Efficacy of seventh generation bonding agents as desensitizers in patients with dentin hypersensitivity: a randomized clinical trial.

BACKGROUND: Dentin hypersensitivity (DH) is one of the most challenging and persistent dental complaints characterized by transient, intense pain triggered by various stimuli. It affects a significant portion of the global population, predominantly those aged 20-40. This study aims to evaluate the desensitizing efficacy of seventh-generation dentin bonding agents (Single Bond Universal by 3 M ESPE and Xeno-V + by Dentsply) against a control group using Bifluorid 12 by Voco in mitigating DH within a month of the follow-up period. METHODS: This was a single-center, parallel-group, double-blind, controlled randomized clinical trial conducted at Dow University of Health Sciences, Karachi, Pakistan. A total of 105 patients with DH were allocated into three groups for this study. The patients were divided into three groups (Single Bond Universal by 3 M ESPE and Xeno-V + by Dentsply) and the control group containing fluoride varnish (Bifluorid 12 by Voco). Discomfort Interval Scale scores and Schiff Cold Air Sensitivity Scale scores were recorded at baseline, immediately after the intervention, after 01 weeks, and after 01 month. RESULTS: All the materials demonstrated a statistically significant reduction in discomfort and sensitivity (DIS scores p-value 0.01) immediately after 01 week and over a period of 01 month after treatment compared with the baseline scores before application, with no single material proving superior over the one-month observation period. The study also provided insights into dental hygiene practices, with a significant majority using a toothbrush and sensitivity patterns, with cold stimuli being the most common cause of sensitivity. CONCLUSION: The study demonstrates that Single Bond Universal, Xeno V+, and Bifluorid 12 are equally effective in reducing dentin hypersensitivity, with no distinct superiority observed over a one-month period. The findings highlight the potential of fluoride varnishes as a less technique-sensitive and cost-effective option for treating DH, offering valuable insights for future research and clinical practice. TRIAL REGISTRATION: NCT04225247 ( https://clinicaltrials.gov/study/NCT04225247 ), Date of Registration: 13/01/2020. (Retrospectively registered).

Insight into the interaction of isochroman with bovine serum albumin: extensive experimental and computational investigations.

The way therapeutic compounds interact with serum protein provides valuable information on their pharmacokinetics, toxicity, effectiveness, and even their structural-related information. Isochroman (IC) is a phytochemical compound obtained from the leaves of Olea europea plant. The derivatives of IC have various pharmacological properties including antidepressants, antihistamines, antiinflammation, anticonvulsants, appetite depressants, etc. The binding of small molecules to bovine serum albumin (BSA) is useful to ensure their efficacy. Thus, in this study, we have found out the binding mode of IC with BSA using several spectroscopic and in silico studies. UV and fluorescence spectroscopy suggested the complex formation between IC and BSA with a binding constant of 103 M-1. IC resulted in fluorescence quenching in BSA through static mechanism. The microenvironmental and conformational changes in BSA were confirmed using synchronous and three-dimensional studies. Site marker experiment revealed the IC binding in site-III of BSA. The influence of vitamins, metals and ß-cyclodextrin (ß-CD) on binding constant of IC-BSA complex was also examined. Circular dichroism spectra showed that α-helical of BSA decreased upon interaction with IC. Computational and experimental results were complimentary with one another and assisted in determining the binding sites, nature of bonds and amino acids included in the IC-BSA complex formation.Communicated by Ramaswamy H. Sarma.

Phytocompounds screening of Nigella sativa in terms of human cancer by targeting sphingosine kinase-1 and pyruvate kinase-M2: a study based on in silico analysis.

Cancer is a multifactorial disease that can cause morbidity and mortality in humans. An altered gene expression in cancer leads to a change in the overall activity of the human cell. Overexpression of cancer protein may give a piece of wide information about the specific type of tumor. Sphingosine kinase-1 (SK-1) is a metabolic enzyme that is mainly overexpressed in several types of cancer and other inflammatory diseases. Similarly, pyruvate kinase-M2 (PK-M2) is an important oncogenic ATP-producing glycolytic enzyme that is upregulated in most cancer cells. The phytocompound of medicinal plants such as Nigella sativa contains a variety of micronutrients that inhibit the proliferation and activity of tumor cells. In this study, the role of phytocompounds in combating cancer was studied against the model kinase proteins, that is, PK-M2 and SK-1. In silico tool like the PASS-Way2Drug server was used to predict the anticancer properties of phytocompounds. Moreover, the CLC-Pred web server provided the cytotoxicity prediction of chemical compounds against several human cancer cell lines. The pharmacokinetics and toxicity profiles were predicted by the SwissADME and pkCSM software. The binding energies were obtained by molecular docking to confirm the intermolecular interaction of selected phytocompounds with proteins. Consequently, molecular dynamics (MD) simulation confirmed the stability, conformational changes, and dynamic behavior of the kinase proteins complexed with the lead phytocompounds, that is, epicatechin, apigenin, and kaempferol.Communicated by Ramaswamy H. Sarma.

Preparation of nanoformulation of 5-fluorouracil to improve anticancer efficacy: integrated spectroscopic, docking, and MD simulation approaches.

Nanoformulations (NFs) can be used as a novel drug delivery system to treat all cancer types. One of the major drawbacks of conventional anticancer drugs is that they have poor specificity and higher toxicity towards normal cells. 5-fluorouracil (5-FU) is a well-studied anticancer drug that has a significant role in various cancers, specifically colorectal cancer therapy. This study was performed to determine the functional groups, particle size, surface charge, heterogeneity, and stability of the NF. The NFs of 5-FU were prepared through the ultrasonication technique by increasing the surfactant (Tween-80) concentrations. Among all three NFs, nanoformulated 5-FU (n5-FU) showed the most effective particle size (10.72 nm) with a zeta potential of (-4.57 mV). The cytotoxicity and apoptosis profiles confirmed that n5-FU enhanced the anticancer effect of the pure drug in HCT-116 cells, as evident from MTT assay, fluorescence microscopy, and FACS analysis. In HCT-116 cells, the IC50 values of pure and n5-FU were obtained as 41.3 µM and 18.8 µM, respectively, indicating that n5-FU was more effective against the cancer cell line. The cellular uptake study was performed to check the intake of NF in cancer cells. However, the microtubule-affinity regulating kinase-4 (MARK-4), a cancer-target protein, was purified to study the inhibition and interaction studies. The inhibition assay confirmed the inhibitory potential of 5-FU against MARK-4 protein. the multi-spectroscopic, molecular docking and MD simulation studies were performed to analyse the conformational changes, binding studies, intermolecular interactions, and stability of MARK-4 protein upon binding 5-FU. This demonstrates that NF can enhance the effectiveness of anticancer drugs.Communicated by Ramaswamy H. Sarma.

Challenges and Perspectives for Integrating Quinoa into the Agri-Food System.

Quinoa is a highly nutritious and abiotic stress-tolerant crop that can be used to ensure food security for the rapidly growing world population under changing climate conditions. Various experiments, based on morphology, phenology, physiology, and yield-related attributes, are being conducted across the globe to check its adoptability under stressful environmental conditions. High weed infestation, early stand establishment, photoperiod sensitivity, loss of seed viability after harvest, and heat stress during its reproductive stage are major constraints to its cultivation. The presence of saponin on its outer surface is also a significant restriction to its local consumption. Scientists are using modern breeding programs, such as participatory approaches, to understand and define breeding goals to promote quinoa adaptation under marginalized conditions. Despite its rich nutritional value, there is still a need to create awareness among people and industries about its nutritional profile and potential for revenue generation. In the future, the breeding of the sweet and larger-grain quinoa varietals will be an option for avoiding the cleaning of saponins, but with the risk of having more pests in the field. There is also a need to focus on mechanized farming systems for the cultivation, harvesting, and processing of quinoa to facilitate and expand its cultivation and consumption across the globe, considering its high genetic diversity.

Cryo-EM structure of antibacterial efflux transporter QacA from Staphylococcus aureus reveals a novel extracellular loop with allosteric role.

Efflux of antibacterial compounds is a major mechanism for developing antimicrobial resistance. In the Gram-positive pathogen Staphylococcus aureus, QacA, a 14 transmembrane helix containing major facilitator superfamily antiporter, mediates proton-coupled efflux of mono and divalent cationic antibacterial compounds. In this study, we report the cryo-EM structure of QacA, with a single mutation D411N that improves homogeneity and retains efflux activity against divalent cationic compounds like dequalinium and chlorhexidine. The structure of substrate-free QacA, complexed to two single-domain camelid antibodies, was elucidated to a resolution of 3.6 Å. The structure displays an outward-open conformation with an extracellular helical hairpin loop (EL7) between transmembrane helices 13 and 14, which is conserved in a subset of DHA2 transporters. Removal of the EL7 hairpin loop or disrupting the interface formed between EL7 and EL1 compromises efflux activity. Chimeric constructs of QacA with a helical hairpin and EL1 grafted from other DHA2 members, LfrA and SmvA, restore activity in the EL7 deleted QacA revealing the allosteric and vital role of EL7 hairpin in antibacterial efflux in QacA and related members.

A placebo-controlled randomized clinical trial of antibiotics versus probiotics as an adjuvant to nonsurgical periodontal treatment among smokers with Stage III, Grade C generalized periodontitis.

BACKGROUND: Probiotics are viable microorganisms, which if delivered in appropriate dose can provide health benefits. Lactobacillus reuteri (DM17938+ATCC PTA 5289) has been recommended as a safe choice for probiotics. The objective of this study is to compare the improvement in the periodontal parameters amongst smokers with generalized periodontitis with Stage III, Grade C treated with nonsurgical periodontal treatment (NSPT) to which either an antibiotics or probiotics were given as an adjuvant. METHODS: Sixty smokers with Stage III, Grade C generalized periodontitis were randomized in two groups after taking informed consent. Periodontal parameters including bleeding on probing (BOP), probing depth (PD), attachment loss (AL), gingival index (GI), and plaque index (PI) were recorded. Group 1 received (after NSPT and oral hygiene instructions) amoxicillin and metronidazole for 7 days and a placebo for probiotics for 30 days. Group 2 was provided (after NSPT and oral hygiene instructions) with one tablet of Lactobacillus reuteri probiotics (2 × 108 CFU) twice daily for 30 days and placebo antibiotics for 7 days. The periodontal parameters were recorded again at 1- and 3-month follow-ups as outcome variables. Mean, standard deviation, and confidence interval were reported using SPSS 20.0. RESULTS: A statistically significant clinical improvement in the PD, BOP, PI, and GI were observed in both the groups at 3-month follow-up. However, the AL remained unchanged in both the groups. CONCLUSIONS: Administration of probiotics and antibiotics along with NSPT yield statistically significant differences in PD and BOP from baseline to 3-month follow-up. However, between the group differences were not statistically significant for the periodontal parameters (AL, PD, and BOP).

Correlation of index finger length to vertical dimensions of occlusion for edentulous patients and their satisfaction: a randomized controlled trial.

The prevalence of edentulism is pandemic and people resort to complete dentures for the restoration of missing teeth and esthetics. However, the determination of the correct occlusal vertical dimensions (OVD) constitutes to play an important role in overall patient satisfaction. The objective of this study was to apply anthropometric methods to correlate the length of index finger (2D) to measure the OVD from base of the nose to the base of the chin (Sn-Me) and to assess satisfaction by comparing both the methods. A total of 80 edentulous patients were randomized and controlled for this trial into experimental and control groups. A correlation was found between Sn-Me and finger measurements, dentures' satisfaction was assessed after a 1-week follow-up and marked according to the Visual Analog Scale. Our findings established that finger measurements are greater among males, and in both genders, positive, and statistically significant correlations exist between the facial and finger length measurements. Moreover, 97.0% patients from experimental group were satisfied with the use of complete dentures through the new anthropometric method. Hence measuring the length of index finger can be an adjunct method for the restoration of OVD and is a relatively time-effective and simple method with a satisfactory follow-up.Trial registration: ID: NCT05153213 ( https://clinicaltrials.gov/ct2/show/NCT05153213 ).

Structural and functional insights into Spns2-mediated transport of sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) is an important signaling sphingolipid that regulates the immune system, angiogenesis, auditory function, and epithelial and endothelial barrier integrity. Spinster homolog 2 (Spns2) is an S1P transporter that exports S1P to initiate lipid signaling cascades. Modulating Spns2 activity can be beneficial in treatments of cancer, inflammation, and immune diseases. However, the transport mechanism of Spns2 and its inhibition remain unclear. Here, we present six cryo-EM structures of human Spns2 in lipid nanodiscs, including two functionally relevant intermediate conformations that link the inward- and outward-facing states, to reveal the structural basis of the S1P transport cycle. Functional analyses suggest that Spns2 exports S1P via facilitated diffusion, a mechanism distinct from other MFS lipid transporters. Finally, we show that the Spns2 inhibitor 16d attenuates the transport activity by locking Spns2 in the inward-facing state. Our work sheds light on Spns2-mediated S1P transport and aids the development of advanced Spns2 inhibitors.

A technical note on percutaneous fixation of trapezium fracture under gravitational traction.

Trapezium fractures rarely present as isolated fractures and warrant anatomical fixation to minimize post-traumatic arthritis. Fixation techniques reported include open approach to trapezium fracture and fixation, percutaneous fixation under image intensifier guidance or percutaneous arthroscopic fixation under gravitational traction. We have reported a novel method of percutaneous fixation of a trapezium Walker type IV coronal split fractures under gravitational traction and per-operative image intensifier guidance. Two percutaneous headless differential pitch screws were used for fixation, which was off-loaded using an inter-metacarpal K-wire. Patient was discharged on the same day with a light-weight cast. The fixation yielded acceptable reduction of the articular surface as confirmed by radiographs. The cast and off-loading K-wire were removed at four weeks post-surgery and hand therapy was commenced. 3-month review showed optimal radiological healing with excellent return to function without pain. In conclusion, using gravitational traction can allow percutaneous fixation of trapezium fractures with excellent results and also make an otherwise complex procedure simpler. LEVEL OF EVIDENCE: IV.

Effect of Propolis on Root Dentine Microhardness When Used as an Intracanal Medicament: An In Vitro Study.

Application of intracanal medicaments may affect the physical properties of root dentine. Calcium hydroxide (CH), a gold standard intracanal medicament, has proven to decrease root dentine microhardness. A natural extract, propolis, has been shown to be superior to CH in eradicating endodontic microbes, but its effect on the microhardness of root dentine is still not known. This investigation aims to evaluate the effect of propolis on root dentine microhardness compared to calcium hydroxide. Ninety root discs were randomly divided into three groups and treated with CH, propolis, and a control. A Vickers hardness indentation machine with a load of 200 g and dwell time of 15 s at 24 h, 3, and 7 days was used for microhardness testing. ANOVA and Tukey's post hoc test were used for statistical analysis. A progressive decrease in microhardness values was observed in CH (p < 0.01), whereas a progressive increase was observed in the propolis group (p < 0.01). At 7 days, propolis demonstrated the highest microhardness value (64.43 ± 1.69), whereas CH demonstrated the lowest value (48.46 ± 1.60). The root dentine microhardness increased over time when propolis was applied, while it decreased over time after application of CH on root dentine sections.

In vitro and in silico binding studies of phytochemical isochroman with calf thymus DNA using multi-spectroscopic and computational modelling techniques.

A wide range of therapeutic molecules uses deoxyribonucleic acid (DNA) as an intracellular target. The interaction of small molecules to DNA is a key feature in pharmacology and plays a vital role in the development of novel and more efficient drugs with increased selective activity and enhanced therapeutic effectiveness. Isochroman (IC) is a constituent of Olea europea plant, which has been shown to exhibit several beneficial pharmacological activities. At present, its interaction studies using calf thymus DNA (ct-DNA) have not been explained. A set of multi-spectroscopic techniques has been performed to determine the interaction mechanism of isochroman with ct-DNA. Absorption spectra and quenching in fluorescence studies show that isochroman and ct-DNA form a complex. The static mode of quenching was determined by the Stern-Volmer plot. The value of binding constant, Kb = 4.0 × 103 M-1 revealed moderate type of binding. Effects of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) and ionic strength were studied to examine the isochroman binding to ct-DNA. Potassium iodide (KI) quenching effects and competitive binding studies clearly showed that isochroman binds in the minor groove of ct-DNA. Circular dichroic and DNA melting experiments also confirmed these results. The experimental outputs were further corroborated via in silico computational modelling studies. Lipinski's rule of 5 and SwissADME showed drug-likeness and oral bioavailability scores. Protox ІІ online software predicts oral and organ toxicity.Communicated by Ramaswamy H. Sarma.

Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics.

Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA-binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.

Nizatidine interacts with ct-DNA causing genotoxicity and cytotoxicity: an assessment by in vitro, in vivo, and in silico studies.

H2 receptor antagonists are the medication given for treating stomach ulcers, but lately, reports have shown their role in healing several malignant ulcers. The present work entails the interaction of H2 blocker nizatidine with calf thymus (ct)-DNA for determining the binding mode and energetics of the interaction. Multi-spectroscopic, calorimetric, viscometric and bioinformatic analysis revealed that nizatidine interacted with ct-DNA via groove-binding mode and is characterised by exothermic reaction. Moreover, assessment of genotoxic potential of nizatidine in vitro was carried out in peripheral human lymphocytes by alkaline comet assay. DNA damage occurred at high concentrations of nizatidine. Genotoxicity of nizatidine was also evaluated in vivo by assessing cytogenetic biomarkers viz. micronuclei formation and chromosomal aberration test. Nizatidine was able to induce micronuclei formation and chromosomal damage at high dose. Additionally, cytotoxic activity of nizatidine was determined in cancer cell lines, namely HeLa and HCT-116 and compared with the normal human cell line HEK-293 employing MTT assay. It was observed that nizatidine was more toxic towards HeLa and HCT-116 than HEK-293. Cell morphology analysis by compound inverted microscopy further strengthens the finding obtained through MTT assay.

Effectiveness of Single Dose Premedication of Piroxicam and Prednisolone on Post Endodontic Pain in One Visit Root Canal Treatment: A Randomized Clinical Trial.

OBJECTIVE: The objective of this study was to compare the effectiveness of premedication drugs including single dose Piroxicam and Prednisolone in regard to post endodontic pain at different time intervals (24, 48, 72 and 96 hours) after single visit root canal treatment. METHODS: This randomized clinical trial (registration no. NCT04124822) was performed in operative dentistry department of a private clinical institute. One hundred twenty patients identified with symptomatic irreversible pulpitis were included in the study. The pain intensity levels were marked through the use of visual analog scale (VAS) before the commencement of treatment. The participants were randomly allocated in three groups, Group I (n=40) received no medication (control), Group II (n=40) received Piroxicam (20 mg) and Group III (n=40) received Prednisolone (20 mg). The drugs were administered thirty minutes before the endodontic procedure was initiated. Root canal treatmentwas carried out followed by placement of provisional restoration in a single appointment. The patients were instructed to continue marking their pain intensity levels after 24, 48, 72 and 96 hours using VAS. All patients were called for follow up after 4 days for clinical evaluation and the placement of permanent restoration. The effectiveness of each drug over different time interval was studied employing ANOVA test. The significance level was set at P≤0.05. RESULTS: The results of the present study revealed that a higher percentage of patients in all 3 groups, reported no post-operative pain at all evaluated time durations (24, 48, 72, and 96 hours). However, the long term effectiveness (96 hours) of both drugs to reduce post-endodontic pain was observed to be statistically insignificant. There was no significant difference in demographic data in terms of age (P=0.14), gender (P=0.12), whilst tooth type (P≤0.05) showed statistically significant value. CONCLUSION: Pre-medication with either single dose piroxicam or prednisolone was able to prevent post-endodontic pain in patients with symptomatic irreversible pulpitis.

Combining cysteine scanning with chemical labeling to map protein-protein interactions and infer bound structure in an intrinsically disordered region.

The Mycobacterium tuberculosis genome harbours nine toxin-antitoxin (TA) systems of the mazEF family. These consist of two proteins, a toxin and an antitoxin, encoded in an operon. While the toxin has a conserved fold, the antitoxins are structurally diverse and the toxin binding region is typically intrinsically disordered before binding. We describe high throughput methodology for accurate mapping of interfacial residues and apply it to three MazEF complexes. The method involves screening one partner protein against a panel of chemically masked single cysteine mutants of its interacting partner, displayed on the surface of yeast cells. Such libraries have much lower diversity than those generated by saturation mutagenesis, simplifying library generation and data analysis. Further, because of the steric bulk of the masking reagent, labeling of virtually all exposed epitope residues should result in loss of binding, and buried residues are inaccessible to the labeling reagent. The binding residues are deciphered by probing the loss of binding to the labeled cognate partner by flow cytometry. Using this methodology, we have identified the interfacial residues for MazEF3, MazEF6 and MazEF9 TA systems of M. tuberculosis. In the case of MazEF9, where a crystal structure was available, there was excellent agreement between our predictions and the crystal structure, superior to those with AlphaFold2. We also report detailed biophysical characterization of the MazEF3 and MazEF9 TA systems and measured the relative affinities between cognate and non-cognate toxin-antitoxin partners in order to probe possible cross-talk between these systems.

Mechanistic insights into global suppressors of protein folding defects.

Most amino acid substitutions in a protein either lead to partial loss-of-function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss-of-function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examined six global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 ß-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. When coupled to inactive mutants, they promote increased in-vivo solubilities as well as regain-of-function phenotypes. In the case of CcdB, where novel suppressors were isolated, we determined the crystal structures of three such suppressors to obtain insight into the specific molecular interactions responsible for the observed effects. While most individual suppressors result in small stability enhancements relative to wildtype, which can be combined to yield significant stability increments, thermodynamic stabilisation is neither necessary nor sufficient for suppressor action. Instead, in diverse systems, we observe that individual global suppressors greatly enhance the foldability of buried site mutants, primarily through increase in refolding rate parameters measured in vitro. In the crowded intracellular environment, mutations that slow down folding likely facilitate off-pathway aggregation. We suggest that suppressor mutations that accelerate refolding can counteract this, enhancing the yield of properly folded, functional protein in vivo.

Analysing root roughness and smear layer relationship by comparing contemporary dental curettes with conventional dental curettes: a randomised controlled trial.

BACKGROUND: Root debridement procedures for the treatment of periodontal diseases, produces root surface irregularities and smear layer on the root surface that can adversely affect the healing of periodontal tissues. The objective of the present study was to evaluate the surface texture of root after hand instrumentation with Gracey curette, After Five curette, and Mini Five curette. METHODS: A randomised, controlled clinical trial was conducted with 120 participants clinically diagnosed with chronic periodontitis. Participants were equally randomised into four groups, with parallel treatment assignment of scaling and root planning using Gracey Curettes, After five and Mini five curette, and a control group with no instrumentation. Mobile teeth of these patients were then extracted atraumatically and analysed under a Scanning Electron Microscope and graded for "Roughness and Loss of Tooth substance index" and "Smear layer index." Cross Tabulation was made between the test groups (Control, Gracey Curette, After five, and Mini Five) versus "Roughness and Loss of Tooth substance Index" and "Smear Layer Index." A Chi-square test with Bonferroni correction was used to determine the graded distribution among the groups. RESULTS: In the control group, 73.3% of the teeth showed grade 1 roughness. In the Gracey group, 56.7% showed grade 2 roughness; in the After 5 group, 70% showed grade 3 roughness; in the Mini 5 group, 76.7% showed grade 3 roughness. A significant association was found between roughness scores and the use of individual instruments. Regarding smear layer formation, 46.7% of teeth showed a grade 4 smear layer in the control group. 50% of teeth showed grade 2 smear layer thickness in the Gracey group. In the After 5 group, 73.3% of teeth showed a grade 1 smear layer, while in the Mini 5 group, 80.0% showed a grade 1 smear layer. The use of individual instruments was significantly associated with the smear layer scores. CONCLUSION: Gracey curettes produced relatively smoother root surfaces with less smear layer formation than After Five and Mini Five curettes, which produced relatively more roughened root surfaces with thicker smear layer formation. TRIAL REGISTRATION: ID: ClinicalTrials.gov Identifier: NCT04216966 Date of Registration: January 3, 2020.

Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies against Delta and Omicron in Mice.

As existing vaccines fail to completely prevent COVID-19 infections or community transmission, there is an unmet need for vaccines that can better combat SARS-CoV-2 variants of concern (VOC). We previously developed highly thermo-tolerant monomeric and trimeric receptor-binding domain derivatives that can withstand 100 °C for 90 min and 37 °C for four weeks and help eliminate cold-chain requirements. We show that mice immunised with these vaccine formulations elicit high titres of antibodies that neutralise SARS-CoV-2 variants VIC31 (with Spike: D614G mutation), Delta and Omicron (BA.1.1) VOC. Compared to VIC31, there was an average 14.4-fold reduction in neutralisation against BA.1.1 for the three monomeric antigen-adjuvant combinations and a 16.5-fold reduction for the three trimeric antigen-adjuvant combinations; the corresponding values against Delta were 2.5 and 3.0. Our findings suggest that monomeric formulations are suitable for upcoming Phase I human clinical trials and that there is potential for increasing the efficacy with vaccine matching to improve the responses against emerging variants. These findings are consistent with in silico modelling and AlphaFold predictions, which show that, while oligomeric presentation can be generally beneficial, it can make important epitopes inaccessible and also carries the risk of eliciting unwanted antibodies against the oligomerisation domain.

Functional and Biochemical Characterization of the MazEF6 Toxin-Antitoxin System of Mycobacterium tuberculosis.

The Mycobacterium tuberculosis genome harbors nine toxin-antitoxin (TA) systems that are members of the mazEF family, unlike other prokaryotes, which have only one or two. Although the overall tertiary folds of MazF toxins are predicted to be similar, it is unclear how they recognize structurally different RNAs and antitoxins with divergent sequence specificity. Here, we have expressed and purified the individual components and complex of the MazEF6 TA system from M. tuberculosis. Size exclusion chromatography-multiangle light scattering (SEC-MALS) was performed to determine the oligomerization status of the toxin, antitoxin, and the complex in different stoichiometric ratios. The relative stabilities of the proteins were determined by nano-differential scanning fluorimetry (nano-DSF). Microscale thermophoresis (MST) and yeast surface display (YSD) were performed to measure the relative affinities between the cognate toxin-antitoxin partners. The interaction between MazEF6 complexes and cognate promoter DNA was also studied using MST. Analysis of paired-end RNA sequencing data revealed that the overexpression of MazF6 resulted in differential expression of 323 transcripts in M. tuberculosis. Network analysis was performed to identify the nodes from the top-response network. The analysis of mRNA protection ratios resulted in identification of putative MazF6 cleavage site in its native host, M. tuberculosis. IMPORTANCE M. tuberculosis harbors a large number of type II toxin-antitoxin (TA) systems, the exact roles for most of which are unclear. Prior studies have reported that overexpression of several of these type II toxins inhibits bacterial growth and contributes to the formation of drug-tolerant populations in vitro. To obtain insights into M. tuberculosis MazEF6 type II TA system function, we determined stability, oligomeric states, and binding affinities of cognate partners with each other and with their promoter operator DNA. Using RNA-seq data obtained from M. tuberculosis overexpression strains, we have identified putative MazF6 cleavage sites and targets in its native, cellular context.