystadenomas) was used for initial proteomic analysis (2D gel electrophoresis (2DE:(3)), PF2D(2)). Initial analysis shows a striking differential protein profile between serous and mucinous samples (by PF2D) in both dimensions (1-D chromatofocussing and 2-D reverse phase). Comparison of two-dimensional maps of the pancreatic serous and mucinous cystadenomas show few overlaps thus suggesting many proteins unique to each cystic neoplasm. Interpretable 2-D electrophoresis gels were obtained from pancreatic cyst fluid. Using 2-D Electrophoresis we have already identified over 20 unique cellular and membrane proteins using LC-MS/MS. Through 2-D electrophoresis gel we have identified cellular and membrane proteins using LC-MS/MS. Chromatofocussed fractions were processed over a second dimension HPRP column to produce over 700 fractions in each experiment. The second dimension reverse phase fractions for serous cyst show a very different protein profile than mucinous cysts. Conclusions: To our knowledge, this is the first report detailing the proteome of pancreatic cystic neoplasm fluid. Differential protein expression was found between pancreatic mucinous and serous cystadenoma fluid. Identification of specific protein patterns or proteins may be used as highly accurate serum based or cyst fluid based biomarkers.

Background:To date, there is limited information on the progression of human infections of avian influenza virus A (H7N9). This study investigated differential blood protein profiling of a H7N9-infected family cluster to find a slice of crucial proteins concerning disease attack and virus clearance.Materials and Methods:Plasma samples from one family cluster (including one index case and one asymptomatic case) were collected at four time points. The protein profiles were identified by isobaric tagging for relative and absolute quantification-based quantitative differential LC/MS/MS, and their functional annotations were analyzed by PANTHER and STRING tools.Results:A total of 1257 nonredundant proteins were identified from 3027 unique peptides. Three differential protein profiles for each subject were generated by comparing relative protein abundance between samples of each of the first three time points and the last time point. Gene ontology analysis indicated that differential protein profiles for the two cases were mainly enriched in the biological processes of response to stimulus, immunity, blood coagulation, lipid transport, and cell adhesion. Two groups of proteins with an upward or downward expression change according to the postinfection time points were detected for each case. STRING analysis further indicated that the hubs in the network of these time-dependent proteins were mostly apolipoproteins.Conclusions:Significant perturbation of the response upon viral infection occurred immediately after confirmation of H7N9 virus infection. The differential protein profiles shed further light on distinguishing the index case from the asymptomatic one. Furthermore, apolipoproteins may play an important role in the progression of the disease.

Salmonella bacteria causes a wide variety of disease and disease syndrome in different animals, birds including human beings and remains as a serious problem with public health significance throughout the world. A suitable vaccine or suitable immunogen detection system is not yet still available. However, it is interesting to characterize of a common immunodominant surface protein from a wide variety of Salmonella serovars to get the protective measures of Salmonellosis. Salmonella surface protein characterization could be useful for development of protective measures against Salmonellosis and for analysis of the protein profile relationship among the Salmonella serovars. A common and immunodominant surface protein of Salmonella serovars was critically important. SDS-PAGE analysis during the period of January 2004 to December 2004 showed a target surface protein of 37.81 kDa among the 54 Salmonella serovars in comparison to some enterobactors. The protein profiles in SDS-PAGE of Salmonella serovars were not different among all Salmonella serovars examined in this study. In contrast to the protein band of 37.81 kDa in all serovars of Salmonella were compared and recorded with those of Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, and Enterobacter cloacae and were detected as 36.5 kDa. SDS-PAGE analysis showed different size of protein of Salmonella serovars and other tested enterobacters. However, it needs further investigation including Western blotting and 2-D PAGE analysis of the specific band of 37.81 kDa and 36.5 kDa protein. Key words: Salmonella serovars, characterization, SDS-PAGE doi: 10.3329/bjvm.v6i2.2331 Bangl. J. Vet. Med. (2008). 6 (2): 169-174 Â Â

Diabetes mellitus (DM) is a disorder of the chronic metabolic system due to insufficiency of insulin function. DM is classified into two main categories, type 1 and type 2. The purpose of this study was to separate proteins using the SDS-PAGE method to determine the potential protein profile as a biomarker contained in the blood of type 2 DM patients, so that the results of the analysis can be used as an indicator of early detection of type 2 DM. This research is a true experimental research. The samples of this study were the blood of 20 patients with type 2 DM in Sukorejo Bangsalsari Village and the blood of 5 patients who non DM in Sukorejo Bangsalsari Village. sampling technique was randomized controlled trial, after the blood collection process, blood serum was then prepared at the Molecular Biology Laboratory, FMIPA UNEJ, then protein analysis was performed using the 1D-gel electrophoresis (SDS-PAGE) method. The results of protein characterization using SDS-PAGE analysis with blood serum of type 2 DM patients and serum of healthy people (non-patients) as negative controls, obtained protein bands that are less specific to potential targets, because the results of running are still not good, so it is not possible to know the biomarker protein profile in the blood of patients with type 2 DM in Sukorejo Bangsalsari Village, and the results of this analysis still cannot be used as an indicator for early detection of type 2 diabetes mellitus. Analysis of potential proteins profile as biomarkers using SDS-PAGE analysis has revealed three bands which had molecular weights of 28, 45 and 235 kDa.Keywords: Diabetes mellitus, Biomarker, SDS-PAGE