Transient excited states of the metamorphic protein Mad2 and their implications for function.

The spindle checkpoint complex is a key surveillance mechanism in cell division that prevents premature separation of sister chromatids. Mad2 is an integral component of this spindle checkpoint complex that recognizes cognate substrates such as Mad1 and Cdc20 in its closed (C-Mad2) conformation by fastening a "seatbelt" around short peptide regions that bind to the substrate recognition site. Mad2 is also a metamorphic protein that adopts not only the fold found in C-Mad2, but also a structurally distinct open conformation (O-Mad2) which is incapable of binding substrates. Here, we show using chemical exchange saturation transfer (CEST) and relaxation dispersion (CPMG) NMR experiments that Mad2 transiently populates three other higher free energy states with millisecond lifetimes, two in equilibrium with C-Mad2 (E1 and E2) and one with O-Mad2 (E3). E1 is a mimic of substrate-bound C-Mad2 in which the N-terminus of one C-Mad2 molecule inserts into the seatbelt region of a second molecule of C-Mad2, providing a potential pathway for autoinhibition of C-Mad2. E2 is the "unbuckled" conformation of C-Mad2 that facilitates the triage of molecules along competing fold-switching and substrate binding pathways. The E3 conformation that coexists with O-Mad2 shows fluctuations at a hydrophobic lock that is required for stabilizing the O-Mad2 fold and we hypothesize that E3 represents an early intermediate on-pathway towards conversion to C-Mad2. Collectively, the NMR data highlight the rugged free energy landscape of Mad2 with multiple low-lying intermediates that interlink substrate-binding and fold-switching, and also emphasize the role of molecular dynamics in its function.

An evaluation of Composite Reference Standard (CRS) for diagnosis of Female Genital Tuberculosis.

BACKGROUND: Female genital tuberculosis (FGTB) is a common cause of infertility in developing countries. Its diagnosis is difficult due to its paucibacillary nature, with no single test having high sensitivity and specificity. This study is to share the experience of using Composite Reference Standard (CRS) for the diagnosis of FGTB. METHODS: This is a prospective study conducted between September 2017 to June 2019, over 100 infertile females found to have FGTB on composite reference standard which consisted of acid-fast bacilli on microscopy or culture, histopathological evidence of epithelioid granuloma, positive gene Xpert on endometrial sample or definite or probable finding of FGTB on laparoscopy. RESULTS: A total of 100 infertile women (78% primary, 22% secondary) found to have FGTB on CRS were enrolled in this study. Mean age, body mass index, parity and duration of infertility were 28.2 years, 23.17 kg/m2, 0.24 ± 0.12 and 2.41 years respectively. Various symptoms were scanty menses (16%), irregular cycle (7%), dysmenorrhea (11%), pelvic pain (11%). Various signs were vaginal discharge (65%), adnexal mass (6%), tubo-ovarian mass on ultrasound (15%), abnormal hysterosalpingography findings (57.14%), positive polymerase chain reaction test (65%) and abnormal hysteroscopy (82.2%). The positive findings on CRS were positive AFB on microscopy or culture (3%), positive gene Xpert (28%) (done in some cases), epithelioid granuloma on histopathology (13%), definite findings on laparoscopy like tubercles, caseous nodules and beaded tubes in (57.19%) patients while probable findings of FGTB like straw colored fluid in POD, extensive dense pelvic, peri-tubal, peri-ovarian adhesions; hydrosalpinx; tubo-ovarian mass; thick fibrosed tubes; mid tubal block; peri hepatic adhesions (Fitz Hugh Curtis Syndrome); hyperemia of tubes/blue uterus on chromotubation were seen in (48.8%) patients. All patients found to be positive on CRS were given 6 months of anti-tubercular therapy. CONCLUSION: This study demonstrates the high reliability of use of composite reference standard for diagnosis of FGTB.

Elucidating the mechanisms underlying protein conformational switching using NMR spectroscopy.

How proteins switch between various ligand-free and ligand-bound structures has been a key biophysical question ever since the postulation of the Monod-Wyman-Changeux and Koshland-Nemethy-Filmer models over six decades ago. The ability of NMR spectroscopy to provide structural and kinetic information on biomolecular conformational exchange places it in a unique position as an analytical tool to interrogate the mechanisms of biological processes such as protein folding and biomolecular complex formation. In addition, recent methodological developments in the areas of saturation transfer and relaxation dispersion have expanded the scope of NMR for probing the mechanics of transitions in systems where one or more states constituting the exchange process are sparsely populated and 'invisible' in NMR spectra. In this review, we highlight some of the strategies available from NMR spectroscopy for examining the nature of multi-site conformational exchange, using five case studies that have employed NMR, either in isolation, or in conjunction with other biophysical tools.

Unequal allotment of patients in phase III oncology clinical trials.

Patient enrollment in cancer clinical trials has traditionally been limited to an equal distribution between cases and controls, however recently some clinical trials have utilized an unequal distribution between the case and control arms. Trends and proportion of phase 3 cancer clinical trials that have an unequal allocation between the years 2010 and 2019 were studied from data extracted from clinicaltrials.gov. 323 trials with two arms and 35 trials with 3 arms were identified as randomized control trials with the primary purpose of a cancer-related treatment that provided allocation data. Amongst the trials with two arms, 238 trials had equal allocation and 85 trials had unequal allocation. Therefore, cancer clinical trials with unequal allocation represent about one in four 2-arm phase 3 trials. Amongst the eligible trials with three arms, 26 trials had equal allocation and 9 trials had unequal allocation. There was no significant difference in the annual proportion of trials with unequal allocation from 2010 to 2019. The categories of cancer which had the highest number of unequally allotted two-arm clinical trials were: gastrointestinal, breast, and genitourinary malignancies. This shift may represent a new trend in clinical trial design to help enhance closer monitoring of adverse events despite higher costs and lower statistical power attached to this method.

Adapting Education at the Medical College of Georgia at Augusta University in Response to the COVID-19 Pandemic: the Pandemic Medicine Elective.

The Medical College of Georgia (MCG) responded to the COVID-19 pandemic's challenges to medical education with a novel, comprehensive curriculum. The Pandemic Medicine Elective was an effective solution with a safe, virtual alternative to traditional clinical experiences. As the elective evolved to include pre-clinical students and service initiatives across Georgia, students and faculty navigated online platforms to execute critical community-based projects. This curricular development utilized an interdisciplinary approach by faculty across each of MCG's regional campuses. We describe the curriculum of the electives, the student initiatives, and lessons learned while quickly adapting curriculum during the COVID-19 pandemic.

Evaluation of Gene Xpert as compared to conventional methods in diagnosis of Female Genital Tuberculosis.

To evaluate Gene Xpert for diagnosis of Female Genital Tuberculosis (FGTB) as compared to conventional methods. STUDY DESIGN: It was a prospective study conducted over 167 cases of infertile female genital tuberculosis (FGTB) diagnosed on composite reference standard (CRS) (smear for AFB, histopathological evidence of epithelioid granuloma or definite or possible findings of tuberculosis on laparoscopy). All women underwent endometrial biopsy for AFB microscopy, culture, gene Xpert, PCR and histopathology) and laparoscopy and hysteroscopy for diagnosis and prognostication of disease. The results of Gene Xpert were compared with conventional methods in detection of FGTB. All patients were treated with 6 months course of rifampicin (R), isoniazid (H), pyrazinamide (Z) and ethambutol (E) (RHZE for 2 months, RHE for 4 months) using directly observed treatment short course strategy. RESULTS: Mean age, parity, body mass index and history of contact was 28.3 years, 0.28, 22.9 Kg/m2 and 38.92% respectively. Primary infertility was seen in 87.42% cases with mean duration of 2.42 years. Menstrual dysfunctions, abdominal or pelvic pain and lump were seen in 38.92%, 14.37% and 10.77% cases. Abnormal vaginal discharge and adnexal mass were seen in 28.14% and 13.17 % cases. On diagnostic laparoscopy, definite findings of tuberculosis (beaded tubes, tuebrcles and caseous nodules) were seen in 96 (57.48%) women while probable findings of tuberculosis (pelvic or abdominal adhesions, hydrosaplinx, tubo-ovarian mass, pyosalpinx) were seen in 81 (48.50%) women. On laboratory investigations, positive AFB on microscopy or culture was seen in 2.99% casess, PCR was positive in 47.90% gene Xpert was positive in 18.56% cases while epitheloid granuloma was seen on histopathology in 16 (9.58%) cases. Gene Xpert had sensitivity of 35.63%, specificity of 100%, positive predictive value of 100% and negative predictive value of 58.82% and diagnostic accuracy of 66.47% in the present study. CONCLUSION: Gene Xpert is a very useful test to rule in tuberculosis whereas when it is negative it is not a good test to rule out tuberculosis.

Molecular capsular typing and multi locus sequence typing of invasive, non-invasive and commensal Streptococcus pneumoniae isolates from North India.

Purpose: The purpose of this study is to determine the antimicrobial susceptibility pattern, serotype distribution and sequence type (ST) of Streptococcus pneumoniae isolates from invasive and non-invasive infection and correlate it with isolates from commensal nasopharyngeal flora to ascertain their role in infection. Materials and Methods: S. pneumoniae isolates from blood, cerebrospinal fluid, pleural fluid and respiratory secretions (sputum, bronchoalveolar lavage and nasopharyngeal swab/throat swab) were analysed to determine ST, serotype and antimicrobial susceptibility pattern. Serotyping was performed by multiplex polymerase chain reactions as well as by quellung reaction. Antimicrobial susceptibility testing was determined using Kirby Bauer's disc diffusion method as per the Clinical Laboratory Standards Institute guidelines. Minimum inhibitory concentration was determined using E-test for penicillin. Multilocus sequence typing (MLST) was done to understand genetic relatedness and evolutionary relationship among strains. Results: A total of 125 S. pneumoniae isolates were collected, including 25 from invasive pneumococcal disease, 25 from non-invasive and 75 from nasopharyngeal swab of healthy children (Commensal). Resistance to penicillin, erythromycin, and co-trimoxazole was observed in 14.4%, 12% and 81.6% of the isolates, respectively, by KirbyBauer's disc diffusion method. Serotype 14 was found to be the most prevalent in invasive and non-invasive isolates, while serotype 6 was the most common in commensal isolates. New STs were found among invasive (ST13826, ST13827), non-invasive (ST13823, ST13824, and ST13961) and commensal (ST13825) isolates. Conclusion: MLST sequence analysis shows that invasive isolates were found to be clustered with non-invasive and commensal isolates. Analysis of MLST suggests the possibility of genetic relatedness and exchange of genetic material between invasive, non-invasive and commensal isolates.

Structural Modelling of Hybrid ZnO-CdSe Nano-Compounds Using X-ray Photoelectron Spectroscopy Depth-Profiling Technique.

In the present work, ZnO nanoparticles were synthesized using chemical route method. Composition ratio for Zn:O and various oxidation states were determined using XPS (X-ray photoelectron spectroscopy) technique. The 1 hr calcined ZnO nanoparticles were found to the best due to their monodispersed nature (size ˜20 nm) and high purity. These nanoparticles were then used for synthesizing ZnO-CdSe nano-compounds. These nanocrystals were integrated with CdSe qdots (synthesized using Hot-Injection technique) of varied size (5 nm and 8 nm) via MPA (mercaptopropionic acid) as a linker to develop hybrid nano-compounds for photoactive applications like Quantum Dot sensitized solar cells (QDSSC's) etc. Here, the main objective of the work was to explore the structure of hybrid nano-compounds. XPS-depth-profiling technique was used as an investigation technique for the compositional and structural analyses of ZnO-CdSe nano-compounds. The compositional structure was analyzed layer wise (obtained by etching at different time of sputtering) for the exact position of ZnO and CdSe nanoparticles. On the basis of above study utilizing depth profiling technique of XPS and TEM images, it was found that high quality of hybrid nano-compounds can be synthesized with smaller sized quantum dots, as compared to larger sized quantum dots because of unbounded phosphorus (P) and selenium (Se). Also, it confirms the role of linker that strengthens the binding of CdSe quantum dots on the surface of ZnO thus making it hard to separate the anchored, interstitial CdSe completely which paves the way for the development of stoichiometric, structurally and morphologically-stable ZnO-CdSe nano-compounds.

An electrochemical biosensor based on novel butylamine capped CZTS nanoparticles immobilized by uricase for uric acid detection.

Quaternary chalcopyrite, i.e., Cu2ZnSnS4 (CZTS) nanoparticles films have been proposed as a novel matrix system for enzyme-based electrochemical biosensors providing a non-toxic, low-cost alternative for the fabrication of bioelectrodes. The easy tuneability of the band gap of CZTS by varying the cation ratio and size of nanoparticles facilitate to impart desirable electrical properties in the material. Butylamine capped spherical CZTS nanoparticles of size 15-16 nm and band gap 2.65 eV have been synthesized by colloidal hot injection technique. The films of CZTS onto ITO substrates are deposited using dip coating technique, and uricase enzyme have been immobilized onto CZTS films using EDC-NHS binding chemistry. Electrochemical analyses of this bioelectrode revealed that the uricase/CZTS/ITO/glass electrode exhibits good linearity over a wide range of 0-700 µM uric acid concentration with a limit of detection (LOD) of 0.066 µM. The low value of 0.13 × 10-4 M of Michaelis-Menten constant (Km) indicate the enhanced affinity of immobilized enzyme (uricase) towards uric acid. Thus, the present report confirms the promising application of the p-type CZTS thin film matrix for the realization of an electrochemical biosensor.

Surgical Management of a Separated Endodontic Instrument using Second Generation Platelet Concentrate and Hydroxyapatite.

Fractured endodontic instrument is an unfortunate endodontic mishap which may obstruct thorough cleaning and shaping of the root canals with potential impact on the endodontic prognosis and treatment outcome. When the fractured segment lies apical to canal curvature, overzealous removal of tooth structure is required to gain access to the separated segment which in turn increases the likelihood of root fracture. In infected cases, the stage at which instrument separation occurs is crucial as root canal disinfection is jeopardized. This case report describes the surgical retrieval of a fractured endodontic file from the mesiobuccal canal of mandibular first molar by limited resection of mesial root. Second generation platelet concentrate called Platelet Rich Fibrin (PRF) and nanocrystalline hydroxyapatite and ß-tricalcium phosphate bone graft was placed to fill the surgical defect as the combination enhances the regenerative effect of PRF by exerting an osteoconductive effect in the bony defect area. The clinical and radiographic examination after eighteen months revealed complete periapical healing.

Anesthetic efficacy of the supplemental X-tip intraosseous injection using 4% articaine with 1:100,000 adrenaline in patients with irreversible pulpitis: An in vivo study.

INTRODUCTION: Pain management remains the utmost important qualifying criteria in minimizing patient agony and establishing a strong dentist-patient rapport. Symptomatic irreversible pulpitis is a painful condition necessitating immediate attention and supplemental anesthetic techniques are often resorted to in addition to conventional inferior alveolar nerve block. AIM: The purpose of the study was to evaluate the anesthetic efficacy of X-tip intraosseous injection in patients with symptomatic irreversible pulpitis, in mandibular posterior teeth, using 4% Articaine with 1:100,000 adrenaline as local anesthetic, when the conventional inferior alveolar nerve block proved ineffective. MATERIALS AND METHODS: X-tip system was used to administer 1.7 ml of 4% articaine with 1:100,000 adrenaline in 30 patients diagnosed with irreversible pulpitis of mandibular posterior teeth with moderate to severe pain on endodontic access after administration of an inferior alveolar nerve block. RESULTS: The results of the study showed that 25 X-tip injections (83.33%) were successful and 5 X-tip injections (16.66%) were unsuccessful. CONCLUSION: When the inferior alveolar nerve block fails to provide adequate pulpal anesthesia, X-tip system using 4% articaine with 1:100,000 adrenaline was successful in achieving pulpal anesthesia in patients with irreversible pulpitis.