Changes in the Fluorescence Tracking of NaV1.6 Protein Expression in a BTBR T+Itpr3tf/J Autistic Mouse Model.

The axon initial segment (AIS), the site of action potential initiation in neurons, is a critical determinant of neuronal excitability. Growing evidence indicates that appropriate recruitment of the AIS macrocomplex is essential for synchronized firing. However, disruption of the AIS structure is linked to the etiology of multiple disorders, including autism spectrum disorder (ASD), a condition characterized by deficits in social communication, stereotyped behaviors, and very limited interests. To date, a complete understanding of the molecular components that underlie the AIS in ASD has remained elusive. In this research, we examined the AIS structure in a BTBR T+Itpr3tf/J mouse model (BTBR), a valid model that exhibits behavioral, electrical, and molecular features of autism, and compared this to the C57BL/6J wild-type control mouse. Using Western blot studies and high-resolution confocal microscopy in the prefrontal frontal cortex (PFC), our data indicate disrupted expression of different isoforms of the voltage-gated sodium channels (NaV) at the AIS, whereas other components of AIS such as ankyrin-G and fibroblast growth factor 14 (FGF14) and contactin-associated protein 1 (Caspr) in BTBR were comparable to those in wild-type control mice. A Western blot assay showed that BTBR mice exhibited a marked increase in different sodium channel isoforms in the PFC compared to wild-type mice. Our results provide potential evidence for previously undescribed mechanisms that may play a role in the pathogenesis of autistic-like phenotypes in BTBR mice.

Biomarkers as Independent Predictors of Bone Regeneration around Biomaterials: A Systematic Review of Literature.

BACKGROUND: Biomarkers are detected during bone formation and resorption associated with the dynamics of bone metabolism and are gaining importance as preferential indicators of bone healing in comparison with conventional methodologies. Current literature suggests that the usage of bone turnover markers for monitoring bone regeneration in association with biomaterials is limited. AIM: To systematically review literature and evaluate whether bone-biomarkers can independently predict bone regeneration following implantation of various bone biomaterials. MATERIALS AND METHODS: An electronic search was conducted in PubMed (MEDLINE) database from 1980 to January 2017. The articles for systematic review were selected based on formulated inclusion and exclusion criteria Results: Upon database searching, 443 articles were retrieved and thoroughly reviewed based on the inclusion and exclusion criteria. In all, 41 studies were finally included for evaluation out of which 4 were clinical studies and the remaining 37 studies utilized animal models. On further evaluation, 12 studies reported the presence of biomarkers in association with cellular response during bone regeneration around bio-materials. Moreover, biomarkers related to enzyme activity and matrix protein derivatives were enhanced during bone-matrix deposition as reported in 14 studies. Inorganic skeletal matrix biomarkers indicative of bone mineralization showed positive expression in eight studies. CONCLUSION: Several biomarkers appear to be useful for the assessment of bone regeneration around biomaterials. Although biomarkers are capable of independently predicting bone regeneration, lack of substantial evidence in the literature limits their true clinical utility. CLINICAL SIGNIFICANCE: Noninvasive and inexpensive methods of isolating and characterization of biomarkers from cellular and extracellular skeletal matrix during bone regeneration have proven value in evaluating success of bone biomaterials.

FTY720 Reduces the Biomass of Biofilms in Pseudomonas aeruginosa in a Dose-Dependent Manner.

Pseudomonas aeruginosa, a nosocomial pathogen, has strong biofilm capabilities, representing the main source of infection in the human body. Repurposing existing drugs has been explored as an alternative strategy to combat emerging antibiotic-resistant pathogens. Fingolimod hydrochloride (FTY720), an immunomodulatory drug for multiple sclerosis, has shown promising antimicrobial effects against some ESKAPE pathogens. Therefore, the effects of FTY720 on the biofilm capabilities of Pseudomonas aeruginosa were investigated in this study. It was determined that FTY720 inhibited the growth of P. aeruginosa PAO1 at 100 µM. The significant reduction in PAO1 cell viability was observed to be dose-dependent. Additional cytotoxicity analysis on human cell lines showed that FTY720 significantly reduced viabilities at sub-inhibitory concentrations of 25-50 µM. Microtiter assays and confocal analysis confirmed reductions in biofilm mass and thickness and the cell survivability ratio in the presence of FTY720. Similarly, virulence production and biofilm-related gene expression (rhlA, rhlB, pilA, pilI, fliC, fliD and algR) were determined. The results demonstrate that pigment production was affected and quantitative real-time PCR analysis showed a variable degree of reduced gene expression in response to FTY720 at 12.5-50 µM. These findings suggest that FTY720 could be repurposed as an alternative antibiofilm agent against Pseudomonas aeruginosa.

Exploring glutathione transferase and Cathepsin L-like proteinase for designing of epitopes-based vaccine against Fasciola hepatica by immunoinformatics and biophysics studies.

Fasciolosis is a zoonotic infection and is considered a developing deserted tropical illness threatening ruminant productivity and causing financial losses. Herein, we applied immunoinformatics and biophysics studies to develop an epitopes vaccine against Fasciola hepatica using glutathione transferase and Cathepsin L-like proteinase as possible vaccine candidates. Using the selected proteins, B- and T-cell epitopes were predicted. After epitopes prediction, the epitopes were clarified over immunoinformatics screening, and only five epitopes, EFGRWQQEKCTIDLD, RRNIWEKNVKHIQEH, FKAKYLTEMSRASDI, TDMTFEEFKAKYLTE, and YTAVEGQCR were selected for vaccine construction; selected epitopes were linked with the help of a GPGPG linker and attached with an adjuvant through another linker, EAAAK linker. Cholera toxin B subunit was used as an adjuvant. The ExPASy ProtParam tool server predicted 234 amino acids, 25.86257 kDa molecular weight, 8.54 theoretical pI, 36.86 instability index, and -0.424 grand average of hydropathicity. Molecular docking analysis predicted that the vaccine could activate the immune system against F. hepatica. We calculated negative binding energy values. A biophysics study, likely molecular docking molecular dynamic simulation, further validated the docking results. In molecular dynamic simulation analysis, the top hit docked compounds with the lowest binding energy values were subjected to MD simulation; the simulation analysis showed that the vaccine and immune cell receptors are stable and can activate the immune system. MMGBSA of -146.27 net energy (kcal/mol) was calculated for the vaccine-TLR2 complex, while vaccine-TLR4 of -148.11 net energy (kcal/mol) was estimated. Furthermore, the C-ImmSim bioinformatics tool predicted that the vaccine construct can activate the immune system against F. hepatica, eradicate the infection caused by F. hepatica, and reduce financial losses that need to be spent while protecting against infections of F. hepatica. The computational immune simulation unveils that the vaccine model can activate the immune system against F. hepatica; hence, the experimental scientist can validate the finding accomplished through computational approaches.

High molecular weight hyaluronic acid reduces the growth and biofilm formation of the oral pathogen Porphyromonas gingivalis.

Background: Porphyromonas gingivalis (P. gingivalis) is viewed as a keystone microorganism in the pathogenesis of periodontal and peri-implant diseases. Hyaluronic acid (HA) is believed to exert antimicrobial activity. The aim of this study is to assess the in-vitro growth and biofilm formation of P. gingivalis under HA and compare the effect of HA to that of azithromycin (AZM) and chlorhexidine (CHX). Materials and methods: In each material, the minimum inhibitory concentration (MIC), 50% MIC, 25% MIC, and 12.5% MIC were tested. The growth of P. gingivalis was evaluated by absorbance spectrophotometry after 48 h. A biofilm inhibition assay was performed on a 72-hour culture by washing planktonic bacterial cells, fixing and staining adherent cells, and measuring the variation in stain concentrations relative to the untreated control using absorbance spectrophotometry. Results: The results show that the overall growth of P. gingivalis after 48 h was 0.048 ± 0.030, 0.008 ± 0.013, and 0.073 ± 0.071 under HA, AZM, and CHX, respectively, while the untreated control reached 0.236 ± 0.039. HA was also able to significantly reduce the biofilm formation of P. gingivalis by 64.30 % ± 22.39, while AZM and CHX reduced biofilm formation by 91.16 %±12.58 and 88.35 %±17.11, respectively. Conclusions: High molecular-weight HA significantly inhibited the growth of P. gingivalis. The overall effect of HA on the growth of P. gingivalis was similar to that of CHX but less than that of AZM. HA was also able to significantly reduce the biofilm formation of P. gingivalis. However, the ability of HA to prevent the biofilm formation of P. gingivalis was generally less than that of both AZM and CHX.

Vitamin C influences antioxidative, anti-inflammatory and wound healing markers in smokers' gingival fibroblasts in vitro.

Background: Saudi Arabia has an overall smoking rate of 15.9%. The link between smoking and periodontal disease has been studied extensively. It is possible for human gingival fibroblasts to accumulate nicotine intracellularly over a period of four hours. Additionally, unmetabolized nicotine is released into the environment. Tobacco presence can impair tissue inflammation, wound healing, and organ development. To counterbalance tobacco toxins, vitamin C has been added to a variety of products. Aim: This study aims to analyze the RNA expression of antioxidant, anti-inflammatory, and wound healing proteins in human gingival fibroblasts from smokers and nonsmokers using polymerase chain reaction. Materials and Methods: hGFs were extracted from clinically healthy periodontium sites of adult male subjects. Both heavy cigarette smokers and never-smokers participated as subjects. Cells were cultured and subcultured in supplemented growth medium. Vitamin C was inducted in the medium at the experimental 6th passage. RNA expression analysis (qRT-PCR) was performed to analyze adhesion, proliferation, and extracellular matrix expression. Results: The results revealed marked expression of a wound healing gene (VEGF-A) in never-smokers (p value = 0.016). GPX3 and SOD3 represent antioxidants that are highly expressed in treated never-smoker cells. SOD2 significantly increased (p value = 0.016) in smokers after vitamin C exposure. The anti-inflammatory markers IL-6 and IL-8 were lower among smokers than among nonsmokers (p < 0.0001). Conclusion: Tobacco smoking suppressed gingival fibroblasts' abilities to regenerate, heal, combat inflammation, and resist free radicals. Vitamin C at cellular levels was beneficial and should be considered in the treatment component of smokers in the dental clinic.

LuxS quorum sensing system and biofilm formation of oral microflora: A short review article.

The LuxS quorum sensing system is considered as the main system that most of the oral bacteria use to communicate in order to create biofilms. Here we identified 11 of the most important biofilm formers that utilize the LuxS system and presented current and recent information regarding this system. Though different bacterial species are able to communicate thorough the LuxS system, it was also found that cross kingdom communication can occur between bacteria and fungi and bacteria and epithelial cells. Immune response also plays and important role in mitigating the effects of biofilms. Here we identified 6 of the most important molecules that are involved in the immune response to biofilms. These immune molecules maintain the stability in the oral cavity by preventing bacteria from overwhelming the space and simultaneously minimizing the immune response in order not to cause tissue damage. Here we also discuss current research being done in order to maintain the balance in the oral cavity via inhibiting biofilm formation without eradicating oral bacteria in order to prevent the overgrowth of other organisms such as Candida albicans. One approach being used is inhibiting AI-2 intermediates which leads to lack of quorum sensing communication between bacteria through the use of intermediate analogues. Another approach that found success is the utilization of D forms of sugars where D-ribose and D-galactose have been proven to inhibit the LuxS system and subsequently preventing the process of quorum sensing leading to the reduction in biofilm formation.

Histological and Histomorphometric Analyses of Bone Regeneration in Osteoporotic Rats Using a Xenograft Material.

We evaluated the effect of osteoporotic induction after eight weeks of initial healing of bone defects grafted with a xenograft material in a rat model. Bone defects were created in the femoral condyles of 16 female Wistar rats (one defect per rat). The defects were filled with bovine bone (Inter-Oss) granules. After eight weeks of bone healing, rats were randomly ovariectomized (OVX) or sham-operated (SHAM). At 14 weeks of bone healing, all animals were euthanized. Bone specimens were harvested and processed for histological and histomorphometric analyses to assess new bone formation (N-BF%), remaining bone graft (RBG%) and trabecular bone space (Tb.Sp%) within the defect area. After 14 weeks of bone healing, histological evaluation revealed a significant alteration in trabecular bone in OVX rats compared to SHAM rats. There was lower N-BF% in OVX rats (22.5% ± 3.0%) compared to SHAM rats (37.7% ± 7.9%; p < 0.05). Additionally, the RBG% was significantly lower in OVX (23.7% ± 5.8%) compared to SHAM (34.8% ± 9.6%; p < 0.05) rats. Finally, the Tb.Sp% was higher in OVX (53.8% ± 7.7%) compared to SHAM (27.5% ± 14.3%; p < 0.05) rats. In conclusion, within the limitations of this study, inducing an osteoporotic condition in a rat model negatively influenced bone regeneration in the created bone defect and grafted with a xenograft material.

Impact of Single or Combined Drug Therapy on Bone Regeneration in Healthy and Osteoporotic Rats.

Complications in bone regeneration in patients with systemic impaired bone metabolism (e.g., osteoporosis) represent a rapidly increasing clinical challenge. Alendronate and simvastatin are drugs commonly used to promote bone metabolism in osteoporotic conditions. The aim of this study was to evaluate initial bone regeneration within osseous defects grafted with beta-tricalcium phosphate (ß-TCP) in adjunction with systemic coadministrations of alendronate and simvastatin (i.e., daily subcutaneous injection for 3 weeks) in healthy and osteoporotic rats. Eighty Wistar female rats were ovariectomized (OVX; n = 40) or sham operated (n = 40). Six weeks later, osseous defects (a 3-mm critical-sized defect) were created in the left femoral condyles and then grafted with ß-TCP. From the day following graft installation, OVX and sham animals received for 3 weeks a daily subcutaneous injection of alendronate (50 µg/kg of body weight) and simvastatin (5 mg/kg of body weight), alone or in combination. A control group was included, which received subcutaneous saline administration. At the end of the 3 weeks, rats were euthanized and specimens (femoral condyles) were retrieved for histological evaluation and histomorphometric measurements, that is, bone area (BA%) and remaining bone graft (RBG%). In osteoporotic rats, 3 weeks of daily subcutaneous injection of combined therapy (alendronate plus simvastatin) led to a significant (p < 0.05) increase in BA% and a significant decrease in RBG% compared to healthy controls in osseous defects grafted with ß-TCP (BA%: 28.6 ± 12.0 vs. 18.2 ± 7.6, RBG% 61.3 ± 11.1 vs. 70.7 ± 7.3). No significant differences in BA% and RBG% were found in the OVX rats for single treatments. Furthermore, healthy controls showed similar BA% and RBG% upon single or combined therapy compared to nontreated control rats. Daily coinjections (for 3 weeks) of alendronate plus simvastatin result in a significant enhancement of bone regeneration within osseous defects grafted with ß-TCP in osteoporotic rats. Despite the expected effects on osteoporotic bone, our study did not confirm the hypothesized benefit of alendronate and simvastatin on bone regeneration in osseous defects in healthy conditions. The efficacy of the combination drug therapy on bone regeneration demands further investigation to elucidate molecular and cellular aspects underlying this therapy.

Probing the surface ultrastructure of Brevibacillus laterosporus using atomic force microscopy.

One of the main obstacles to studying the surface ultrastructure of microbial cells by atomic force microscopy (AFM) is determining how to immobilize live cells on the AFM substrates. Each method has its own advantages and disadvantages. The aim of this study was to characterize a new simple and inexpensive method using two types of polyethersulfone (PES) membrane filters that differ in pore size (micropore and nanopore) to immobilize live and dead Brevibacillus laterosporus for AFM imaging. B. laterosporus was easily trapped by the microporous PES membrane, facilitating the successful AFM scanning of the bacterial surface ultrastructure. In addition, B. laterosporus strongly attached to the nanoporous membranes and withstood the pulling forces exerted by the AFM tip during scanning. These methods of immobilization did not affect the cell viability. The nanostructure and roughness of the bacterial surface were also observed for live, fixed, and air-dried cells. Live and dead bacteria displayed similar morphologies at low resolution, while at high resolution, live bacteria displayed a more convoluted surface ("brain-like structure").

Histomorphometric Evaluation of Peri-Implant Bone Response to Intravenous Administration of Zoledronate (Zometa®) in an Osteoporotic Rat Model.

We evaluated the response to peri-implant bone placed in the femoral condyle of osteoporotic rats, following intravenous zoledronate (ZOL) treatment in three settings: pre-implantation (ZOL-Pre), post-implantation (ZOL-Post), and pre- + post-implantation (ZOL-Pre+Post). Twenty-four female Wistar rats were ovariectomized (OVX). After 12 weeks, the rats received titanium implants in the right femoral condyle. ZOL (0.04 mg/kg, weekly) was administered to six rats 4 weeks pre-implantation and was stopped at implant placement. To another six rats, ZOL was given post-implantation and continued for 6 weeks. Additional six rats received ZOL treatment pre- and post-implantation. Control animals received weekly saline intravenous injections. At 6 weeks post-implantation, samples were retrieved for histological evaluation of the percentage of bone area (%BA) and of the percentage of bone-to-implant contact (%BIC). BA% for ZOL-Pre (29.6% ± 9.0%) and ZOL-Post (27.9% ± 5.6%) rats were significantly increased compared to that of the controls (17.3% ± 3.9%, p < 0.05). In contrast, ZOL-Pre+Post rats (20.4% ± 5.0%) showed similar BA% compared to Saline controls (p = 0.731). BIC% revealed a significant increase for ZOL-Post (65.8% ± 16.9%) and ZOL-Pre+Post (68.3% ± 10.0%) rats compared with that of Saline controls (43.3% ± 9.6%, p < 0.05), while ZOL-Pre rats (55.6% ± 19%) showed a BIC% comparable to that of Saline controls (p = 0.408). Our results suggest that receiving intravenous ZOL treatment before or after implant placement enhances peri-implant bone responses in terms of bone area. However, the effect of different ZOL treatment regimens on BIC% was found to be inconclusive.

Surface modification of pH-responsive poly(2-(tert-butylamino)ethyl methacrylate) brushes grafted on mesoporous silica nanoparticles.

Poly(2­(tert-butylamino)ethyl methacrylate) brushes (PTBAEMA) are grown from mesoporous silica nanoparticles via surface-initiated atom transfer radical polymerization (SI-ATRP). Linear PTBAEMA brushes are protonated and highly swollen at low pH; brushes are collapsed at pH higher than 7.7 due to deprotonation, as determined by dynamic light scattering (DLS). Quaternization of these brushes is conducted using 2-iodoethanol in alkali media. DLS measurement of nanoparticles shows that surface-confined quaternization occurs and produces pH-responsive brushes with a hydrophobic upper surface. Variation of the 2-iodoethanol reaction time enables the mean degree of surface quaternization. The pH-responsive behaviour of quaternized PTBEAMA brushes at 1 h reaction time indicates low degrees of surface quaternization, dictated by the spatial location of 2-iodoethanol. Almost uniformly quaternized brushes prepared when the conducted for 3 h and became less swollen at low pH than brushes that conducted for 1 h. The intensity of the C - C - O component (286.5 eV) in the C1s X-ray photoelectron spectrum increased, suggesting that the reaction with iodoethanol was successful occurred.

Titanium Oxide (TiO2)/Polymethylmethacrylate (PMMA) Denture Base Nanocomposites: Mechanical, Viscoelastic and Antibacterial Behavior.

Currently, polymethylmethacrylate (PMMA) is the most popular denture base material. Most fractures of dentures that occur during function are due to its insufficient mechanical strength. The major drawbacks of PMMA are insufficient ductility, strength, and viscoelastic behavior. The purpose of this study was to evaluate a polymethylmethacrylate denture base material modified with TiO2 nanoparticles in terms of nanomechanical, creep-recovery, and relaxation. Additionally, the effects of addition TiO2 nanoparticles on the thermal and antimicrobial adhesion behaviors were investigated. Differential scanning calorimetry and thermogravimetric analysis indicated that the effect of small amounts of TiO2 nanoparticles (1 wt. %, 2 wt. %, and 3 wt. %) on the degradation behavior of PMMA denture bases was insignificant. The nanomechanical test results of the PMMA and PMMA/TiO2 nanocomposites indicated that the hardness and modulus in the nanoscale range improved due to TiO2 addition. At a 1200-nm penetration depth, the modulus increased by 10%, 16%, and 29% and hardness increased by 18%, 24%, and 35% with the addition of 1 wt. %, 2 wt. %, and 3 wt. % TiO2, respectively. Furthermore, the creep-recovery and relaxation behaviors of PMMA were significantly improved due to the addition of TiO2. The creep strain decreased from 1.41% to 1.06%, 0.66%, and 0.49% with the addition of 1 wt. %, 2 wt. %, and 3 wt. % TiO2, respectively. The relaxation test results showed that the initial stress under 1% strain improved to 19.9, 21.2, and 22 MPa with the addition of 1 wt. %, 2 wt. %, and 3 wt. % TiO2, respectively. The improvement in the nanohardness, modulus, creep recovery, and relaxation behavior of PMMA due to the addition of TiO2 nanoparticles indicated the role of the nanoparticles in increasing the PMMA matrix stiffness by reducing its mobility and free volume. TiO2 nanoparticles also improved the antimicrobial behavior of PMMA by significantly reducing bacterial adherence with increasing TiO2 ratio.

Antibacterial activity of trimetal (CuZnFe) oxide nanoparticles.

BACKGROUND: The increasing resistance of pathogenic bacteria to antibiotics is a challenging worldwide health problem that has led to the search for new and more efficient antibacterial agents. Nanotechnology has proven to be an effective tool for the fight against bacteria. METHODS: In this paper, we present the synthesis and traits of trimetal (CuZnFe) oxide nanoparticles (NPs) using X-ray diffraction, high-resolution transmission electron microscopy, and energy dispersive x-ray spectroscopy. We evaluated the antibacterial activity of these NPs against gram-negative Escherichia coli and gram-positive Enterococcus faecalis and then compared it to that of their pure single-metal oxide components CuO and ZnO. RESULTS: Our study showed that the antibacterial activity of the trimetal oxide NPs was greater against E. coli than against E. faecalis. Overall, the antimicrobial effect of trimetal NPs is between those of pure ZnO and CuO nanoparticles, which may mean that their cytotoxicity is also between that of pure ZnO and CuO NPs, making them potential antibiotics. However, the cytotoxicity of trimetal NPs to mammalian cells needs to be verified. CONCLUSION: The combination of three metal oxide NPs (ZnO, CuO, and Fe2O3) in one multimetal (CuZnFe) oxide NPs will enhance the therapeutic strategy against a wide range of microbial infections. Bacteria are unlikely to develop resistance against this new NP because bacteria must go through a series of mutations to become resistant to the trimetal oxide NP. Therefore, this NP can combat existing and emerging bacterial infections.