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Purpose. ITP is the most prevalent autoimmune blood disorder. The lack of predictive biomarkers for therapeutic response is a major challenge for physicians caring of chronic ITP patients. This study is aimed at identifying predictive biomarkers for drug therapy responses. Methods. 2D gel electrophoresis (2-DE) was performed to find differentially expressed proteins. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) analysis was performed to identify protein spots. The Cytoscape software was employed to visualize and analyze the protein-protein interaction (PPI) network. Then, enzyme-linked immunosorbent assays (ELISA) were used to confirm the results of the proteins detected in the blood. The DAVID online software was used to explore the Gene Ontology and pathways involved in the disease. Results. Three proteins, including APOA1, GC, and TF, were identified as hub-bottlenecks and confirmed by ELISA. Enrichment analysis results showed the importance of several biological processes and pathway, such as the PPAR signaling pathway, complement and coagulation cascades, platelet activation, vitamin digestion and absorption, fat digestion and absorption, cell adhesion molecule binding, and receptor binding. Conclusion and Clinical Relevance. Our results indicate that plasma proteins (APOA1, GC, and TF) can be suitable biomarkers for the prognosis of the response to drug therapy in ITP patients. [ABSTRACT FROM AUTHOR]

This study is aimed at evaluating whether the occupational exposure to TiO2 during the industrial production process is able to induce genotoxic, oxidative, and inflammatory effects on blood, biomonitoring the same workers that showed micronucleus induction in the exfoliated buccal cells, as previous published. The final aim was to find sensitive and suitable biomarkers to evaluate potential early toxicity of occupational exposure to TiO2. On the same 40 workers involved in the manufacture of TiO2 pigment, 5 office workers, and 18 controls previously studied, we used formamidopyrimidine glycosylase- (Fpg-) comet assay on lymphocytes to evaluate genotoxic/oxidative effects and detected cytokine (IL-6, IL-8, and TNFα) release by ELISA to evaluate proinflammation. Moreover, we studied the possible influence of single nucleotide polymorphisms of XRCC1 and hOGG1 DNA repair genes and of GST metabolism-related genes (GSTT1 and GSTM1) on the evaluated effects. We did not find statistically significant differences in the mean values of the analysed Fpg-comet assay parameters; only the percentage of DNA damaged cells appearing in the test as comets (% comets) resulted higher in the exposed workers compared to controls. Also, the data analysed taking into account the specific task (bagging, industrial cleaning, mobile operations, maintaining, and production) showed differences only for % comets which resulted higher in industrial cleaners compared to controls. We found variations of IL-6 and IL-8 levels in the exposed workers with concentrations that were lower for IL-6 and higher for IL-8 compared to the control group. XRCC1, hOGG1, and GSTT1 polymorphisms did not influence neither comet parameters nor cytokine release. These findings demonstrate that TiO2 production process is able to induce slight proinflammatory effects in terms of IL-8 increased release but not significant genotoxic/oxidative effects on lymphocytes, which do not seem to be a target of TiO2, prevalently inhalable particles, generated in the studied production site. [ABSTRACT FROM AUTHOR]

Knee osteoarthritis (OA) is a common problem in elderly patients. They are often troubled with altered knee function, such as pain and weakness. However, not all these patients are able to receive autologous platelet-rich plasma (PRP) injections as they may be taking antiplatelet or anticoagulant medications. Their physical condition may not allow them to receive total knee replacement surgery as well. Long-term oral intake of nonsteroidal anti-inflammatory drugs may be detrimental to the gastrointestinal tract. As a result, it is crucial to discover new treatment options that can alleviate painful knee symptoms in elderly knee OA patients. In this study, 19 elderly patients diagnosed with moderate degree of knee OA as well as suprapatellar bursitis were recruited. They received low-level laser therapy (LLLT) to their affected knees. Under ultrasound guidance, flexible fiber optic wire was inserted intra-articularly into the knee joint. Red laser followed by infrared irradiation was performed once every 2 weeks for a total of 3 times. The Lequesne index for knee OA and the volume of suprapatellar synovial fluid (SF) were measured. SF proteomic analyses were also performed up to a period of 6 months. The results revealed that after 3 LLLT, the Lequesne index significantly decreased, signifying improvement in the knee joint functional status. The volume of suprapatellar SF and SF proteins associated with inflammation also decreased significantly in the SF. These findings lasted up to a period of at least 3 months. Therefore, LLLT may be considered as a feasible option in treating elderly patients with knee OA who are not suitable for surgical interventions or intra-articular PRP injections. [ABSTRACT FROM AUTHOR]

Effluents discharged by poultry meat industries are heavily polluted with raw materials, such as fat, blood residues, and proteins. Thus, untreated effluents directly discharged into the environment may constitute a public health threat. This study aims to evaluate the bacterial diversity of three water qualities: industrial poultry wastewater (PWW), tap water (TW), and PWW diluted with TW (50 : 50) (V/V) (TWPWW) by the combination of culture-independent and culture-dependent approaches. The total bacterial DNA was extracted using phenol/chloroform method. The hypervariable 16S rRNA region V3-V5 was amplified by PCR using universal primers. The amplicons were separated by vertical electrophoresis on a polyacrylamide gel of increasing denaturing gradient according to their richness in GC bases. Selected bands were reamplified and sequenced. Pure isolated bacteria from nutrient agar medium were characterized according to their morphological and biochemical characteristics. Genomic DNA from pure strains was extracted by boiling method, and a molecular amplification of the 16S–23S ITS region of the 16S rRNA gene was performed using the universal primers. Selected isolates were identified by sequencing. Results showed a high bacterial load and diversity in PWW in comparison with TW and TWPWW. A collection of 44 strains was obtained, and 25 of them were identified by sequencing. Proteobacteria represented 76% of isolated bacteria Gamma-Proteobacteria was the predominate isolate (68%). Other isolates were Firmicutes (8%), Bacteroidetes (12%), and Actinobacteria (8%). These isolates belong to different genera, namely, Pseudomonas, Acinetobacter, Proteus, Empedobacter, Corynebacterium, Enterobacter, Comamonas, Frondibacter, Leclercia, Staphylococcus, Atlantibacter, Klebsiella, and Microbacterium. [ABSTRACT FROM AUTHOR]

Objective. To investigate the genotoxicity of metformin on planarian with different concentrations and exposure times. Methods. The planarians were treated, respectively, with 10 mmol/L and 50 mmol/L metformin for 1, 3, and 5 days, and then, the comet assay and random amplified polymorphic DNA (RAPD) analysis were performed. 13 random primers were used for PCR amplification with the genomic DNAs as templates. Planarians cultured in clear water were used as the control. Genomic template stability (GTS) was calculated by comparing and analyzing the RAPD patterns of the control group and the treatment groups. Results. In the comet assay, DNA damage of planarians treated with 10 mmol/L metformin for 1, 3, and 5 days was 10.2%, 25.4%, and 36.8%, respectively, and that of planarians treated with 50 mmol/L metformin was 40.6%, 62.8%, and 65.4%, respectively. GTS values of planarians exposed to 10 mmol/L metformin for 1, 3, and 5 days were 64.1%, 62.8%, and 52.6%, respectively, and those of planarians exposed to 50 mmol/L metformin for 1, 3, and 5 days were 52.6%, 51.3%, and 50%, respectively. DNA damage increased and GTS values decreased with the increasing metformin exposure concentration and exposure time. Conclusion. Metformin has certain genotoxicity on planarian in a dose- and time-related manner. The comet assay and RAPD analysis are highly sensitive methods for detecting genotoxicity with drugs. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a major human pathogen that is sometimes resistant to vancomycin. In this study, the prevalence of vancomycin-resistant Staphylococcus aureus (VRSA) was studied. 100 isolates of S. aureus were identified based on biochemical and molecular evidence. The antibiotic susceptibility of the studied isolates was tested against 13 antibiotics by the disc diffusion method that showed 24 vancomycin-resistant isolates. The minimum inhibitory concentrations (MICs) were estimated by the agar dilution method to determine vancomycin intermediate-resistant S. aureus (VISA) and VRSA. The resistance gene cluster (vanA, vanR, vanH, and vanY) was amplified by PCR and then sequenced. Amplification of vanA and vanR genes showed that they are present in 21.4% and 14.3% of VRSA isolates, respectively, whereas none of the studied genes has been detected in VISA strains. A significant antimicrobial effect toward VRSA isolates using silver nanoparticles (AgNPs) synthesized from S. aureus and rosemary leaves was recorded. This study confirmed the existence of VRSA strains in Egypt. Furthermore, the use of silver nanoparticles inhibits these vancomycin-resistant S. aureus strains in vitro. [ABSTRACT FROM AUTHOR]

Aim. To investigate and compare the cell cytotoxicity, proliferation, cell attachment, and morphology of human fetal osteoblasts (hFOB) cells of coated samples (titanium nanocopper (Ti Cu), titanium nanohydroxyapatite (Ti HA) and titanium nanocopper ion doped hydroxyapatite (Ti Cu/HA) and uncoated samples (Ti) in order to assess the suitability of these surface modifications on Ti-6Al-7Nb for dental implant application. Materials and Methods. The cytotoxicity was studied by examining the hFOB cell response by MTT assessment. The cell morphology was evaluated by inverted microscopy and observed under scanning electronic microscopy (SEM). Results. MTT assay results displayed that the Cu content on the surface of Ti-6Al-7Nb alloys did not produce any cytotoxic effect on cell viability. The cell viability rate in all samples ranges from 97% to 126%, indicating that hFOB cells grew at a high proliferation rate. However, no significant differences in cell viability were observed between Ti and Ti Cu and between Ti HA and Ti Cu/HA groups. Microscopic examination demonstrated no difference in the cell morphology of hFOB among all samples. In addition, SEM observation indicated favorable adhesion and spreading of the cells on the coated and uncoated samples. Conclusions. The surface modification of Ti-6Al-7Nb alloy with Cu, HA, and Cu/HA exhibits good cell biocompatibility, and the Cu has no influence on the cell proliferation and differentiation of hFOB. [ABSTRACT FROM AUTHOR]

Amylase producing actinobacteria were isolated and characterized from terrestrial environment. There are a limited number of reports investigating the marine environment; hence, in the present study, four marine enzymes were tested for their amylase production ability. On starch agar plates, the Streptomyces rochei strain showed a higher hydrolytic zone (24 mm) than the other isolates. Growth under optimized culture conditions using Plackett-Burman's experimental design led to a 1.7, 9.8, 7.7, and 3.12-fold increase for the isolates S. griseorubens, S. rochei, S. parvus, and Streptomyces sp., respectively, in the specific activity measurement. When applying the Box-Behnken design on S. rochei using the most significant parameters (starch, K2HPO4, pH, and temperature), there was a 12.22-fold increase in the specific activity measurement 7.37 U/mg. The α-amylase was partially purified, and its molecular weight was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. α-Amylase was particularly active at pH 6 and 65°C. The purified enzyme was most active at 65°C and pH 6, thermal stability of 70°C for 40 min, and salt concentration of 1 M with Km and Vmax of 6.58 mg/ml and 21.93 μmol/ml/min, respectively. The α-amylase was improved by adding Cu+2, Zn+2, and Fe+2 (152.21%, 207.24%, and 111.89%). Increased production of α-amylase enzyme by S. rochei KR108310 leads to production of significant industrial products. [ABSTRACT FROM AUTHOR]

Personal identification of two individuals in mixed semen samples in forensic DNA testing in general usually involves analysis using autosomal and Y chromosome short tandem repeats (STRs). Results may exclude unrelated donors but cannot identify individuals. In this study, sperm cell capture based on ABH antigen differences was used to obtain the cells with the single ABO blood type. Immunohistochemical staining using labeled anti-A, anti-B, and anti-H antibodies and the laser microdissection system can be used to enrich sperm with different ABO types in mixed seminal stains from two individuals. Then, PCR amplification and capillary electrophoresis were performed to genotype the STR loci. To some extent, after sperm cell capture based on ABH antigen differences, autosomal STR typing using enriched single blood group cells can be utilized to partially identify different individuals in a mixed seminal stain sample from two individuals. [ABSTRACT FROM AUTHOR]

Dental caries is a multifactorial disease mainly caused by cariogenic bacteria commonly found in the oral cavity. Dental caries may cause demineralization of the tooth, cavitation, hypersensitivity, pulp inflammation, and even tooth loss if left untreated. Saliva secreted in the oral cavity can serve as a tool for identification of biomarkers for early detection of diseases. In the present study, differential expression of salivary proteins from 33 dental caries patients was compared with 10 control subjects. The unstimulated saliva was analyzed by 12% SDS-PAGE and two-dimensional gel electrophoresis. Gelatin and casein zymography was performed to check for protease activity. Also, salivary IgAs from both groups were compared by sandwich ELISA technique. Dental caries patient's saliva showed decreased caseinolytic and increased gelatinolytic activity probably due to metalloproteases and cathepsins. Mean salivary levels of sIgA were also significantly higher (p < 0.018) in dental caries saliva samples. The 2D electrophoresis profile of both the groups showed regions on gel with visually detectable alterations in protein expression. The present study is among the few initial studies in the locality for identification of protein differences in saliva from dental caries patients and has demonstrated a good potential to identify alterations. However, a large population-based analysis is required to validate these findings to be translated as a tool for indicative applications. [ABSTRACT FROM AUTHOR]

We have previously reported that F1012-2, a sesquiterpene lactone isolated from the Chinese herbal medicine Eupatorium lindleyanum DC., exhibits strong effects against Triple Negative Breast Cancer (TNBC). In this study, we found F1012-2 effectively inhibited cell migration and invasion detected by wound healing and transwell assays. In order to elucidate the potential mechanisms of F1012-2, we further studied its effect on DNA damage in TNBC cell lines. Using single cell gel electrophoresis (comet assay), immunofluorescence, and western blotting assays, we found that F1012-2 treatment induced significant DNA strand breaks and γ-H2AX activation. Moreover, exposure to F1012-2 led to overproduction of reactive oxygen species (ROS). NAC treatment completely eliminated ROS, which may be due to the interaction between NAC and F1012-2. A further study of the molecular mechanisms demonstrated that the MAPK signaling pathway participated in the anti-TNBC effect of F1012-2. Pretreatment with specific inhibitors targeting JNK (SP600125) and ERK (PD98059) could rescue the decrease in cell viability and inhibit expressions of JNK and ERK phosphorylation, but SB203580 had no effects. Finally, in the acute toxicity experiment, there were no obvious symptoms of poisoning in the F1012-2 treatment group. An in vivo study demonstrated that F1012-2 significantly suppressed the tumor growth and induced DNA damage. In conclusion, the activity of F1012-2-induced DNA damage in TNBC was found in vivo and in vitro, which might trigger the MAPK pathway through ROS accumulation. These results indicate that F1012-2 may be an effective anti-TNBC therapeutic agent. [ABSTRACT FROM AUTHOR]

To investigate the effects of ficin on biofilm formation of conditionally cariogenic Streptococcus mutans (S. mutans). Biomass and metabolic activity of biofilm were assessed using crystal violet assay, colony-forming unit (CFU) counting, and MTT assay. Extracellular polysaccharide (EPS) synthesis was displayed by SEM imaging, bacteria/EPS staining, and anthrone method while acid production was revealed by lactic acid assay. Growth curve and live/dead bacterial staining were conducted to monitor bacterial growth state in both planktonic and biofilm form. Total protein and extracellular proteins of S. mutans biofilm were analyzed by protein/bacterial staining and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), severally. qRT-PCR was conducted to detect acid production, acid tolerance, and biofilm formation associated genes. Crystal violet assay, CFU counting, and MTT assay showed that the suppression effect of ficin on S. mutans biofilm formation was concentration dependent. 4 mg/mL ficin had significant inhibitory effect on S. mutans biofilm formation including biomass, metabolic activity, EPS synthesis, and lactic acid production (p < 0.05). The growth curves from 0 mg/mL to 4 mg/mL ficin were aligned with each other. There was no significant difference among different ficin groups in terms of live/dead bacterial staining result (p > 0.05). Protein/bacterial staining outcome indicated that ficin inhibit both total protein and biofilm formation during the biofilm development. There were more relatively small molecular weight protein bands in extracellular proteins of 4 mg/mL ficin group when compared with the control. Generally, ficin could inhibit biofilm formation and reduce cariogenic virulence of S. mutans effectively in vitro; thus, it could be a potential anticaries agent. [ABSTRACT FROM AUTHOR]

The study focuses on engineering of recombinant Aspergillus niger to produce highly active xylanase. The xylanase G2 encoding gene originating from Aspergillus oryzae VTCC-F187 was cloned, amplified, and inserted into the pAN7.1GluA vector with specific primers possessing BamHI. The recombinant plasmid was introduced into Aspergillus niger VTCC-F017 by chemical methods. The recombinant strain was checked by polymerase chain reaction method and Southern blot. Next, the recombinant protein was expressed and purified by His-tag column. The molecular mass of the purified xylanase G2, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), was 21 kDa with a specific activity of 1025 IU/mg towards 0.5% (w/v) of birchwood xylan. The optimal temperature and pH were 55°C and pH 6.5, respectively. The enzyme was stable in a temperature ranges 25–40°C and a pH ranges 5–7. The presence of Tween 80 enhanced xylanase activity. Triton X-100, however, had no impact on the function of the enzyme. The xylanase activity was reduced by Tween 20, SDS, and organic solvents. The enzyme was completely inhibited by Hg2+ and partially by Zn2+, Fe2+, and Ag+, while it was slightly stimulated by K+ and EDTA. [ABSTRACT FROM AUTHOR]

Bone marrow-derived mesenchymal stem cells (BM-MSCs) display high tumor tropism and cause indirect effects through the cytokines they secrete. However, the effects of BM-MSCs on the biological behaviors of glioblastoma multiforme remain unclear. In this study, the conditioned medium from BM-MSCs significantly inhibited the proliferation of C6 cells (P < 0.05) but promoted their migration and invasion (P < 0.05). Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) proteomic analysis revealed 17 proteins differentially expressed in C6 cells exposed to the BM-MSC-conditioned medium including five upregulated proteins and 12 downregulated proteins. Among these, six differentially expressed proteins (Calr, Set, Oat, Npm1, Ddah1, and Tardbp) were closely related to cell proliferation and differentiation, and nine proteins (Pdia6, Sphk1, Anxa4, Vim, Tuba1c, Actr1b, Actn4, Rap2c, and Tpm2) were associated with motility and the cytoskeleton, which may modulate the invasion and migration of tumor cells. Above all, by identifying the differentially expressed proteins using proteomics and bioinformatics analysis, BM-MSCs could be genetically modified to specifically express tumor-suppressive factors when BM-MSCs are to be used as tumor-selective targeting carriers in the future. [ABSTRACT FROM AUTHOR]

High-density lipoprotein- (HDL-) cholesterol measurements are generally used in the diagnosis of cardiovascular diseases. However, HDL is a complicated heterogeneous lipoprotein, and furthermore, it can be converted into dysfunctional forms during pathological conditions including inflammation. Therefore, qualitative analysis of pathophysiologically diversified HDL forms is important. A recent study demonstrated that serum amyloid A (SAA) can remodel HDL and induce atherosclerosis not only over long periods of time, such as during chronic inflammation, but also over shorter periods. However, few studies have investigated rapid HDL remodeling. In this study, we analyzed HDL samples from patients undergoing orthopedic surgery inducing acute inflammation. We enrolled 13 otherwise healthy patients who underwent orthopedic surgery. Plasma samples were obtained on preoperative day and postoperative days (POD) 1-7. SAA, apolipoprotein A-I (apoA-I), and apolipoprotein A-II (apoA-II) levels in the isolated HDL were determined. HDL particle size, surface charge, and SAA and apoA-I distributions were also analyzed. In every patient, plasma SAA levels peaked on POD3. Consistently, the HDL apoA-I : apoA-II ratio markedly decreased at this timepoint. Native-polyacrylamide gel electrophoresis and high-performance liquid chromatography revealed the loss of small HDL particles during acute inflammation. Furthermore, HDL had a decreased negative surface charge on POD3 compared to the other timepoints. All changes observed were SAA-dependent. SAA-dependent rapid changes in HDL size and surface charge were observed after orthopedic surgery. These changes might affect the atheroprotective functions of HDL, and its analysis can be available for the qualitative HDL assessment. [ABSTRACT FROM AUTHOR]

Poultries including chickens, ducks, geese, and pigeons are widely used in the biological and medical research in many aspects. The genetic quality of experimental poultries directly affects the results of the research. In this study, following electrophoresis analysis and short tandem repeat (STR) scanning, we screened out the microsatellite loci for determining the genetic characteristics of Chinese experimental chickens, ducks, geese, and pigeons. The panels of loci selected in our research provide a good choice for genetic monitoring of the population genetic diversity of Chinese native experimental chickens, ducks, geese, and ducks. [ABSTRACT FROM AUTHOR]

Peony (Paeonia) has high ornamental, edible, and medicinal values. In order to distinguish seeds varieties, describe the proteomic profiles correlated with stress tolerance, and evaluate peony seed protein (PSP) as a functional food product, we characterized the seed protein profiles of these three species and their glucosidase inhibition activities. Results showed that the intensity of protein bands in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and specific protein ID (especially for specifically expressed proteins (SEPs)) was effective to distinguish these peony seed varieties. Proteomic analysis of the three species showed that P. ostii "Fengdan" has heat and pathogen tolerance-related proteins, while P. rockii has higher content of proteins related to cold resistance, which were all highly consistent with their adaptation of heat or cold habitat. Moreover, stress-related proteins were also accumulated in P. lactiflora Pall "Hangshao" seeds, showing its potential for stress resistance. Further protein analysis showed that the primary composition of PSP was albumin and globulin. And the solubility of PSP was good. Furthermore, PSP also showed high glucosidase inhibition activity, indicating that PSP might have some potential function for the remission of hyperglycemia. And P. ostii "Fengdan" seeds may be a better source for protein production than seeds of the other two species in terms of protein solubility and the content of total protein, albumin, and globulin. In addition, an optimal protocol of microwave-assisted alkali extraction was developed to produce PSP. In conclusion, the evaluated stress-related proteins in three peony seed species by proteomic analysis quite agreed with their adaptation of heat or cold stress; proteomics could also be a very useful tool for distinguishing species in the production; and peony seeds may be a good source for protein production. [ABSTRACT FROM AUTHOR]

Purpose. β-Defensin 118 (DEFB118) is a novel host defense peptide (HDP) identified in humans. To evaluate its potentials for future utilization, the DEFB118 gene was expressed in Escherichia coli (E. coli) and the recombinant protein was fully characterized. Methods. The DEFB118 protein was obtained by heterologous expression using E. coli Rosetta (DE3). Antibacterial activity of DEFB118 was determined by using various bacterial strains. IPEC-J cells challenged by E. coli K88 were used to determine its influences on inflammatory responses. Results. The E. coli transformants yielded more than 250 μg/mL DEFB118 protein after 4 h induction by 1.0 mM IPTG. The DEFB118 was estimated by SDS-PAGE to be 30 kDa, and MALDI-TOF analysis verified that it is a human β-defensin 118. Importantly, the DEFB118 showed antimicrobial activities against both Gram-negative bacteria (E. coli K88 and E. coli DH5α) and Gram-positive bacteria (S. aureus and B. subtilis), with a minimum inhibitory concentration (MIC) of 4 μg/mL. Hemolytic assays showed that DEFB118 had no detrimental impact on cell viability. Additionally, DEFB118 was found to elevate the viability of IPEC-J2 cells upon E. coli K88 challenge. Moreover, DEFB118 significantly decreased cell apoptosis in the late apoptosis phase and downregulated the expression of inflammatory cytokines such as IL-1β and TNF-α in IPEC-J2 cell exposure to E. coli K88. Conclusions. These results suggested a novel function of the mammalian defensins, and the antibacterial and anti-inflammatory properties of DEFB118 may allow it as a potential substitute for conventionally used antibiotics or drugs. [ABSTRACT FROM AUTHOR]

The aim of this study was to detect oral bacteremia and offer the antibiotic resistance patterns. Bacterial resistance pattern was evaluated in 50 patients. A spectrophotometer device equipped with UV and electrophoresis of the extracted samples on agarose gel for antibiogram test were used. PCR test 15 minutes after tooth extraction showed that bacterial strains were extracted from 16 patients. Lactobacillus, Enterococcus faecalis (E. faecalis), Streptococcus sanguinis (S. sanguinis), Streptococcus salivarius (S. salivarius), and Streptococcus mutans (S. mutans) were extracted from 5, 4, 4, 4, and 6 patients. 100% of Lactobacillus, E. faecalis, S. sanguinis, S. salivarius, and S. mutans were sensitive to tigecycline. Most of the Lactobacillus antibiotic resistance was against tetracycline and ciprofloxacin. Antibiotic resistance in S. salivarius was observed in 75% of the cases against piperacillin-tazobactam, ciprofloxacin, and cefotaxime, while in S. mutans was 84% of the cases against ceftriaxone. The results of the current study showed that tooth extraction causes bacteremia before, during, and after tooth extraction. Generally, the highest antibiotic resistance occurred against tetracycline, ciprofloxacin, and ampicillin-sulbactam. In most cases, the bacteria showed partial resistance to these antibiotics; however, tigecycline showed 100% efficacy on all types of bacteria. Streptococcus strains (salivarius, mutans, and sanguinis) were sensitive to most of the antibiotics while antibiotic sensitivity was less evident in Lactobacillus and E. faecalis. Antibiotic resistance has become a critical issue, since it leads to treatment failure when there is a need for antibiotic therapy. [ABSTRACT FROM AUTHOR]

A new peptidase designated as SAPV produced from a moderately halophilic Virgibacillus natechei sp. nov., strain FarDT was investigated by purification to homogeneity followed by biochemical and molecular characterization purposes. Through optimization, it was determined that the optimum peptidase activity was 16,000 U/mL. It was achieved after 36 h incubation at 35°C in the optimized enzyme liquid medium (ELM) at pH 7.4 that contains only white shrimp shell by-product (60 g/L) as sole energy and carbon sources. The SAPV enzyme is a monomer protein with a molecular mass of 31 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) gel filtration chromatography. The sequence of its NH2-terminal amino-acid residues showed homology with those of Bacillus peptidases S8/S53 superfamily. The SAPV showed optimal activity at pH 9 and 60°C. Irreversible inhibition of enzyme activity by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine peptidases. Considering its interesting biochemical characterization, the sapV gene was cloned, sequenced, and heterologously overexpressed in the extracellular fraction of E. coli BL21(DE3)pLysS. The biochemical properties of the recombinant peptidase (rSAPV) were similar to those of the native one. The highest sequence identity value (97.66%) of SAPV was obtained with peptidase S8 from Virgibacillus massiliensis DSM 28587, with 9 amino-acid residues of difference. Interestingly, rSAPV showed an outstanding and high resistance to several organic solvents than SPVP from Aeribacillus pallidus VP3 and Thermolysin type X. Furthermore, rSAPV exhibited an excellent detergent stability and compatibility than Alcalase 2.4 L FG and Bioprotease N100L. Considering all these remarkable properties, rSAPV has attracted the interest of industrialists. [ABSTRACT FROM AUTHOR]

Prefractionation is a prerequisite step for deep plasma proteomics. Highly abundant proteins, particularly human serum albumin (HSA) and immunoglobulin G (IgG), typically interfere with investigation of proteins with lower abundance. A relatively simple preparation method based on high temperature can precipitate thermolabile proteins, providing a strategic window to access the thermostable plasma subproteome. This study aimed to optimize thermal treatment as a reliable prefractionation method and to compare it with two commercial kits, including HSA and IgG immunodepletion (IMDP) and combinatorial peptide ligand libraries (CPLL), using untreated plasma as a control condition. By varying the temperature and the incubation period, the optimal condition was found as treatment at 95°C for 20 min, which maintained about 1% recovery yield of soluble proteins. Consistency and reproducibility of thermal treatment-derived plasma subproteome were checked by two-dimensional electrophoresis. The coefficient of variation regarding protein spot numbers was less than 10% among three independent specimens. Highly abundant protein depletion of the thermal treatment was evaluated by immunoblotting against HSA and IgG as compared to the untreated plasma, IMDP, and CPLL. Multidimensional comparison based on 489 unique peptides derived from the label-free quantitative mass spectrometry revealed that the thermal treatment, IMDP, and CPLL provided distinct sets of plasma subproteome compared to untreated plasma, and these appeared to be complementary to each other. Comparing the characteristics of the three procedures suggested that thermal treatment was more cost-effective and less time-consuming than IMDP and CPLL. This study proposes the use of thermal treatment as a reliable and cost-effective method for plasma prefractionation which provides benefits to large-scale proteomic projects and biomarker studies. [ABSTRACT FROM AUTHOR]

Sweet potato (Ipomoea batatas L. Lam.), known as "Shakarqandi" in Pakistan, is an imperative root vegetable with large size, traditionally used as aphrodisiac, antiprostatic, anti-inflammatory, antidiabetic, cardiotonic, and anticancer agent. Present study was conducted to gauge aphrodisiac potential of Ipomoea batatas ethyl acetate (IPT-EA, IPA-EA) and methanol (IPT-M, IPA-M) extracts from tuber and aerial part, respectively, via behavioral and biochemical tests and their possible protective role in BPA-induced gonadotoxicity at the dose 300 mg/kg in male Sprague Dawley rats. Phytochemical analysis was done qualitatively and quantitatively through total phenolic and flavonoid content (TPC and TFC) and high performance liquid chromatographic (HPLC-DAD) fingerprinting while antioxidant profiling used multimode in vitro assays. To calculate sexual excitement mount latency, intromission latency, mount frequency, intromission frequency, ejaculatory latency, and postejaculatory interval were examined while for biochemical ratification semen characteristics, levels of testosterone, follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol were measured. Gonadoprotective ability was assessed through comet assay and histomorphological examination of testes. Qualitative analysis ensured the presence of phenols, flavonoids, tannins, anthocyanin, saponins, coumarins, terpenoids, and betacyanin. Quantitatively maximal TPC (304.32±7.20 μg GAE/mg dry extract) and TFC (214.77±4.09 μg QE/mg DE) were estimated in IPA-EA extract. IPT-EA yielded maximum rutin (7.3±0.12) and myricetin (2.7±0.14 μg/mg DE) while IPA-EA and IPA-M yielded maximum caffeic acid (4.05±0.22 and 1.92±0.17 μg/mg DE, respectively) in HPLC-DAD analysis. Extracts enhanced sexual excitement, improved semen quality, levels of testosterone, FSH, LH, and estradiol, and successfully attenuated toxic effects of BPA. Levels of endogenous antioxidant enzymes (CAT, SOD, POD, and GSH) were restored and NO abundance was minimized. Significant stimulation in sexual behavior, amelioration of toxicity symptoms, elevated spermatic production, raised viability, vitalized levels of gonadal hormones, maintained endogenous enzymes, genoprotection, and reformed testicular histology endorsed I. batatas as a better aphrodisiac alternative and gonadoprotective agent. [ABSTRACT FROM AUTHOR]

This in vitro study was designed to evaluate the effect of sol-gel derived TiO2 coating on blood coagulation, blood protein adsorption, and platelet response on zirconia surfaces. Square-shaped zirconia (n=96) (10x10x2 mm) was cut, ground, sintered, and finally cleansed ultrasonically in each of acetone and ethanol for 5 minutes. Three experimental groups (n=32) were fabricated: (a) zirconia coated with sol-gel derived TiO2, (b) zirconia coated with sol-gel derived TiO2 and treated with ultraviolet (UV) irradiation for 1 hour, and (c) non-coated zirconia as control. The coatings were prepared from tetraisopropyl orthotitanate solution by dip-coating. The thrombogenicity of the specimens was evaluated using a whole blood kinetic clotting time method where the extent of blood clotting was evaluated at 10, 20, 30, 40, 50, and 60 minutes (n=4/time point, total n=24/group). Scanning electron microscope images were taken to observe platelet morphologies after 1-hour incubation with platelet-rich plasma (PRP) (n=5/group). Surface characteristics were visualized using atomic force microscopy (n=1/group). Adsorption of plasma proteins and fibronectin on each surface was studied by gel electrophoresis (n=2/group). Significant differences were observed in blood coagulation between the test groups at 20-, 30-, 40-, and 50-minute time points (p<0.005). UV treated TiO2 coated specimens showed fastest blood coagulation followed by TiO2 coated and non-coated specimens. Furthermore, platelets appeared at a higher activation state on coated specimens. Gel electrophoresis revealed no difference in protein adsorption among the experimental groups. In summary, TiO2 coatings promoted blood coagulation, and it was further enhanced by UV treatment, which has the potential to hasten the wound healing process in vivo. [ABSTRACT FROM AUTHOR]

Ribonuclease L (RNase L) is an important antiviral endoribonuclease regulated by type I IFN. RNase L is activated by viral infection and dsRNA. Because the role of swine RNase L (sRNase L) is not fully understood, in this study, we generated a sRNase L knockout PK-15 (KO-PK) cell line through the CRISPR/Cas9 gene editing system to evaluate the function of sRNase L. After transfection with CRISPR-Cas9 followed by selection using puromycin, sRNase L knockout in PK-15 cells was further validated by agarose gel electrophoresis, DNA sequencing, and Western blotting. The sRNase L KO-PK cells failed to trigger RNA degradation and induced less apoptosis than the parental PK-15 cells after transfected with poly (I: C). Furthermore, the levels of ISGs mRNA in sRNase L KO-PK cells were higher than those in the parental PK-15 cells after treated with poly (I: C). Finally, both wild type and attenuated pseudorabies viruses (PRV) replicated more efficiently in sRNase L KO-PK cells than the parental PK-15 cells. Taken together, these findings suggest that sRNase L has multiple biological functions including cellular single-stranded RNA degradation, induction of apoptosis, downregulation of transcript levels of ISGs, and antiviral activity against PRV. The sRNase L KO-PK cell line will be a valuable tool for studying functions of sRNase L as well as for producing PRV attenuated vaccine. [ABSTRACT FROM AUTHOR]

Skin secretions of frogs have a high chemical complexity. They have diverse types of biomolecules, such as proteins, peptides, biogenic amines, and alkaloids. These compounds protect amphibians' skin against growth of bacteria, fungi, and protozoa and participate in defense system against attack from predators. Therewith, this work performed biochemical and biological profile of macroglands parotoid secretion from cane toad. For poison analysis, we performed molecular exclusion and reverse phase chromatography, electrophoresis, and mass spectrometry. Antimicrobial, antiplasmodial, leishmanicidal, cytotoxicity, genotoxicity, and inflammatory activity of crude and/or fractions of R. marina secretion were also evaluated. Fractionation prior to filtration from poison showed separation of low mass content (steroids and alkaloids) and high molecular mass (protein). Material below 10 kDa two steroids, marinobufagin and desacetylcinobufagin, was detected. Crude extract and fractions were active against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Plasmodium falciparum, Leishmania guyanensis, and Leishmania braziliensis. Crude extract was also active against cancer cells although it was not cytotoxic for normal cells. This extract did not show significant DNA damage but it showed an important inflammatory effect in vivo. The information obtained in this work contributes to the understanding of the constituents of R. marina secretion as well as the bioactive potential of these molecules. [ABSTRACT FROM AUTHOR]

Background. Mouse models of hypercholesterolaemia have been used to identify arterial proteins involved in atherosclerosis. As the liver is extremely sensitive to dyslipidemia, one might expect major changes in the abundance of liver proteins in these models even before atherosclerosis develops. Methods. Lipid levels were measured and a proteomic approach was used to quantify proteins in the livers of mice with an elevated low-density lipoprotein (LDL) and the presence of lipoprotein(a) [Lp(a)] but no atherosclerosis. Results. The livers of Lp(a) mice showed an increased triglyceride but reduced phospholipid and oxidised lipid content. Two-dimensional gel electrophoresis and mass spectrometry analysis identified 24 liver proteins with significantly increased abundance in Lp(a) mice (P<0.05). A bioinformatic analysis of the 24 proteins showed the major effect was that of an enhanced antioxidant and lipid efflux response with significant increases in antioxidant (Park7, Gpx1, Prdx6, and Sod1) and lipid metabolism proteins (Fabp4, Acaa2, apoA4, and ApoA1). Interestingly, human liver cells treated with Lp(a) showed significant increases in Gpx1 and Prdx6 but not Sod1 or Park7. Conclusions. The presence of human LDL and Lp(a) in mice promotes an enhanced flux of lipids into the liver which elicits an antioxidant and lipid export response before the onset of atherosclerosis. The antioxidant response can be reproduced in human liver cells treated with Lp(a). [ABSTRACT FROM AUTHOR]

Excess body weight has been causally linked to an increased risk of different cancer types, including colorectal cancer (CRC) but the mechanisms underlying this association are practically unknown. We investigate redox state-superoxide (SO) generation rate, activity of complex I in electron transport chain (ETC) of mitochondria and of dinitrosyl iron complexes by electron paramagnetic resonance; activity of matrix metalloproteinase (gelatinase) MMP-2 and MMP-9 by gel zymography of adipose tissues (AT) from 46 patients (64.0 ± 1.6 y.o.) with CRC (II–III stages, pT2–3N0–2M0) in the AT adjacent to tumor (ATAT) and at a distance of 3 cm from the tumor (ATD) to follow the connection of the AT redox state with some of the tumor microenvironment indicators. We have incubated the AT species with the tumor necrosis factor α (TNF-α) to follow its influence on the measured values. As a control, normal AT (NAT) obtained during the liposuction is used. Tumor-induced changes in mitochondrial ETC of ATAT, particularly for Complex I, lead to the enhanced SO generation and consequent oxidative modifications of DNA in ATAT (up to 6.1 times higher than that in NAT and 3.7 times higher than that in ATD, p < 0.05). Gelatinase activity in ATAT is significantly higher than in ATD. A considerable effect of TNF-α on ATAT and ATD (but not on NAT, i.e., only on the tissues where the reprogramming of metabolism has already occurred under the influence of tumor) manifested in increase of cellular hypoxia, gelatinase activity, and SO generation rate is observed. The results can be used for better understanding the mechanism(s) of metabolic symbiosis of tumor and AT as well as serving as a basis for new therapeutic approaches. [ABSTRACT FROM AUTHOR]

Several studies have compared molecular components between red and white skeletal muscles in mammals. However, mammalian skeletal muscles are composed of mixed types of muscle fibers. In the current study, we analyzed and compared the distributions of titin, lipid, phosphate ions, and fatty acid levels in red and white muscles using a fish model (Tilapia), which is rich in red and white muscles, and these are well separated. Oil-red O staining showed that red muscle had more-abundant lipids than did white muscle. A time-of-flight secondary-ion mass spectrometric (TOF-SIMS) analysis revealed that red muscle possessed high levels of palmitic acid and oleic acid, but white muscle contained more phosphate ions. Moreover, elastica-van Gieson (EVG) and Mito-Tracker green FM staining showed that collagen and elastic fibers were highly, respectively, distributed in connective tissues and mitochondria in red muscle. An electron micrographic analysis indicated that red muscle had a relatively higher number of mitochondria and longer sarcomere lengths and Z-line widths, while myofibril diameters were thicker in white muscle. Myofibrillar proteins separated by SDS-PAGE showed that the major giant protein, titin, was highly expressed in white muscle than in red muscle. Furthermore, ratios of titin to myosin heavy chain (MHC) (titin/MHC) were about 1.3 times higher in white muscle than red muscle. We postulated that white muscle is fit for short and strong contractile performance due to high levels of titin and condensed sarcomeres, whereas red muscle is fit for low intensity and long-lasting activity due to high levels of lipids and mitochondria and long sarcomeres. [ABSTRACT FROM AUTHOR]

Silymarin (SM), a standardized extract derived from Silybum marianum (L.) Gaertn, is primarily composed of flavonolignans, with silibinin (SB) as its major active constituent. The present study aimed to evaluate the antigenotoxic activities of SM and SB using the alkaline comet assay in whole blood cells and to assess their effects on the expression of genes associated with carcinogenesis and chemopreventive processes. Different concentrations of SM or SB (1.0, 2.5, 5.0, and 7.5 mg/ml) were used in combination with the DNA damage-inducing agent methyl methanesulfonate (MMS, 800 μM) to evaluate their genoprotective potential. To investigate the role of SM and SB in modulating gene expression, we performed quantitative real-time PCR (qRT-PCR) analysis of five genes that are known to be involved in DNA damage, carcinogenesis, and/or chemopreventive mechanisms. Treatment with SM or SB was found to significantly reduce the genotoxicity of MMS, upregulate the expression of PTEN and BCL2, and downregulate the expression of BAX and ABL1. We observed no significant changes in ETV6 expression levels following treatment with SM or SB. In conclusion, both SM and SB exerted antigenotoxic activities and modulated the expression of genes related to cell protection against DNA damage. [ABSTRACT FROM AUTHOR]

Objective. To quantitatively assess the influence of reducing agents on biological macromolecules and on the possible repair of oxidative damage. Methods. Samples (antibody, enzyme, DNA, and diluted serum) were treated with reducing agents (ammonium ferrous sulfate, ascorbic acid, potassium iodide, and sodium hyposulfite) in the experimental group and with NaCl in the control group. Enzyme-linked immunosorbent assay and quantitative PCR were used to determine the activity of antibody, enzyme, and DNA. Native gel electrophoresis (Native-PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to determine protein structure. Reducing agents that had no inhibitory effect on biological macromolecules were selected. Antibodies were treated with oxidants to caused oxidative damage and then treated with reducing agents, and the possible repair of oxidative damage was assessed. Results. Certain concentrations of ammonium ferrous sulfate resulted in significant inhibition of antibody, enzyme, DNA, and diluted serum. Certain concentrations of ascorbic acid resulted in significant inhibition of antibody. Sodium hyposulfite and potassium iodide had no effect on antibody, enzyme, DNA, and diluted serum. The OD values in group A (in which HBsAb was treated by oxidation and then a reductant) were significantly higher than those in group B (HBsAb treated by oxidation). Conclusion. Ammonium ferrous sulfate, ascorbic acid, sodium hyposulfite, and potassium iodide had different effects on antibody, enzyme, DNA, and diluted serum. The reduction in antibody activity due to an oxidant was partially repaired by a reductant. [ABSTRACT FROM AUTHOR]

Oxytetracycline and copper are the common residues in animal manures. Meanwhile, anaerobic digestion is considered as a clean biotechnology for the disposal of animal manures. In this paper, the performance of anaerobic digesters and the dynamics of bacterial communities under the different treatments of oxytetracycline and copper were discussed. The parameters of methane production and pH values were studied to reflect the performance of anaerobic digester. Results showed that the changes of methane production and pH values were not obvious compared with the control. This means that the treatments of oxytetracycline and copper almost have no effects on the performance of anaerobic digesters. This phenomenon might be due to the chelation reaction between oxytetracycline and copper. This chelation reaction might reduce the toxicity of oxytetracycline. The study on the dynamics of bacterial communities was based on the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method. Results indicated that the bacterial communities had significant differences under the different treatments of oxytetracycline and copper. Uncultured Bacteroidetes bacterium (CU922272.1) and uncultured Bacteroidetes bacterium (AB780945.1) showed adaptability to the different treatments of oxytetracycline and copper and were the dominant bacterial communities. [ABSTRACT FROM AUTHOR]

Saliva gained interest as a potential noninvasive source of biomarkers in humans and that interest starts to be extended also to other animal species. For this purpose, the knowledge of the salivary proteome in healthy conditions and the factors that affect it and how they affect it are necessary. The aim of the present study was to assess the effect that gender and breed have in saliva proteome and the changes in it induced by stimulation with acid. Saliva from 4 different purebred dogs (Portuguese Podengo, Greyhound, Rafeiro Alentejano, and Beagle) of both genders was collected without and after stimulation with lemon juice. SDS-PAGE and two-dimensional gel electrophoresis (2-DE) profiles were compared and the proteins of interest in-gel digested and identified by mass spectrometry. Acid stimulation decreased total protein concentration and the relative amounts of some protein bands/spots. Gender appeared to have minimal effect in saliva proteome, whereas the influence of breed varies. Beagles and Portuguese Podengos were the two breeds with higher differences. In conclusion, stimulation procedures and dog breed should be considered in data analysis when using salivary proteins for diagnostic purposes. [ABSTRACT FROM AUTHOR]

Objective. This work aimed to determine if cataractous changes associated with EMT occurring in the K14E6 mice lenses are associated with TGF-β and Wnt/β-catenin signaling activation. Materials and Methods. Cataracts of K14E6 mice were analysed histologically; and components of TGF-β and Wnt/β-catenin signaling were evaluated by Western blot, RT-qPCR, in situ RT-PCR, IHC, or IF technics. Metalloproteinases involved in EMT were also assayed using zymography. The endogenous stabilisation of Smad7 protein was also assessed using an HDAC inhibitor. Results. The K14E6 mice, which displayed binocular cataracts in 100% of the animals, exhibited loss of tissue organisation, cortical liquefaction, and an increase in the number of hyperproliferative-nucleated cells with mesenchymal-like characteristics in the lenses. Changes in lenses’ cell morphology were due to actin filaments reorganisation, activation of TGF-β and Wnt/β-catenin pathways, and the accumulation of MTA1 protein. Finally, the stabilisation of Smad7 protein diminishes cell proliferation, as well as MTA1 protein levels. Conclusion. The HPV16-E6 oncoprotein induces EMT in transgenic mice cataracts. The molecular mechanism may involve TGF-β and Wnt/β-catenin pathways, suggesting that the K14E6 transgenic mouse could be a useful model for the study or treatment of EMT-induced cataracts. [ABSTRACT FROM AUTHOR]

It is well established that, during development of malignancies, metabolic changes occur, including alterations of enzyme activities and isoenzyme expression. Arginase and nitric oxide (NO) synthase (NOS) are two of those enzymes considered to be involved in tumorigenesis. The goal of this article was to study the involvement of arginase and NOS in the development of different stages of breast cancer. Our results have shown that human serum arginase activity and NO (resp., and NOS activity) and polyamines quantities increased in parallel with cancer stage progression and decreased after neoadjuvant chemotherapy. For breast cancer, the only isoenzyme of arginase expressed in serum before and after chemotherapy was in a cationic form. The data of Lineweaver-Burk plot with a Km value of 2 mM was calculated, which is characteristic for human liver type isoform of arginase. During electrophoresis at pH 8.9, the enzyme exhibited high electrophoretic mobility and was detected near the anode. The presented results demonstrated that arginase in human serum with breast cancer and after chemotherapy is not polymorphic. We suggest that arginase and NOS inhibition has antitumor effects on cancer development, as it can inhibit polyamines and NO levels, a precursor of cancer cell proliferation, metastasis, and tumor angiogenesis. [ABSTRACT FROM AUTHOR]

N-lined glycosylation is one of the critical quality attributes (CQA) for biotherapeutics impacting the safety and activity of drug product. Changes in pattern and level of glycosylation can significantly alter the intrinsic properties of the product and, therefore, have to be monitored throughout its lifecycle. Therefore fast, precise, and unbiased N-glycan mapping assay is desired. To ensure these qualities, using analytical methods that evaluate completeness of deglycosylation is necessary. For quantification of deglycosylation yield, methods such as reduced liquid chromatography–mass spectrometry (LC-MS) and reduced capillary gel electrophoresis (CGE) have been commonly used. Here we present development of two additional methods to evaluate deglycosylation yield: one based on LC using reverse phase (RP) column and one based on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE gel) with offline software (GelAnalyzer). With the advent of rapid deglycosylation workflows in the market for N-glycan profiling replacing overnight incubation, we have aimed to quantify the level of deglycosylation in a selected rapid deglycosylation workflow. Our results have shown well resolved peaks of glycosylated and deglycosylated protein species with RP-LC method allowing simple quantification of deglycosylation yield of protein with high confidence. Additionally a good correlation, ≥0.94, was found between deglycosylation yields estimated by RP-LC method and that of reduced SDS-PAGE gel method with offline software. Evaluation of rapid deglycosylation protocol from GlycanAssure™ HyPerformance assay kit performed on fetuin and RNase B has shown complete deglycosylation within the recommended protocol time when evaluated with these techniques. Using this kit, N-glycans from NIST mAb were prepared in 1.4 hr and analyzed by hydrophilic interaction chromatography (HILIC) ultrahigh performance LC (UHPLC) equipped with a fluorescence detector (FLD). 37 peaks were resolved with good resolution. Excellent sample preparation repeatability was found with relative standard deviation (RSD) of <5% for peaks with >0.5% relative area. [ABSTRACT FROM AUTHOR]

This study explores determining the sex of humans from blood stains taken from different surfaces and compares the time course of detection with the conventional PCR, Conventional Loop Mediated Isothermal Amplification (LAMP), and LAMP-Lateral Flow Dipstick (LFD). For the DNA templates, 7 male and 7 female blood stained samples were extracted and added to LAMP and PCR reaction solution to amplify the SRY gene. The DNA samples were extracted from the following blood stained materials: cloth, wood, clay, and tile. Then, the samples were stored at room temperature for 1, 7, 30, and 60 day(s). After the DNA amplification, the gel electrophoresis process was applied to detect LAMP product. The LFD was combined with the LAMP to detect LAMP product on the male cloth samples. For the male samples, the time course of detection on the first and seventh days indicated positive for both LAMP and PCR products on all the surfaces while no DNA amplification was found on any of the female samples. On day 30, positive LAMP product was still found on all the male samples. However, it had faded on the tiles. Moreover, all the male samples, which had tested positive for PCR product, were blurred and unclear. On day 60, LAMP product was still found on all the male samples. Conversely, the PCR method resulted in no bands showing for any of the male samples. However, the LAMP-LFD method detected product on all the male samples of cloth. The results show that the LAMP is an effective, practical, and reliable molecular-biological method. Moreover, the LFD can increase the efficiency and sensitivity of the LAMP, making it more suitable for field studies because gel electrophoresis apparatus is not required. [ABSTRACT FROM AUTHOR]

The Lactobacillus paracasei subsp. paracasei M5 strain exerted potential anticancer activity through the cell wall. In this study, whole peptidoglycan (WPG) was extracted from the Lactobacillus paracasei subsp. paracasei M5 strain and was evaluated for anticancer effects as well as its properties. SDS-PAGE analysis confirmed the presence of WPG with dominant bands of approximately 14.4 kDa. Further analysis revealed that the amino acids present in the WPG consisted of alanine, glycine, glutamic acid, and lysine in a molar ratio of approximately 8 : 5 : 3 : 3.5. In addition, the cell viability of HT-29 cells with WPG addition was investigated with methyl thiazolyl tetrazolium (MTT) and trypan blue exclusion (TBE) assays, and cell apoptosis was analyzed with a transmission electron microscope, flow cytometry, and semiquantitative RT-PCR. These results showed that WPG exerted cytotoxic effects on colon cancer HT-29 cells in a dose-dependent manner and upregulated proapoptotic genes, while downregulating antiapoptotic genes. The gene expression study definitively revealed that WPG induced a mitochondria-mediated apoptotic pathway. [ABSTRACT FROM AUTHOR]

Ruminants derived products have a prominent role in diets and economy worldwide; therefore, the capability to control the rumen microbial ecosystem, for ameliorating their quality, is of fundamental importance in the livestock sector. The aim of this study was to evaluate the effect of dietary supplementation with chestnut and quebracho tannins on microbial community and fatty acid profile, in the rumen fluid of dairy ewes. Multivariate analysis of PCR-DGGE profiles of rumen microbial communities showed a correlation among the presence of chestnut or quebracho in the diet, the specific Butyrivibrio group DGGE profiles, the increase in 18:3 cis9, cis12, and cis15; 18:2 cis9 and cis12; 18:2 cis9 and trans11; 18:2 trans11 and cis15; and 18:1 trans11 content, and the decrease in 18:0 concentration. Phylogenetic analysis of DGGE band sequences revealed the presence of bacteria representatives related to the genera Hungatella, Ruminococcus, and Eubacterium and unclassified Lachnospiraceae family members, suggesting that these taxa could be affected by tannins presence in the diets. The results of this study showed that tannins from chestnut and quebracho can reduce the biohydrogenation of unsaturated fatty acids through changes in rumen microbial communities. [ABSTRACT FROM AUTHOR]

The goal of the present study were (1) to investigate the pathological characteristics of gastrocnemius muscle (GM) and quantitatively assess GM tissue stiffness in rat models with spinal cord injury (SCI) and (2) to explore the correlation between pathological characteristics changes and Young’s modulus value of GM. 24 Sprague Dawley male rats were allocated into normal control groups and SCI model subgroups, respectively. GM stiffness was assessed with shear wave sonoelastography technology. All GMs were further analyzed by pathological examinations. GM weights were decreased, the ratio of type I fibers was decreased, and the ratio of type II fibers was increased in the GM in the model group. MyHC-I was decreased, while MyHC-II was increased according to the electrophoretic analysis in model subgroups. The elastic modulus value of GM was increased in the model group. A significant negative correlation was found between Young’s modulus value of GM and the ratio of type I fibers of GM in model subgroup. Our studies showed that the stiffness of GM is correlated with pathological characteristics during the initial stages of SCI in rats. We also identified shear wave sonoelastography technology as a useful tool to assess GM stiffness in SCI rat models. [ABSTRACT FROM AUTHOR]

Our objective was to determine if whole genome amplification (WGA) provides suitable DNA for qPCR-based genotyping for human embryos. Single blastomeres (Day 3) or trophoblastic cells (Day 5) were isolated from 342 embryos for WGA. Comparative Genomic Hybridization determined embryo sex as well as Trisomy 18 or Trisomy 21. To determine the embryo’s sex, qPCR melting curve analysis for SRY and DYS14 was used. Logistic regression indicated a 4.4%, 57.1%, or 98.8% probability of a male embryo when neither gene, SRY only, or both genes were detected, respectively (accuracy = 94.1%, kappa = 0.882, and p<0.001). Fluorescent Capillary Electrophoresis for the amelogenin genes (AMEL) was also used to determine sex. AMELY peak’s height was higher and this peak’s presence was highly predictive of male embryos (AUC = 0.93, accuracy = 81.7%, kappa = 0.974, and p<0.001). Trisomy 18 and Trisomy 21 were determined using the threshold cycle difference for RPL17 and TTC3, respectively, which were significantly lower in the corresponding embryos. The Ct difference for TTC3 specifically determined Trisomy 21 (AUC = 0.89) and RPL17 for Trisomy 18 (AUC = 0.94). Here, WGA provides adequate DNA for PCR-based techniques for preimplantation genotyping. [ABSTRACT FROM AUTHOR]

Here we aimed to develop a capillary electrophoresis-based high-throughput multiplex polymerase chain reaction (PCR) system for the simultaneous detection of nine pathogens in swine. Nine pairs of specific primers and a set of universal primers were designed; the multiplex PCR was established. The specificity and cross-reactivity of this assay were examined, and the detection limit was determined using serial 10-fold dilutions of plasmids containing the target sequences. The assay was further tested using 144 clinical samples. We found that the nine specific amplification peaks were observed, and the assay had a high degree of specificity, without nonspecific amplification. The simultaneous detection limit for the nine viruses reached 10000 copies μL−1 when all of the premixed viral targets were present. Seventy-seven of the clinical samples tested positive for at least one of the viruses; the principal viral infections in the clinical samples were porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus. This approach has much potential for further development of high-throughput detection tools for the diagnosis of diseases in animals. [ABSTRACT FROM AUTHOR]

Metal nanoparticles have been extensively used in industry as well as in biomedical application. In this work, we have evaluated the toxic potential of manganese dioxide (MnO2) nanoparticles (MNPs) on human neuronal (SH-SY5Y) cells. Cellular toxicity due to MNPs (0, 10, 30, and 60 μg/ml) on the SH-SY5Y cell was observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red uptake (NRU) tests. MNPs produced reactive oxygen species (ROS) and declined in mitochondrial membrane potential in the SH-SY5Y cell in dose and duration dependent manner. Moreover, lipid peroxide (LPO), superoxide dismutase (SOD), and catalase (CAT) activities were increased and glutathione was reduced in dose and time dependent manner. A significant upgrade in Hoechst 33342 fluorescence intensity (chromosome condensation) and phosphatidylserine translocation (apoptotic cell) was visualized in cells treated with MNPs for 48 h. On the other hand, caspase-3 activity was increased due to MNPs in SH-SY5Y cells. DNA strand breaks were determined by alkaline single cell gel electrophoresis assay (Comet Assay) and maximum fragmentation of DNA produced due to MNPs (60 μg/ml) for 48 hours. This result provides a basic mechanism of induction of apoptosis and toxicity by MNPs in SH-SY5Y cells. [ABSTRACT FROM AUTHOR]

Fat mass and obesity-associated protein is an enzyme that oxidatively demethylates DNA. Although there are numerous studies regarding the catalytic function of FTO, the overall existence or absence of FTO on cellular proteome has not been investigated. This study investigated the changes in the soluble proteome of 3T3-L1 cells upon expression of the WT and the mutant (R316Q) FTO proteins. Protein extracts prepared from 3T3-L1 cells expressing either the WT or the mutant FTO proteins were used in DIGE experiments. Analysis of the data revealed the number of spots matched to every member and there were 350 ± 20 spots with 30.5% overall mean coefficient of variation. Eleven regulated protein spots were excised from the gels and identified by MALDI-TOF/TOF. One of the identified proteins was heterogeneous nuclear ribonucleoprotein K, which displayed more than 2.6- and 3.7-fold increases in its abundance in the WT and the mutant FTO expressing cells, respectively. Western blot analysis validated these observations. This is the first study revealing the presence of a parallel increase in expressions of FTO and HNRNPK proteins. This increase may codictate the metabolic changes occurring in the cell and may attribute a significance to HNRNPK in FTO-associated transformations. [ABSTRACT FROM AUTHOR]

Porcine circovirus virus type II (PCV2) is the etiology of postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis, nephropathy syndrome (PDNS), and necrotizing pneumonia. Rapid diagnosis tool for detection of PCV2 plays an important role in the disease control and eradication program. Recombinase polymerase amplification (RPA) assays using a real-time fluorescent detection (PCV2 real-time RPA assay) and RPA combined with lateral flow dipstick (PCV2 RPA LFD assay) were developed targeting the PCV2 ORF2 gene. The results showed that the sensitivity of the PCV2 real-time RPA assay was 102 copies per reaction within 20 min at 37°C and the PCV2 RPA LFD assay had a detection limit of 102 copies per reaction in less than 20 min at 37°C. Both assays were highly specific for PCV2, with no cross-reactions with porcine circovirus virus type 1, foot-and-mouth disease virus, pseudorabies virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, and classical swine fever virus. Therefore, the RPA assays provide a novel alternative for simple, sensitive, and specific identification of PCV2. [ABSTRACT FROM AUTHOR]

Outdoor microalgae cultivation process is threatened by many issues, such as pest pollution and complex, changeable weather. Therefore, it is difficult to have identical growth rate for the microalgae cells and to keep their continuous growth. Outdoor cultivation requires the algae strains not only to have a strong ability to accumulate oil, but also to adapt to the complicated external environment. Using 18S rRNA technology, one wild strain Scenedesmus sp. FS was isolated and identified from the culture of Chlorella zofingiensis. Upon contamination by Scenedesmus sp., the species could quickly replace Chlorella zofingiensis G1 and occupy ecological niche in the outdoor column photobioreactors. The results indicated that Scenedesmus sp. FS showed high alkali resistance. It also showed that even under the condition of a low inoculum rate (OD680, 0.08), Scenedesmus sp. FS could still grow in the outdoor raceway pond under a high alkaline environment. Even under unoptimized conditions, the oil content of Scenedesmus sp. FS could reach more than 22% and C16–C18 content could reach up to 79.68%, showing that this species has the potential for the biodiesel production in the near future. [ABSTRACT FROM AUTHOR]

HS628 has been developed as a proposed biosimilar product of originator tocilizumab (Actemra®). An extensive physicochemical and biological characterization was conducted to assess similarity between HS628 and originator tocilizumab. The amino acid sequence was shown to be identical between HS628 and originator tocilizumab. The higher order structure was found to be indistinguishable from originator tocilizumab. Concerning purity and heterogeneity, HS628 was demonstrated to have similar posttranslational modifications, charge heterogeneity, size heterogeneity, and glycosylation to originator tocilizumab. Moreover, HS628 exhibited highly similar binding affinity and antiproliferative activity as well as capability of inhibiting STAT3 phosphorylation compared to originator tocilizumab. Taken together, HS628 can be considered as a highly similar molecule to originator tocilizumab in terms of physicochemical and biological properties. [ABSTRACT FROM AUTHOR]

The present study was conducted to evaluate both antioxidant and anti-inflammatory activity of Banhasasim-tang (BHSST) on chronic acid reflux esophagitis (CRE) model. Rat CRE model was established operatively and then treated with BHSST (1 g/kg body weight per day) for 15 days Esophageal pathological changes were analyzed using macroscopic examination and hematoxylin/eosin staining. The antioxidant and inflammatory protein levels were determined using Western blotting. The administration of BHSST significantly reduced both the overexpression of serum reactive oxygen species (ROS) and an excessive formation of thiobarbituric acid-reactive substances (TBARS) in esophagus tissue. Thus, the severity of esophageal ulcer was lower in BHSST treated rats than control rats on the gross and histological evaluation. Nuclear factor-erythroid 2-related factor 2 (Nrf2) led to the upregulation of antioxidant enzyme including SOD, GPx-1/2, and HO-1 by binding to antioxidant response element (ARE). Moreover, BHSST administration markedly reduced the expression of inflammatory proteins through mitogen-activated protein kinase- (MAPK-) related signaling pathways and decreased significantly the protein expressions of inflammatory mediators and cytokines by inhibition of nuclear factor-kappa B (NF-κB) activation. Taken together, these results support the fact that BHSST administration can suppress the development of esophageal mucosal ulcer via regulating inflammation through the activation of the antioxidant pathway. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate the contribution of SHISA3 promoter methylation to laryngeal squamous cell carcinoma (LSCC). SHISA3 promoter methylation status and expression were determined using methylation-specific polymerase chain reaction (MSP) and quantitative real-time PCR (qRT-PCR) in 93 paired LSCC and adjacent normal tissues, respectively. Furthermore, the regulatory function of the SHISA3 promoter fragment was analyzed using a luciferase reporter assay. The results reveal that there is a significant increase in SHISA3 methylation in LSCC tissues compared with corresponding nontumor tissues P=4.58E-12. The qRT-PCR results show a significant association between SHISA3 methylation and expression in LSCC P=1.67E-03. In addition, the area under the receiver operating characteristic curve was 0.91. Consequently, a log-rank test and multivariate Cox analysis suggest that SHISA3 promoter hypermethylation is a predictor of poor overall survival for LSCC (log-rank P = 0.024; HR = 2.71; 95% CI = 1.024–7.177; P = 0.047). The results indicate that SHISA3 promoter hypermethylation might increase the risk of LSCC through regulation of gene expression and is a potential diagnostic and prognostic biomarker for LSCC. [ABSTRACT FROM AUTHOR]

JPJD was an ideal alternative traditional Chinese medicine compound in the prevention and treatment of CRC, but its underlying mechanisms has not been fully elucidated. In this study, we demonstrated in vitro that TGF-β-induced EMT promoted the invasion and metastasis of CRC cells, reduced the expression of E-cadherin, and elevated the expression of Vimentin. However, JPJD could inhibit the invasive and migratory ability of TGF-β-stimulated CRC cells in a concentration-dependent manner through increasing the expression of E-cadherin and repressing the expression of Vimentin, as well as the inhibition of TGF-β/Smad signaling pathway. Meanwhile, JPJD reduced the transcriptional activities of EMT-associated factors Snail and E-cadherin during the initiation of TGF-β-induced EMT. In vivo, the results demonstrated that JPJD can significantly inhibit the liver and lung metastasis of orthotopic CRC tumor in nude mice, as well as significantly prolonging the survival time of tumor-bearing in a dose-dependent manner. Additionally, JPJD can upregulate the expression of E-cadherin and Smad2/3 in the cytoplasm and downregulate the expression of Vimentin, p-Smad2/3, and Snail in the orthotopic CRC tumor tissues. In conclusions, our new findings provided evidence that JPJD could inhibit TGF-β-induced EMT in CRC through TGF-β/Smad mediated Snail/E-cadherin expression. [ABSTRACT FROM AUTHOR]

Background. Neuroinflammation which presents as a possible mechanism of delirium is associated with MCP-1, an important proinflammatory factor which is expressed on astrocytes. It is known that dexmedetomidine (DEX) possesses potent anti-inflammatory properties. This study aimed to investigate the potential effects of DEX on the production of MCP-1 in lipopolysaccharide-stimulated astrocytes. Materials and Methods. Astrocytes were treated with LPS (10 ng/ml, 50 ng/ml, 100 ng/ml, and 1000 ng/ml), DEX (500 ng/mL), LPS (100 ng/ml), and DEX (10, 100, and 500 ng/mL) for a duration of three hours; expression levels of MCP-1 were measured by real-time PCR. The double immunofluorescence staining protocol was utilized to determine the expression of α2-adrenoceptors (α2AR) and glial fibrillary acidic protein (GFAP) on astrocytes. Results. Expressions of MCP-1 mRNA in astrocytes were induced dose-dependently by LPS. Administration of DEX significantly inhibited the expression of MCP-1 mRNA (P<0.001). Double immunofluorescence assay showed that α2AR colocalize with GFAP, which indicates the expression of α2-adrenoceptors in astrocytes. Conclusions. DEX is a potent suppressor of MCP-1 in astrocytes induced with lipopolysaccharide through α2A-adrenergic receptors, which potentially explains its beneficial effects in the treatment of delirium by attenuating neuroinflammation. [ABSTRACT FROM AUTHOR]