A review on snake venom extracellular vesicles: Past to present.

Around 95% of snake venom is protein. Along with the soluble proteins, snake venom also contains proteins encapsulated in vesicles known as Snake Venom Extracellular Vesicles (SVEV). SVEVs are nano-sized membrane-bound vesicles released from the snake venom gland cells. The available published research works on SVEVs are minimal. Extracellular vesicles in the Snake Venom gland were initially discovered during the histopathological analysis of the Crotalus durissus terrificus snakes' venom gland. Later, various techniques were employed to isolate and characterize the SVEVs. The cargo of SVEV consists of a variety of proteins like Phospholipase A-2, C-type Lectins, L-Amino Acid Oxidase, Cysteine-Rich Secretory Proteins, Serine Proteinases, Dipeptidyl Peptidase-IV, Aminopeptidase-A, Ecto-5'-nucleotidases, Disintegrins. Proteomic data revealed the presence of some exclusive proteins in the SVEVs, and the other proteins are in varying concentrations in the SVEVs compared to their whole Venom. Interaction of SVEVs with mammalian cell lines showed the disruption of primary physiological functions leads to host immune modulation, and long-term effects of envenoming. Snakebite victim's blood showed variations in the specific Extracellular vesicle concentration. It has been hypothesized that SVEVs are responsible for long-term toxicity. The current review focuses on the various techniques adopted to isolate and characterize SVEVs and discusses the exclusiveness and variations of SVEV proteins and their role in snakebites.

4-Year Pulp Survival in a Randomized Trial on Direct Pulp Capping.

INTRODUCTION: This study aimed to assess pulp survival in a randomized trial on pulp lavage in adult nonpainful posterior teeth with carious pulp exposure. The treatment included complete caries excavation, direct pulp capping with mineral trioxide aggregate, and immediate restoration with composite resin. METHODS: Fluid was collected from the pulp wound to assess matrix metalloproteinase-9 (MMP-9) and total protein values. Before pulp capping, cavities were randomly (block randomization, n = 48) washed with a physiological saline or a sodium hypochlorite solution (2.5% NaOCl). Treatment outcome was assessed clinically (cold test) and radiographically after at least 1 year and again after at least 3 years. Painful failures were differentiated from nonpainful failures. Pulp survival was estimated using the Kaplan-Meier method including 95% confidence intervals (CIs) up to 1500 days. RESULTS: From the 96 patients originally enrolled, 73 individuals could be followed continuously. The clinical observations indicated a beneficial and sustained effect of pulp lavage with 2.5% NaOCl over a control treatment with physiological saline solution on estimated pulp survival 1500 days postintervention, with 7% (95% CI, 1%-40%) in the saline group versus 55% (95% CI, 30%-100%) in the NaOCl group. High MMP-9/total protein values in pulpal fluid collected from the exposed site indicated early and painful treatment failures yet were not associated with failures that occurred more than 250 days after intervention. CONCLUSIONS: The low 4-year success rates reported here challenge the concept of direct pulp capping in the cases that were included. NaOCl lavage did not only increase the survival of affected pulps substantially but also particularly diminished painful failures (33% in the NaOCl group vs 62% in the saline group). The lack of the predictive value of MMP-9 assessments beyond early treatment failures points to inflammatory states of the pulp tissue under deep caries, which are not related to neutrophil infiltration.

Extracellular vesicles as novel drug delivery systems to target cancer and other diseases: Recent advancements and future perspectives.

Extracellular vesicles (EVs) are lipid-bound vesicles produced into the extracellular space by cells. Apoptotic bodies (ApoBD), microvesicles (MVs), and exosomes are examples of EVs, which act as essential regulators in cell-cell communication in both normal and diseased conditions. Natural cargo molecules such as miRNA, messenger RNA, and proteins are carried by EVs and transferred to nearby cells or distant cells through the process of circulation. Different signalling cascades are then influenced by these functionally active molecules. The information to be delivered to the target cells depends on the substances within the EVs that also includes synthesis method. EVs have attracted interest as potential delivery vehicles for therapies due to their features such as improved circulation stability, biocompatibility, reduced immunogenicity, and toxicity. Therefore, EVs are being regarded as potent carriers of therapeutics that can be used as a therapeutic agent for diseases like cancer. This review focuses on the exosome-mediated drug delivery to cancer cells and the advantages and challenges of using exosomes as a carrier molecule.

Anti-infective Efficacy of Mechanical Debridement with Adjunctive Modalities on Clinical and Cytokine Parameters in Treatment of Chronic Periodontitis: Randomized Controlled Clinical Trial.

OBJECTIVES: Periodontal debridement involves conventional scaling and root planing (SRP) along with variant forms of adjunctive therapies. In the present clinical trial, we investigated if the adjunctive use of HybenX gel or diode laser along with SRP could provide a favorable outcome for the treatment of chronic periodontitis. MATERIALS AND METHODS: The present study involved 60 subjects diagnosed with chronic periodontitis who were randomly assigned as test groups (laser or HybenX) or control group (SRP alone). The primary outcomes of the clinical trial were pocket probing depth (PPD) and clinical attachment level (CAL), which was evaluated at baseline and at third-month time interval. Additionally, secondary outcomes included estimation of reduction in inflammatory mediators interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) in gingival crevicular fluid using enzyme-linked immunosorbent assay at baseline and third-month intervals. STATISTICAL ANALYSIS: Normality determination was checked using Shapiro-Wilk test. Since the data was not normally distributed, nonparametric tests were applied. The comparison of clinical parameters between the groups was analyzed with Kruskal-Wallis test. Wilcoxon sign rank test was used to compare the pairwise comparison of clinical parameters among the groups from baseline to third-month follow-up. The inflammatory mediators at various time points were compared using a One-way analysis of variance test, and the inflammatory mediators in each study group were compared using a paired t-test. RESULTS: Both the test groups demonstrated a decrease in PPD and CAL. However, the HybenX group exhibited statistically significant reduction at the end of the third-month study interval compared to the laser group and SRP alone. Further, the secondary outcome IL-1ß and TNF-α analysis exhibited statistically significant reduction in all the groups posttherapy. CONCLUSION: The adjunctive application of HybenX gel yielded an advantage compared to laser and SRP for the treatment of chronic periodontitis. CLINICAL RELEVANCE: Adjunctive use of the oral tissue desiccant (HybenX gel) combined with SRP improved the periodontal pocket disinfection process and enhanced tissue healing devoid of adverse effects.

Evaluation of paraoxonase I and hemoglobin levels in farmers and agricultural workers in relation to organophosphorus and carbamate levels in their blood and urine samples: A cross sectional study.

Background: Pesticides are chemicals that have become common household products in developing countries. The purpose of pesticides is to manage agricultural work. The majority of pesticides for indoor and agricultural use are carbamate and organophosphorus. Toxicity is caused due to excess and improper use or disposal of these chemical agents. Slow exposure to pesticides causes chronic poisoning whereas rapid exposure causes acute poisoning. The paraoxonase I (PON 1) enzyme has a role in detoxifying some of the oxon derivatives which thereby inhibit acetylcholinesterase and butyrylcholinesterase. Methods: This study analyzed farmers who were exposed intermittently to organophosphorus and carbamates pesticides during farming for more than five years. Serum paraoxonase I was evaluated by colorimetry method, and hemoglobin levels were evaluated using portable Fresenius Kabi haemoglobinometer. Results: The study showed that the pesticides were found in the blood and urine samples of farmers and there was an alteration of paraoxonase I and hemoglobin levels in them due to the exposure of pesticides in large quantities over some time. The present study showed around 81% of the participants who were intermittently exposed to pesticides for more than five years were detected with pesticide toxicity. The paraoxonase I level was altered in farmers who were positive for organophosphorus and carbamate pesticides. The hemoglobin level did not show much variation among the farmers exposed to pesticides. This may be due to the lifestyle of the subjects, climatic variations and also their eating habits. Conclusions: The study suggested that there was alteration in the levels of PON1 and hemoglobin in farmers and agricultural workers with positive organophosphorus and carbamates in their blood and urine samples. As our study was done without quantifying the amount of pesticides, further studies can be done by quantifying the pesticide level and comparing it with the paraoxonase I level.

Effect of Maleic Acid Root Conditioning on Release of Transforming Growth Factor Beta 1 from Infected Root Canal Dentin.

INTRODUCTION: Chemically released growth factors play a vital role in regenerative endodontics. Transforming growth factor beta 1 (TGF-ß1) is 1 of the most extensively studied bioactive molecules that promotes cell proliferation, differentiation, and chemotaxis. The goal of the current research was to analyze the effect of 7% maleic acid (MA) root conditioning of an infected root canal on the release of TGF-ß1. METHODS: Single-rooted human teeth were decoronated, and the canals were enlarged with a Peeso reamer. The samples were divided into biofilm and nonbiofilm groups. Subsequently, all the samples of both groups were flushed with 10 mL of each irrigant, namely, 1.5% sodium hypochlorite (NaOCl), 7% MA, 17% EDTA, and a combination of 1.5% NaOCl with 17% EDTA or 7% MA, for 10 minutes each. TGF-ß1 was estimated quantitatively using an enzyme-linked immunosorbent assay kit. RESULTS: TGF-ß1 release was lowest among the biofilm samples compared with nonbiofilm among all the groups. MA 7% with 1.5% NaOCl rendered higher amounts of growth factor release in contrast to the combination of 17% EDTA and 1.5% NaOCl in both the biofilm and nonbiofilm groups (P < .048). The nonbiofilm samples treated with 7% MA alone illustrated higher growth factor release compared with 17% EDTA only (P < .006), but there was no significant difference in growth factor release among the biofilm samples treated with 7% MA and 17% EDTA. CONCLUSIONS: Bacterial biofilms modified the release of TGF-ß1. MA 7% was observed to be significantly more efficacious than 17% EDTA in TGF-ß1 growth factor release from radicular dentin.

Correlation of Carbonic Anhydrase VI Enzyme, Total Proteins, Antioxidant Levels of Saliva and Dental Caries in Caries-Free and Caries-Active Children - A Case-Control Study.

Background and Objectives: Factors in the biofilm influence the induction and advancement of the carious process. This study was done to relate and assess the levels of enzyme carbonic anhydrase VI, total protein, and antioxidants (superoxide dismutase [SOD], catalase, lipid peroxidase, and uric acid) in caries-free and caries-active children. Methods: This case-control study comprised 60 children of age group 6-12 years who were evaluated for decayed missing filled teeth (dmft)/DMFT criteria and distributed into two groups: Group 1 - caries active (case) and Group 2 - caries free (control) for saliva collection with 30 participants in each of the above groups. Stimulated saliva was obtained, and the samples were then evaluated using biochemical lab tests. The data were then statistically evaluated using independent t-test. Results: Catalase in the caries-free group was significantly higher, but the concentration of carbonic anhydrase (CAVI) enzyme, total protein, and other antioxidant enzyme activity was enhanced in caries-active children in which uric acid demonstrated a statistically significant difference with higher levels in caries-active group. Conclusion: There is an increased concentration of CAVI enzyme in caries-active group and total protein showed a linear relation with caries activity. Antioxidant parameters such as SOD and lipid peroxidase were increased with caries activity. Uric acid was significantly higher in the caries-active group, whereas catalase showed an indirect relation with dental caries. Significant variations in the levels of these parameters imply that the levels of these components of saliva can act as strong markers of caries status in children.

Salivary 8-hydroxyguanosine levels in smokers and non-smokers with chronic periodontitis.

This case-controlled clinical trial was performed on the salivary 8-hydroxyguanosine (8-OHdG) levels in smokers and non-smokers with chronic periodontitis after non-surgical periodontal therapy. Subjects (N = 40) with periodontitis (smokers and non-smokers) and with clinically healthy conditions (smokers and non-smokers) were assigned to this study. At baseline, clinical periodontal parameters (plaque index, gingival index, pocket probing depth and clinical attachment levels) were evaluated. Saliva samples were obtained pre- and post-treatment to quantify the 8-OHdG levels using Elisa technique. Subjects diagnosed with chronic periodontitis with smoking habit (CPs) and non-smokers (CPns) received scaling and root planing. In clinically healthy subjects with smoking habit (CHs) and non-smokers (CHns), only oral hygiene tutoring was performed. All clinical measurements and salivary collection were repeated in a 3-month recall interval. Data were analyzed using Anova, Tukey post hoc test and Mann-Whitney 'U' tests (P < 0.05). At baseline, CPs and CPns groups showed significantly higher PI, GI, PD and CAL values than those of CHns and CHs (P < 0.001). Baseline salivary levels of 8-OHdG were significantly higher in CPs group (14.775 pg/mL) (P < 0.001) compared to the other groups. All clinical parameters in chronic periodontitis group improved at the 3rd month recall interval, however, with regards to 8-OHdG values, the CP smoker category still had a higher level compared to CP non-smoker. This study reflects an on-going periodontal destructive status in smokers and salivary 8-OHdG levels could be recognized as an oxidative biomarker for determining periodontal tissue destruction.

Evaluation of a mouthrinse containing guava leaf extract as part of comprehensive oral care regimen- a randomized placebo-controlled clinical trial.

BACKGROUND: The control of biofilm adherence on tooth surface has always been the keystone of periodontal therapeutic systems. However, prevalence of gingivitis suggest inadequacy of self-performed oral hygiene measures and need for adjunctive aid for mechanical plaque control. Oral rinses containing chlorhexidine, has been widely used however, with certain limitations. Herbal products have been used widely reflecting its action as alternative and complementary remedy. Hence, the purpose of the present study was to evaluate the antimicrobial and antioxidant efficacy of a Guava leaf extract based mouthrinse in patients with chronic generalized gingivitis as an adjunct to oral prophylaxis. METHODS: Sixty subjects (n = 20) in compliance with the inclusion criteria were randomly assigned to one of the 3 study groups i.e. Group A- 0.15%Guava mouth rinse, Group B- 0.2% Chlorhexidine (CHX) mouth rinse, Group C- Distilled water (placebo). All the participants received professional oral prophylaxis and were dispensed with experimental mouth rinses and instructed to use for period of 30 days. Clinical parameters such as gingival index, plaque index along with microbial colony forming units using plaque samples and antioxidant levels in saliva were estimated at baseline, 30 and 90 days' time intervals. RESULTS: All 3 groups showed gradual reduction in GI, PI and microbial counts. Considering the mean scores of recorded parameters at the scheduled time intervals, notable changes were observed between chlorhexidine and guava mouth rinse compared to placebo group. Although there was improvement in the antioxidant status in all study participants, yet there was no statistically significant difference observed. CONCLUSION: Guava mouth rinse can be used as an empirical adjunct to professional oral prophylaxis owing to its multifactorial properties and favourable acceptance. However, long term studies need to be conducted to validate its use for an extended period of time. TRIAL REGISTRATION: The clinical trial has been prospectively registered on 17th February 2017 by the Clinical Trials Registry-India (CTRI/2017/02/007898).

The effect of nonsurgical periodontal therapy on pentraxin 3 levels in smokers and nonsmokers with chronic periodontitis.

The purpose of this study was to determine the pentraxin 3 (PTX3) levels in the gingival crevicular fluid (GCF) and saliva of smokers and nonsmokers with chronic periodontitis and to compare these levels before and after initial nonsurgical periodontal therapy. Forty subjects were divided into 2 groups with chronic periodontitis (smokers and nonsmokers) and 2 clinically healthy groups (smokers and nonsmokers). At baseline, clinical periodontal parameters, including plaque index, gingival index, probing depth, and clinical attachment levels, were assessed. Saliva and GCF samples were procured to quantify the PTX3 levels. All subjects with periodontitis, smokers and nonsmokers, received scaling and root planing. The 2 treated groups were examined 2 weeks after therapy, and any changes in the clinical parameters or PTX3 levels were recorded. At baseline, PTX3 levels in both groups of patients with chronic periodontitis were found to be significantly higher (smokers had the highest level, followed by nonsmokers) than levels in both groups of clinically healthy subjects (nonsmokers, followed by smokers) (P < 0.05). Five patients with chronic periodontitis (3 smokers and 2 nonsmokers) were lost to follow-up and therefore excluded from the statistical analysis. Scaling and root planing led to an improvement in the clinical parameters and a statistically significant reduction of PTX3 levels (P < 0.05) in both chronic periodontitis groups at the 2-week follow-up, but the changes were greater in the smokers than in the nonsmokers. In the present study, smoking was found to play a contributory role in the alteration of PTX3 levels in GCF and saliva in patients with chronic periodontitis. The role of PTX3 as a prognostic tool for resolution of periodontal inflammation still remains obscure.

MMP-9 in Dentinal Fluid Correlates with Caries Lesion Depth.

The analysis of molecular cues in dentinal fluid from an excavated cavity could improve diagnostics in the context of minimally invasive caries treatment. In the current clinical trial we assessed whether the dentinal fluid levels of MMP-9 (matrix metalloproteinase-9; neutrophil gelatinase) would increase with the progression of carious lesions. MMP-9 is associated with neutrophil-related tissue breakdown in the pulp. Absolute MMP-9 levels were contrasted against the levels of MMP-2, an enzyme related to normal tissue turnover. Dentinal fluid was collected below deep and shallow caries from molars and premolars within the same patients aged 18 years and older (n = 30, 1 tooth per group/patient). Experimental teeth were isolated under a rubber dam prior to excavation. Dentinal fluid was collected from the bottom of the cavity using a size 25 paper point. MMP levels were assessed using an enzyme-linked immunosorbent assay. Nonparametric methods were applied to test for differences between groups. Significantly more (p < 0.05, Wilcoxon test) MMP-9 was collected from the deep carious lesions than from the shallow counterparts. Pairwise comparison of MMP-9 values within patients revealed that there was more MMP-9 collected from deep lesions than from shallow counterparts in 27 of the 30 individuals under investigation (pairwise Wilcoxon test, p < 0.001). In contrast, no such difference existed for MMP-2. There was a high correlation between MMP-9 from deep and shallow lesions (Spearman's ρ = 0.72, p < 0.001), indicating that patients with more MMP-9 in the deep carious lesion also tended to have more MMP-9 in the shallow lesion.

Serum copper, ceruloplasmin, protein thiols and thiobarbituric acid reactive substance status in liver cancer associated with elevated levels of alpha-fetoprotein.

Serum copper, ceruloplasmin, protein thiols and thiobarbituric acid reactive substances (TEARS) were estimated in 25 patients of liver cancer. The copper to ceruloplasmin ratio was moderately increased (P<0.05) but the copper (P<0.001) and ceruloplasmin (P<0.001) levels were significantly increased in liver cancer patients when compared to controls. Protein thiols levels were found to be highly significant (P<0.001). Where as the TEARS levels were not found to be significant. Trace elements and free radicals have been implicated in the etiology of cancer. Hence the estimation of ceruloplasmin and protein thiols along with the copper may be of high value in the early diagnosis of cancer.

Serum copper, ceruloplasmin and thiobarbituric acid reactive substance status in patients with ovarian cancer.

Serum copper, ceruloplasmin and thiobarbituric acid reactive substances (TBARS) were estimated in 30 patients of ovarian cancer. The copper to ceruloplasmin ratio was moderately increased (P<0.05) but the copper (P<0.01) and ceruloplasmin (P<0.001) levels were significantly increased in ovarian cancer patients as compared to controls. TBARS levels were also found to be highly significant (P<0.001). Trace elements and free radicals have been implicated in the etiology of cancer. Hence the estimation of antioxidants like ceruloplasmin and TBARS along with the trace element like copper may be of value in the early diagnosis of cancer.

Serum copper, ceruloplasmin and thiobarbituric acid reactive substance status in patients with ovarian cancer.

Serum copper, ceruloplasmin and thiobarbituric acid reactive substances (TBARS) were estimated in 30 patients of ovarian cancer. The copper to ceruloplasmin ratio was moderately increased (P<0.05) but the copper (P<0.01( and ceruloplasmin (P<0.001) levels were significantly increased in ovarian cancer patients as compared to controls. TBARS levels were also found to be highly significant (P<0.001). Trace elements and free radicals have been implicated in the etiology of cancer. Hence the estimation of antioxidants like ceruloplasmin and TBARS along with the trace element like copper may be of value in the early diagnosis of cancer.

Copper and ceruloplasmin status in serum of prostate and colon cancer patients.

Serum copper and ceruloplasmin levels were estimated in 20 patients each of prostate and colon cancer. Although copper to ceruloplasmin ratio was not significantly altered, copper and ceruloplasmin levels were increased significantly in the cancer patients as compared to controls. Trace elements and free radicals have been implicated in the etiology of cancer. Hence determination of specific antioxidants (like ceruloplasmin) and trace elements (like copper) may be of value in the early diagnosis of prostate and colon cancer.