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.

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.

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.

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).

Identification of stabilizing point mutations through mutagenesis of destabilized protein libraries.

Although there have been recent transformative advances in the area of protein structure prediction, prediction of point mutations that improve protein stability remains challenging. It is possible to construct and screen large mutant libraries for improved activity or ligand binding. However, reliable screens for mutants that improve protein stability do not yet exist, especially for proteins that are well folded and relatively stable. Here, we demonstrate that incorporation of a single, specific, destabilizing mutation termed parent inactivating mutation into each member of a single-site saturation mutagenesis library, followed by screening for suppressors, allows for robust and accurate identification of stabilizing mutations. We carried out fluorescence-activated cell sorting of such a yeast surface display, saturation suppressor library of the bacterial toxin CcdB, followed by deep sequencing of sorted populations. We found that multiple stabilizing mutations could be identified after a single round of sorting. In addition, multiple libraries with different parent inactivating mutations could be pooled and simultaneously screened to further enhance the accuracy of identification of stabilizing mutations. Finally, we show that individual stabilizing mutations could be combined to result in a multi-mutant that demonstrated an increase in thermal melting temperature of about 20 °C, and that displayed enhanced tolerance to high temperature exposure. We conclude that as this method is robust and employs small library sizes, it can be readily extended to other display and screening formats to rapidly isolate stabilized protein mutants.

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.

Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment.

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic-acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the receptor-binding domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan-engineered RBD protein fragment that is expressed at a purified yield of 214 mg/l in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over 4 weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ∼415 against replicative virus, comparing favorably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance, and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

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.

A comparative study on the effect of propolis and dentine bonding agent in treating dentine hypersensitivity.

OBJECTIVE: To find out if there is a difference in the efficacy of propolis and seventh-generation dentine bonding agent in reducing dentine hypersensitivity. METHODS: The randomised, single-blind study was conducted at the Department of Periodontology, Dow International Dental College, Dow University of Health Sciences, Karachi, from December 2018 to November 2019, and comprised patients with complaint of dentine hypersensitivity who were divided in group A which received 30% ethanolic extract of propolis, and group B which received dentine bonding agent. Recordings of dentine hypersensitivity were obtained at baseline, before and after the application of experimental agents, and on days 7, 15 and 30. The response was measured using the Schiff Cold Air Sensitivity Scale. Data was analysed using SPSS 20. RESULTS: Of the 52 patients, 19 (36.5%) were males and 33 (63.5%) were females. The overall mean age was 29.9 ± 6.5 years. Majority of the subjects were students i.e. 16 (30.8%) and housewives i.e. 11 (21.2%), while drivers, teachers, businessmen etc. constituted of 25 (48%) subjects. Significant reduction of dentine hypersensitivity was observed in both groups (p<0.05). Intergroup comparison showed non-significant differences (p>0.05). CONCLUSIONS: Propolis and dentine bonding agent had significant effect in reducing dentine hypersensitivity. The difference between the two was not significant.

Case-control study for assessment of factors associated with periodontitis among adults attending a university hospital in Karachi, Pakistan.

OBJECTIVE: To compare the physiological and biochemical markers in healthy and periodontitis subjects, and to relate these markers with the periodontal health condition. METHOD: The case-control study was conducted at the Dow University of Health Sciences, Karachi, from April 2017 to March 2018, and comprised systematically healthy controls and periodontitis cases. Periodontal probing depth, clinical attachment loss, oral hygiene indices, educational status and body mass index were recorded for all the subjects. Serum levels of biochemical markers, including calcium, phosphate and interleukin-6, were also measured. Data was analysed using SPSS 16. RESULTS: Of the 150 subjects, 75(50%) each were in the case and control groups. The overall mean age was 31.23±3.7 years (range: 22-42 years). The cases had relatively poor oral hygiene indices and educational status compared to the controls (p<0.05). Serum calcium level was lower, whereas mean body mass index was higher in the cases compared to the controls (p<0.05). No significant difference was found in interleukin-6 and phosphate levels (p>0.05). Clinical attachment loss showed significant correlation (p<0.05). CONCLUSIONS: Low serum calcium and educational levels, higher body mass index and poor oral hygiene were found to be the risk factors for the progression of periodontitis.

Effect of pre-cooling agent on intensity of pricking pain at intraoral injection site in adults: An experimental study.

OBJECTIVE: To determine the effect of pre-cooling agent on the intensity of pricking pain at the intraoral injection site in adult patients. METHODS: The in-vivo interventional study was conducted at the Department of Operative Dentistry, Dr. Ishrat-ul-Ebad Khan Institute of Oral Health Sciences, Dow University of Health Sciences, Karachi, from September 2018 to August 2019, and comprised adult patients of either gender. The pricking pain perception during needle administration was assessed using split-mouth technique. Topical anaesthesia benzocaine gel was applied on the left side, which was treated as controls, for 1 min, while on the right side, which was treated as the experimental side, refrigerated cartridge was placed for 2 min. Infiltration anaesthesia was then administered on both sides. Pain perception ratings were measured through visual analogue scale. After profound anaesthesia was achieved, restorative treatment was performed under rubber dam isolation. Data was analysed using SPSS 24. RESULTS: Of the 152 subjects, 77(50.65%) were females and 75(49.34%) were males. The overall mean age was 35.97±8.669 years (range: 21-50 years). The effect of refrigerated cartridge was significant on the intensity of pricking pain at the intraoral injection site in patients aged 41-50 years, and in female patients aged 21-30 years (p<0.05), whereas its effect was non-significant in males aged 21-30 years and patients aged 31-40 years (p>0.05). CONCLUSIONS: Pre-cooling agent was found to be effective in decreasing pricking pain felt by patients.

Monte Carlo commissioning of radiotherapy LINAC-Introducing an improved methodology.

PURPOSE: Monte Carlo (MC) commissioning of medical linear accelerator (LINAC) is a time-consuming process involving a comparison between measured and simulated cross beam/lateral profiles and percentage depth doses (PDDs) for various field sizes. An agreement between these two data sets is sought by trial and error method while varying the incident electron beam parameters, such as electron beam energy or width, etc. This study aims to improve the efficiency of MC commissioning of a LINAC by assessing the feasibility of using a limited number of simulated PDDs. MATERIALS AND METHODS: Using EGSnrc codes, a Varian Clinac 2100 unit has been commissioned for 6 MV photon beam, and a methodology has been proposed to identify the incident electron beam parameters in a speedier fashion. Impact of voxel size in 3-dimensions and cost functions used for comparison of the measured and simulated data have been investigated along with the role of interpolation. RESULTS: A voxel size of 1 × 1×0.5 cm3 has been identified as suitable for accurate and fast commissioning of the LIANC. The optimum number of simulated PDDs (required for further interpolation) has been found to be five. CONCLUSION: The present study suggests that PDDs alone at times can be insufficient for an unambiguous commissioning process and should be supported by including the lateral beam profiles in the process.

Arthroscopic knotless anterior labral stabilization using labral tape and wide awake anaesthesia-short term results.

BACKGROUND: The shoulder is the least constrained of all joints of the body and is more susceptible to injury including dislocation. The rate of recurrent instability following primary stabilization procedure at 10 years of follow-up ranged from 3.4 to 20%. There is a lack of evidence in the literature regarding use of labral tape and anchors for anterior stabilization despite the growing market for this product. We describe the outcomes of 67 patients who underwent knotless arthroscopic anterior stabilisation under awake anaesthesia using 1.5 mm LabralTape with 2.9 mm Pushlock anchors for primary anterior instability by a single surgeon. METHODS: This was a retrospective analysis of prospectively collected outcome data for adult patients undergoing anterior stabilisation for primary traumatic anterior shoulder instability between 2013 and 2016 at two centres. Patients with > 25% glenoid bone loss, engaging Hill Sach's, and multidirectional instability were excluded. All cases underwent surgery using awake anaesthetic technique. The surgical technique and post-operative physiotherapy was standardized. Outcomes were measured at 6 months and 12 months. RESULTS: Of the 74 patients in our study, 7 were lost to follow up. Outcomes were measured using the Oxford Instability Shoulder Score (OISS) and clinical assessment including the range of motion. The OISS showed statistically significant improvement from a mean score and standard deviation (SD) of 24.72 ± 2.8 pre-surgery to 43.09 ± 3.5 after the procedure at 12 months with good to excellent outcomes in 66 cases (98.5%). The mean abduction was 134.2 ± 6.32 and external rotation was 72.55 ± 5.42 at 60-90 position at 12 months. We report no failures due to knot slippage or anchor pull-out. CONCLUSION: Our case series using the above technique has distinct advantages of combining a small non-absorbable implant with flat, braided, and high-strength polyethylene tape. This technique demonstrates superior medium term results to conventional suture knot techniques for labral stabilization thereby validating its use.

Translating bed total body irradiation lung shielding and dose optimization using asymmetric MLC apertures.

A revised translating bed total body irradiation (TBI) technique is developed for shielding organs at risk (lungs) to tolerance dose limits, and optimizing dose distribution in three dimensions (3D) using an asymmetrically-adjusted, dynamic multileaf collimator. We present a dosimetric comparison of this technique with a previously developed symmetric MLC-based TBI technique. An anthropomor-phic RANDO phantom is CT scanned with 3 mm slice thickness. Radiological depths (RD) are calculated on individual CT slices along the divergent ray lines. Asymmetric MLC apertures are defined every 9 mm over the phantom length in the craniocaudal direction. Individual asymmetric MLC leaf positions are optimized based on RD values of all slices for uniform dose distributions. Dose calculations are performed in the Eclipse treatment planning system over these optimized MLC apertures. Dose uniformity along midline of the RANDO phantom is within the confidence limit (CL) of 2.1% (with a confidence probability p = 0.065). The issue of over- and underdose at the interfaces that is observed when symmetric MLC apertures are used is reduced from more than ± 4% to less than ± 1.5% with asymmetric MLC apertures. Lungs are shielded by 20%, 30%, and 40% of the prescribed dose by adjusting the MLC apertures. Dose-volume histogram analysis confirms that the revised technique provides effective lung shielding, as well as a homogeneous dose coverage to the whole body. The asymmetric technique also reduces hot and cold spots at lung-tissue interfaces compared to previous symmetric MLC-based TBI technique. MLC-based shielding of OARs eliminates the need to fabricate and setup cumbersome patient-specific physical blocks.

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.

A rare cause of hypokalemia.

Hypokalemia is a common electrolyte derangement seen in the inpatient setting, often with multiple plausible explanations. However, for patients with nonobvious causes, obtaining a more thorough history, including dietary history, can yield valuable insight and clues to guide clinicians in their evaluation.

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.

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.

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.

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.