Evaluation of the Impact of Irradiance Lamps and Storage Media on Elution of TEGDMA from Dental Composites.

OBJECTIVES: The aim of this study was to evaluate and compare the effect of irradiance light and storage media on the elution of triethylene glycol dimethacrylate (TEGDMA) from conventional Filtek Z350XT 3M ESPE and two bulk-fill composites Shofu Beautifil-Bulk and Filtek Bulk fill flowable 3M ESPE using high-performance liquid chromatography (HPLC). MATERIALS AND METHODS: Shofu Beautifil-Bulk, Filtek Bulk fill flowable 3M ESPE, and Filtek Z350XT 3M ESPE were the three types of composites used in this study. Disk shaped samples of 4-mm thickness and 10-mm diameter were fabricated using a stainless steel mold and were polymerized using light emitting diode (LED) and quartz tungsten halogen (QTH) lamps. After polymerization, the samples were immersed in ethanol, artificial saliva with betel quid extract, and distilled water for 1, 7, and 30 days, respectively. The elution of monomer TEGDMA was evaluated using HPLC. STATISTICAL ANALYSIS: To evaluate the mean concentration difference, mixed way analysis of variance (ANOVA) was applied. Between different light, materials, and within the time duration, Tukey's post hoc test was used. A p value of 0.05 was considered significant. RESULTS: During the first day of storage, a significant amount of monomer TEGDMA elution was seen in all the materials. The highest values observed to be in the disks cured with QTH lamp. However, the highest elution was seen when the disks were immersed in ethanol/water solution. While the most stable medium was distilled water, artificial saliva with betel nut extract also had a significant effect on the elution of TEGDMA. The highest value obtained was of Filtek Bulk fill flowable 3M ESPE after 30 days of immersion in both LED and QTH cured disks. CONCLUSION: Filtek Bulk fill flowable 3M ESPE shows better properties in relation to the release of monomer TEGDMA as it releases less amount of monomer in the storage media. The release of monomer was highest in ethanol as compared to artificial saliva and distilled water with the passage of time.

Assessment of Salivary MMP-8 and IL-1ß for the Diagnosis of Periodontal Diseases in Pakistani Population.

OBJECTIVE: Clinical methods use the subjective diagnosis of periodontal diseases by visual observation that could result in differences and variability of diagnosis. The addition of specific markers could aid in the accurate diagnosis of the local population. The objective of the study was to target two of the major proteins for possible significance in such an approach. MATERIALS AND METHODS: Unstimulated saliva samples were collected from 60 participants aged between 18 and 70 years. Three groups each with twenty participants were recruited into periodontitis, gingivitis, and healthy control. STATISTICAL ANALYSIS: The samples were analyzed using human enzyme-linked immunosorbent assay kits for matrix metalloproteinase-8 (MMP-8) and interleukin-1ß (IL-1ß). RESULTS: SPSS version 20 was used to analyze the result. Posthoc analysis by Tukey's test revealed that MMP-8 levels were higher in gingivitis and periodontitis groups as compared with healthy controls. The test also revealed that IL-1ß levels were higher in the periodontitis group compared with the healthy control and gingivitis group. Additionally, one-way analysis of variance analysis showed a significant effect on probing depth in gingivitis and periodontitis patients. The mean age of periodontitis group was significantly higher than other groups. CONCLUSION: Salivary biomarkers may provide useful diagnostic information and could be utilized as tests for periodontal disease screening, prognosis, and prediction.

Bioaccumulation of Chloropyrifos Organo-pesticide and Its Toxicogenic Association with Antioxidant GSTP1 in Pakistani Pest Control Workers.

Synthetic pesticides are employed to enhance agricultural production. Chronic exposure to organophosphate (OP) pesticides may be a source of health problems. The present study was designed to examine an association of GSTP1 (rs1695) polymorphism with OP pesticide chronic exposure. A case-control study was recruited with 250 subjects comprising exposed (n = 100) and controls (n = 150). A survey was conducted to determine the pesticide type to which workers had exposed. According to recorded survey assessment, two compounds of OP pesticides chloropyrifos and malathion were investigated in the blood samples of exposed study subjects using high-performance liquid chromatography (HPLC). For screening of genetic polymorphism in GSTP1 (rs1695) polymerase chain reaction, restriction length polymorphism (PCR-RFLP) and agarose gel electrophoresis were performed. Statistically, data were analyzed using SPSS v. 20.0 and MedCal© software. Total chrom© navigator programmer was used for detection of OP residues in serum and local pesticide solution. chloropyrifos-OP pesticide residues were detected in serum of estimated chronically exposed subjects at 206 nm HPLC optimal conditions. The pattern of GSTP1 (rs1695) genotypic frequencies depicted that heterozygous genotype was higher in Chloropyrifos exposed subjects (0.56) when compared with controls (0.44). Statistical outcomes showed an insignificant association with GSTP1 (rs1695) polymorphism and chloropyrifos-OP pesticide toxicity (Fisher's exact test 1.0, p = 0.25). An insignificant allelic investigation reflected a protective effect of mutant allele G against chloropyrifos-OP pesticide toxicity in exposed subjects. Findings may be helpful in identifying bioaccumulated pesticide residues, but in studied Pakistani exposed workers, no significant association of GSTP1 (rs1695) variant with chloropyrifos-OPs was demonstrated.

Utilization of wild Cressa cretica biomass for pectinase production from a halo-thermotolerant bacterium.

Halophytes are the native inhabitants of saline environment. Their biomass can be considered as a potential substrate for the production of microbial enzymes. This study was intended at feasible utilization of a halophytic biomass, Cressia cretica, for pectinase production using a halo- and thermo-tolerant bacterium, Bacillus vallismortis MH 10. The data from fractionation of the C. cretica biomass revealed presence of 17% pectin in this wild biomass. Seven different factors (temperature, agitation, pH, inoculum size, peptone concentration, substrate concentration, and incubation time) affecting pectinase production using C. cretica were assessed through a statistical tool, Plackett-Burman design. Consequently, two significant factors (incubation time and peptone concentration) were optimized using the central composite design. The strain produced 20 IU mL-1 of pectinase after 24 h under optimized conditions. The enzyme production kinetics data also confirmed that 24 h is the most suitable cultivation period for pectinase production. Fourier transform infrared spectroscopy and scanning electron microscopy of C. cretica biomass ascertained utilization of pectin and structural changes after fermentation. The purification of pectinase by using DEAE column yielded specific activity and purification fold of 88.26 IU mg-1 and 3.2, respectively. The purified pectinase had a molecular weight of >65 kDa. This study offers prospects of large-scale production of pectinase by halotolerant strain in the presence of economical and locally grown substrate that makes the enzyme valuable for various industrial operations.

Colloidal gold based immunochromatographic detection of Mycoplasmopsis synoviae infection and its prevalence in avian species of Karachi, Pakistan.

Avian mycoplasmosis is an infection that commonly prevails in birds, particularly in poultry chickens. Among mycoplasmosis causing organisms, Mycoplasmopsis synoviae is a predominant and lethal pathogen to the aves. Considering the increased incidence of infections by M. synoviae, the prevalence of M. synoviae was deduced in poultry chickens and fancy birds of Karachi region. The lungs and tracheal samples from chicken and dead fancy birds and swab samples from live fancy birds were collected and investigated by amplifying 16 s rRNA gene of M. synoviae. Biochemical characteristics of M. synoviae was also evaluated. Furthermore, surface-associated membrane proteins, that represent key antigens for diagnosis of M. synoviae infection was extracted by Triton X- 114 method. Results showed that M. synoviae was detected more frequently in lungs than in trachea, that could be due to its invasion capacity and tissue affinity. SDS PAGE analysis of extracted membrane proteins showed two prominent hydrophobic proteins of different molecular mass including proteins of 150 and 50 kDa. Protein of 150 kDa was purified by size exclusion chromatography and it exhibited agglutinogen activity. Purified protein was used in the development of one-step immunochromatographic (ICT) assay for the detection of antibodies against M. synoviae using gold nanoparticles coated with polyclonal antibodies. Low levels of antibodies were detected by the developed ICT kit, which has 88% sensitivity with 92% specificity.

Isolation of lectin from Musa acuminata for its antibiofilm potential against Methicillin-resistant Staphylococcus aureus and its synergistic effect with Enterococcus species.

Methicillin resistant Staphylococcus aureus (MRSA) is an emerging pathogen posing a considerable burden on the healthcare system due to its involvement in skin and soft tissue infections (SSTIs). Lectins are carbohydrate binding proteins found ubiquitously in animals, plants and microorganisms. Extraction and isolation of proteins from Musa acuminata were performed by using Affinity chromatography with Sephadex G 75 to determine antibiofilm activity against MRSA. Enterococcus strains obtained from dairy products, beans and vegetables were also screened for its potential to inhibit growth and biofilm formation of MRSA by using 96 well microtiter plates. Synergistic effect of cell free supernatant of Enterococcus with proteins from ripe banana were also tested. BanLec was successfully isolated and appeared as 15 KDa band after SDS-PAGE (15%) while multiple bands of unbound protein fractions were observed. The unbound fractions showed inhibition of planktonic cells and biofilm but BanLec exhibited no significant effect. The CFS of Enterococcus faecium (LCM002), Enterococcus lactis (LCM003) and Enterococcus durans (LCM004 and LCM005) displayed antagonistic effects against pathogen. The synergistic effect of CFS from E. lactis (LCM003) and unbound proteins showed inhibition of biofilm and pathogenic growth. This study demonstrates the use of Enterococcus species and plant proteins against pathogens and results suggested that it can inhibit the growth of resistant strains of Staphylococcus aureus and their synergistic effect has opened new ways to tackle emerging resistance. Furthermore, after assessment of Enterococcus as probiotics, this could be used in food industries as well as in treatment of severe skin infections.

Synthesis of methylcellulose-polyvinyl alcohol composite, biopolymer film and thermostable enzymes from sugarcane bagasse.

Agro-industrial wastes and by-products are the natural and abundant resources of biomaterials to obtain various value-added items such as biopolymer films, bio-composites and enzymes. This study presents a way to fractionate and to convert an agro-industrial residue, sugarcane bagasse (SB), into useful materials with potential applications. Initially cellulose was extracted from SB which was then converted into methylcellulose. The synthesized methylcellulose was characterized by scanning electron microscopy and FTIR. Biopolymer film was prepared by using methylcellulose, polyvinyl alcohol (PVA), glutaraldehyde, starch and glycerol. The biopolymer was characterized to exhibit 16.30 MPa tensile strength, 0.05 g/m2 h of water vapor transmission rate, 366 % of water absorption to its original weight after 115 min of immersion, 59.08 % water solubility, 99.05 % moisture retention capability and 6.01 % of moisture absorption after 144 h. Furthermore, in vitro studies on absorption and dissolution of model drug by biopolymer showed 2.04 and 104.59 % of swelling ratio and equilibrium water content, respectively. Biocompatibility of the biopolymer was checked by using gelatin media and it was observed that swelling ratio was higher in initial 20 min of contact. The extracted hemicellulose and pectin from SB were fermented by a thermophilic bacterial strain, Neobacillus sedimentimangrovi UE25 that yielded 12.52 and 6.4 IU mL-1 of xylanase and pectinase, respectively. These industrially important enzymes further augmented the utility of SB in this study. Therefore, this study emphasizes the possibility for industrial application of SB to form various products.

Characterization of a novel end product tolerant and thermostable cellulase from Neobacillus sedimentimangrovi UE25.

Recent advancements in biorefinery processes necessitate search for cost effective and thermostable cellulases. This study was designed to characterize the cellulase obtained from a thermophilic bacterium, Neobacillus sedimentimangrovi UE25. A combined pretreatment of NaOH and methyltrioctylammonium chloride was given to sugarcane bagasse (SB) for lignin removal and the pretreated SB was utilized as a carbon source for the cellulase production. The thermostable cellulase thus obtained was characterized by adopting central composite design which has not been reported earlier for this purpose. Cellulase showed its maximum activity at pH 7 and temperature 60 â„ƒ and it remained active in the presence of many salts and detergents. Endoglucanase (EG) was found to be stable for 30 min at 80 â„ƒ. The purification of EG by using DEAE column yielded specific activity and purification fold of 365.866 IU mg-1 and 4.264, respectively. The purified EG had a molecular weight of ∼45 kDa. End product tolerance of EG was also evident, as an activity of 228.57 IU mL-1 was observed in the presence of 60 mM glucose which revealed that it does not lose its activity upon accumulation of end-product when the reaction is prolonged. The purified EG exhibited Vmax and Km of 294 U mL-1 min-1 and 36 µM, respectively, in the presence of 60 mM glucose. This novel thermostable cellulase can finds its applications in industrial sector.

Differentially Expressed Salivary Proteins in Dental Caries Patients.

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.

Human salivary protein extraction from RNAPro·SAL™, Pure·SAL™, and passive drooling method.

OBJECTIVE: The aim of the current study was to carry out a preliminary validation of devices for standardized collection of whole mouth fluid (WMF) in comparison to the passive drooling method for protein analysis in healthy subjects. MATERIALS AND METHODS: A carefully designed sample collection/pretreatment protocol is crucial to the success of any saliva proteomics project. In this study, WMF was collected from healthy volunteers (n = 10, ages: 18-26 years). Individuals with any oral disease were excluded from the study group. In our study, we evaluated the following collection methods; the classical passive drooling method (unstimulated whole saliva) and standardized tools for saliva collection (Pure·SAL™, and RNAPro·SAL™) from Oasis Diagnostics® Corporation (Vancouver WA, USA). For estimation of protein levels, we used the bicinchoninic acid assay and protein assay kit (Thermo Fisher). The two-dimensional gel electrophoresis sample analysis was carried out for the estimation of proteins in one of the samples. RESULTS: When gels were compared, the difference was seen in the resolution of spots. Protein spots were fading from high- to low-molecular weight masses. Hence, advanced devices in comparison to spitting method resulted in much clearer protein spots which in turn prove the validation of devices. CONCLUSIONS: In this study, we concluded that protein extraction could be possible by both methods such as passive drooling method and through advanced saliva collection devices (Pure·SAL™ and RNAPro·SAL™).

Histatin peptides: Pharmacological functions and their applications in dentistry.

There are many human oral antimicrobial peptides responsible for playing important roles including maintenance, repairing of oral tissues (hard or soft) and defense against oral microbes. In this review we have highlighted the biochemistry, physiology and proteomics of human oral histatin peptides, secreted from parotid and submandibular salivary glands in human. The significance of these peptides includes capability for ionic binding that can kill fungal Candida albicans. They have histidine rich amino acid sequences (7-12 family members; corresponding to residues 12-24, 13-24, 12-25, 13-25, 5-11, and 5-12, respectively) for Histatin-3. However, Histatin-3 can be synthesized proteolytically from histatin 5 or 6. Due to their fungicidal response and high biocompatibility (little or no toxicity), these peptides can be considered as therapeutic agents with most probable applications for example, artificial saliva for denture wearers and salivary gland dysfunction conditions. The objectives of current article are to explore the human histatin peptides for its types, chemical and biological aspects. In addition, the potential for therapeutic bio-dental applications has been elaborated.

The Primary Structure of ß(I)-Chain of Hemoglobin from Snake Sindhi Krait (Bungarus sindanus sindanus).

The amino acid sequence of ß(I)-globin chain from Sindhi Krait (Bungarus sindanus sindanus) was determined to study the molecular evolution among snakes. The hemoglobin was isolated from the red blood cells and was analyzed by ion-exchange chromatography (IEX). The crude globin was subjected to reversed phased-high performance liquid chromatography (RP-HPLC) using C4 column. The N-terminal sequences of intact globin chains and tryptic peptides were determined by Edman degradation in a pulsed liquid gas phase sequencer using an online Phenylthiohydantoin analyzer. Sindhi Krait is expected to express three hemoglobin components that are composed of ß(II), ß(I), α(D) and α(A)-globin chains, as apparent by IEX, RP-HPLC and N-terminal sequence analyses. Sequence alignment and phylogenetic analyses of ß(I) globin chain from Sindhi Krait showed closest relationship with ß(I) globin chain from Rattlesnake, Water snake and Indigo snake. Interestingly, comparison of primary sequence of ß(I) globin chain of Sindhi Krait with human ß chain revealed 63 % similarity along with the retention of all heme contact points. Variations among the two sequences were prominent at αß contact points and in regions directly not important for function.

Laccase enzymes: purification, structure to catalysis and tailoring.

Laccases belong to the multicopper binding protein family that catalysis the reduction of oxygen molecule to produce water. These enzymes are glycosylated proteins and have been isolated and purified from fungi, bacteria, plant, insects and lichens. The variety of commercial and industrial application of laccases has attracted much attention towards the research addressing different aspects of the protein characterization, production and fit for purpose molecule. Here we briefly discuss the purification, catalytic mechanism in light of available understanding of structure-function relationship and the tailoring side of the protein, which has been the subject of recent research. Purification strategy of laccases is a method of choice and is facilitated by increased production of the enzyme. The structure-function relationship has given insights to unfold the catalytic mechanism. Site directed mutagenesis and other modification at C-terminal end or surrounding environment of copper centres have shown promising results to fit for purpose aspect, with a lot remains to be explored in glycosylation status and its alteration.