Structural and functional characterization of mycobacterial PhoH2 and identification of potential inhibitor of its enzymatic activity.

Mycobacterium tuberculosis is composed of a cumbersome signaling and protein network which partakes in bacterial survival and augments its pathogenesis. Mycobacterial PhoH2 (Mt-PhoH2) is a signaling element and a predictive phosphate starvation protein that works in an ATP-dependent manner. Here, we elaborated the characterization of Mt-PhoH2 through biophysical, biochemical, and computational methods. In addition to its intrinsic ATPase activity, the biochemical experiments revealed its GTPase activity and both activities are metal ion dependent. Magnesium, manganese, copper, iron, nickel, zinc, cesium, calcium, and lithium were examined for their effect on activity, and the optimum activity was found with 10 mM of Mg2+ ions. The kinetic parameters of 3 µM Mt-PhoH2 were observed as Km 4.873 ± 0.44 µM, Vmax 12.3817 ± 0.084 µM/min/mg, Kcat 0.0075 ± 0.00005 s-1, and Kcat/Km 0.0015 ± 0.000001 µM-1 s-1 with GTP. In the case of GTP as a substrate, a 20% decrease in enzymatic activity and a 50% increase in binding affinity of Mt-PhoH2 were observed. The substrates ADP and GDP inhibit the ATPase and GTPase activity of Mt-PhoH2. CD spectroscopy showed the dominance of alpha helix in the secondary structure of Mt-PhoH2, and this structural pattern was altered upon addition of ATP and GTP. In silico inhibitor screening revealed ML141 and NAV_2729 as two potential inhibitors of the catalytic activity of Mt-PhoH2. Mt-PhoH2 is essential for mycobacterial growth as its knockdown strain showed a decreased growth effect. Overall, the present article emphasizes the factors essential for the proper functioning of Mt-PhoH2 which is a participant in the toxin-antitoxin machinery and may also play an important role in phosphate starvation.

Identification of G-quadruplex structures in MALAT1 lncRNA that interact with nucleolin and nucleophosmin.

Nuclear-retained long non-coding RNAs (lncRNAs) including MALAT1 have emerged as critical regulators of many molecular processes including transcription, alternative splicing and chromatin organization. Here, we report the presence of three conserved and thermodynamically stable RNA G-quadruplexes (rG4s) located in the 3' region of MALAT1. Using rG4 domain-specific RNA pull-down followed by mass spectrometry and RNA immunoprecipitation, we demonstrated that the MALAT1 rG4 structures are specifically bound by two nucleolar proteins, Nucleolin (NCL) and Nucleophosmin (NPM). Using imaging, we found that the MALAT1 rG4s facilitate the localization of both NCL and NPM to nuclear speckles, and specific G-to-A mutations that disrupt the rG4 structures compromised the localization of both NCL and NPM in speckles. In vitro biophysical studies established that a truncated version of NCL (ΔNCL) binds tightly to all three rG4s. Overall, our study revealed new rG4s within MALAT1, established that they are specifically recognized by NCL and NPM, and showed that disrupting the rG4s abolished localization of these proteins to nuclear speckles.

Biophysical Characterization of Nucleolin Domains Crucial for Interaction with Telomeric and TERRA G-Quadruplexes.

Nucleolin (NCL) is a well-characterized nucleic acid-binding protein; it binds to various canonical and noncanonical structures including single- and double-stranded DNA and RNA, hairpin, loops, and G-quadruplex structures. G-quadruplex structures are majorly formed in promoter, telomeric, and untranslated regions of the genome and affect the process of replication, transcription, and translation. One of the widely studied G-quadruplex-forming regions are telomeres, as these are sites for the recruitment for various proteins providing stability or having an effect on the telomerase activity. NCL is known to bind to both single- and double-stranded telomeric regions and its transcribed telomeric RNA (TERRA). In our study, we show that the 21nt G-quadruplex-forming region of telomeric DNA and TERRA RNA binds to NCL and the domains RRM1234 destabilize the telomeric G-quadruplex structure. We also show the preferential binding of the RNA G-quadruplex over the DNA G-quadruplex by two NCL domains, RRM3 and RRM4. Our findings provide insights into the binding preferences of RRM domains toward G-quadruplex structures and their subsequent effect on the quadruplex stability.

Dorsal Wrist Ganglion: Pilot for Randomized Control Trial Comparing Aspiration Alone or Combined with Injection of Platelet-Rich Plasma.

This pilot study assessed the feasibility of performing a randomized control trial (RCT) investigating injection with platelet-rich plasma (PRP) for dorsal wrist ganglion (DWG). Aspiration alone was compared with aspiration plus injection of PRP. Seventeen patients were enrolled. Nine patients received PRP and eight aspiration alone. Patients were followed up at 6 weeks and 1 year; recurrence of the ganglion and Patient Evaluation Measure scores were measured. At 6 weeks seven patients in the aspiration group had a recurrence and five in the PRP group, but by 1 year, this has increased to seven out of eight in the PRP group whereas in the aspiration group four had resolved leaving three out of eight patients with a ganglion still present. From the basis of our work an RCT would require a minimum of 46 patients per group; however, it is unlikely that PRP will be a panacea for ganglia. This is a Level II study.

Strand 1A variant in neuroserpin shows increased aggregation and no loss of inhibition: implication in ameliorating polymerization to retain activity.

Neuroserpin (NS) is predominantly expressed in the brain and is the primary inhibitor of tissue plasminogen activator (tPA). NS variants are associated with the neurogenerative disease termed familial encephalopathy with neuroserpin inclusion bodies (FENIB). The disease is characterized by variable age of onset and severity. The reactive center loop (RCL) insertion-based inhibitory mechanism of NS requires a coordinated conformational change leading to a shift in the strands of the ß-sheet A and movement of helix F. Strand 1A is connected to the helix F at its C terminal end and with the strand 2A at its N terminal, both these domain move for accommodating the inserting loop; therefore, a variant that influences their movement may alter the inhibition rates. A molecular dynamic simulation analysis of a H138C NS variant from strand 1A showed a large decrease in conformational fluctuations as compared with wild-type NS. H138 was mutated, expressed, purified and a native-PAGE and transmission electron microscopy (TEM) analysis showed that this variant forms large molecular weight aggregates on a slight increase in temperature. However, a circular dichroism analysis showed its secondary structure to be largely conserved. Surprisingly, its tPA inhibition activity and complex formation remain unhindered even after the site-specific labeling of H138C with Alexa fluor C5 maleimide. Further, a helix F-strand 1A (W154C-H138C) double variant still shows appreciable inhibitory activity. Increasingly, it appears that aggregation and not loss of inhibition is the more likely cause of shutter region-based variants phenotypes, indicating that hindering polymer formation using small molecules may retain inhibitory activity in pathological variants of NS.

Alternative splicing modulation mediated by G-quadruplex structures in MALAT1 lncRNA.

MALAT1, an abundant lncRNA specifically localized to nuclear speckles, regulates alternative-splicing (AS). The molecular basis of its role in AS remains poorly understood. Here, we report three conserved, thermodynamically stable, parallel RNA-G-quadruplexes (rG4s) present in the 3' region of MALAT1 which regulates this function. Using rG4 domain-specific RNA-pull-down followed by mass-spectrometry, RNA-immuno-precipitation, and imaging, we demonstrate the rG4 dependent localization of Nucleolin (NCL) and Nucleophosmin (NPM) to nuclear speckles. Specific G-to-A mutations that abolish rG4 structures, result in the localization loss of both the proteins from speckles. Functionally, disruption of rG4 in MALAT1 phenocopies NCL knockdown resulting in altered pre-mRNA splicing of endogenous genes. These results reveal a central role of rG4s within the 3' region of MALAT1 orchestrating AS.

Orf virus infection of the hand in a Scottish sheep farmer. A case report to increase awareness to avoid misdiagnosis.

Awareness of infections which are transmitted between animals and humans have been given prominence following the (COVID-19) pandemic. The Orf infection in humans is rare. Recognition of Orf lesions avoids misdiagnosing and incorrect treatment. We present a case of a Scottish-farmer with pain and discomfort from a lesion on her finger.

A Novel Method of Treatment for a Mal-United Galeazzi Fracture With Dislocation of the Distal Radioulnar Joint Using Scarf Osteotomy.

The Galeazzi fracture is an unstable fracture-dislocation of the forearm. There have been reports of non-union of the radius despite rigid internal fixation with a plate. We present the case of a 25-year-old male who fell from his bicycle, sustaining a closed Galeazzi fracture-dislocation. Definitive surgical fixation involved internal fixation using a six-hole dynamic compression plate. Post-operatively, the patient noted a significant reduction in pronation. The fracture had united but with approximately 5 mm of radial shortening. The operating consultant formulated a surgical plan to resolve the complex nature of this mal-united Galeazzi fracture. A scarf-type osteotomy would correct the deformity and reduce the risk of non-union. Clinically and radiographically, the scarf osteotomy had healed by three months. The patient was very pleased that he underwent revision surgery, as the pain from the wrist resolved and the range of movement improved.

Supra-patellar nailing for isolated closed tibial shaft fractures: Medium term functional outcomes from an Academic Level 1 Trauma centre.

Aims The aim of this study was to assess functional outcome, at medium-term follow up, in patients undergoing intra-medullary nailing of tibial shaft fractures, using the supra-patellar approach. Materials and methods The study included patients with intra-medullary nailing, for diaphysial tibia fractures, between March 2013 to August 2015. An initial audit compared short-term functional outcomes at 15 months, between the supra and infra-patellar approaches, using a cohort of 20 patients (10 in each group). Subsequently, a larger cohort of 22 patients with supra-patellar nailing, were reviewed for medium term assessment of their functional outcomes at average 60 months (50 - 78 months) and the results between all 3 groups were compared. All patients were assessed using the Kujala and Oxford Knee Scores. Results The mean age of the cohorts was 38 years (18-87years). The mean follow-up was 15 months for the initial audit and 60 months for the medium-term results. The initial audit showed that the supra-patellar group had statistically significantly better Oxford and Kujala scores when compared to infra-patellar group (p < 0.05). At 5 years following supra-patellar nailing, the medium-term cohort showed improved outcome scores compared to the infra-patellar group at 1 year (p < 0.01) but showed no difference with the supra-patellar group at 1 year (p value >0.1). Conclusion Our study suggests that patients undergoing supra-patellar tibial nailing have better outcomes at 15 months compared to traditional infra-patellar nailing and more importantly, they continue to do well even at 5 years, despite perceived risks associated with this technique.

Tubercular granuloma mimicking pyogenic liver abscess.

Hepatic Tuberculosis (TB) is extremely rare without miliary involvement in immunocompetent patients. Even in countries like India where TB is a major public health problem only few cases have been diagnosed and treated. We report a case of an immunocompetent patient who presented with undiagnosed pyrexia of 11 days, was initially diagnosed as pyogenic liver abscess, he did not responded to treatment and on liver biopsy was diagnosed as hepatic tuberculoma. Antitubercular treatment (ATT) was started and the patient responded well. We concluded that though hepatic TB is rare in immunocompetent patient, it is important to keep it as a differential diagnosis in patients of liver abscesses who are not responding to treatment in order to avoid needless investigations.

Measuring biopsychosocial risk for back pain disability in chiropractic patients using the STarT back screening tool: a cross-sectional survey.

Background: The Keele STarT Back Screening Tool (SBT), a 9-item questionnaire, screens for pain, physical functioning, fear-avoidance beliefs, catastrophizing, anxious thoughts, low mood, and bothersomeness in persons with back pain. SBT scores designate low, medium, or high risk for developing persistent disabling back pain. The primary study aim was to report the prevalence of SBT-calculated risk for back pain disability in US patients seeking chiropractic care. Methods: The SBT questionnaire was administered to patients ≥18 years in 3 Chiropractic College outpatient teaching clinics in Iowa and Illinois (May 2017). Descriptive statistics were used to analyze respondent characteristics and prevalence of SBT-calculated risk subgroups. Binary logistic regression analysis was used to examine the relationship between respondent characteristics and SBT scores (including psychological subscores). Results: Of 550 respondents, 496 completed the SBT; 392 (79%) scored low-risk, 81 (16%) medium-risk, and 23 (5%) high-risk. Mean (SD) age was 44.8 (15.9), 56.9% were female, 88.2% white, 62.6% employed, mean current pain was 2.9 (2.1) out of 10, and 62% reported symptom duration > 3 months. Eighteen percent of respondents reported anxious thoughts, 32% low mood, 41% ≥ 1 and 21% ≥ 3 SBT psychological risk factors. Respondents reporting higher average pain (OR = 1.8 [1.4, 2.3]) and pain severity (OR = 1.3 [1.0 to 1.6]) were more likely to score with medium or high risk. Respondents reporting mid back versus low back pain (OR = 0.2 [0.1, 0.7]), and those employed less than full-time versus full-time (0.2 [01, 0.5]) were less likely to score with medium or high risk. Respondents reporting higher average pain were more likely to report ≥1 psychological factor (OR = 1.8 [1.5, 2.0]). Respondents employed part-time were less likely to report ≥1 psychological factor than those employed full-time (OR = 0.4 [0.2, 0.7]). Conclusion: The sample surveyed was less likely to score with medium or high risk for back pain disability than previous samples studied, perhaps due to differences in study design and sample characteristics. Rates of low mood and anxious thoughts indicate a need for future research to explore psychological factors among persons seeking chiropractic care.

A graphical clinical decision aid for managing imaging report information.

OBJECTIVE: The purpose of this article is to propose a graphical decision aid for managing radiology report information to assist learners in developing clinical decision-making skills through a structured approach. METHODS: A graphical decision aid informed by learning theories was constructed to manage radiology report information by identifying an overall strategy, specific decision-making steps, and decision goals. A review of radiology reports was performed to demonstrate the rich source of complex information requiring clinical decision making. Radiology report data were descriptively analyzed, and findings were described as definite or indefinite, while recommendations were reported as required or optional. RESULTS: The graphical decision aid involves 4 stages. The 1st 2 stages interpret report information and consider data obtained during the clinical encounter. The following 2 stages guide decisions by answering questions to ensure patient safety and/or to confirm diagnosis and to address broader case management questions. The mean (SD) age of participants whose imaging reports were reviewed was 73.4 (7.0) years. Of 170 reports, common findings included degenerative disc disease (98%), soft tissue or vascular calcification (94%), bone demineralization (92%), and zygapophyseal joint degeneration (86%). Common indefinite findings were spinal stenosis (15%), compression fracture (12%), bony abnormality (12%), radiodensity (12%), and disc degeneration (10%). One hundred twenty-one recommendations suggested follow-up actions. CONCLUSIONS: Information within imaging reports requires identification and interpretation to inform complex clinical decisions. The graphical decision aid proposed in this article is designed to facilitate the development of decision-making skills by providing a structured and evidence-based information management process.

The STarT back tool in chiropractic practice: a narrative review.

BACKGROUND: The Keele STarT Back Tool was designed for primary care medical physicians in the UK to determine the risk for persistent disabling pain in patients with musculoskeletal pain and to tailor treatments accordingly. In medical and physical therapy settings, STarT Back Tool's tailored care plans improved patients' low back pain outcomes and lowered costs. OBJECTIVE: Review studies using the STarT Back Tool in chiropractic patient populations. METHODS: PubMed, The Cochrane Library, Index to Chiropractic Literature, and Science Direct databases were searched. Articles written in English, published in peer-reviewed journals, that studied the STarT Back Tool in patients seeking chiropractic care were included. RESULTS: Seven articles were selected based on inclusion and exclusion criteria. The STarT Back Tool was feasibly incorporated into 19 chiropractic clinics in Denmark. Total STarT Back 5-item score correlated moderately with total Bournemouth Questionnaire score. Two studies reported that the STarT Back Tool's predictive ability was poor, while another reported that the tool predicted outcomes in patients scoring in the medium and high risk categories who completed the STarT Back 2 days after their initial visit. A study examining Danish chiropractic, medical and physical therapy settings revealed that only baseline episode duration affected STarT Back's prognostic ability across all care settings. The tool predicted pain and disability in chiropractic patients whose episode duration was at least 2 weeks, but not in patients with an episode duration <2 weeks. CONCLUSION: While the STarT Back Tool can be incorporated into chiropractic settings and correlates with some elements of the Bournemouth Questionnaire, its prognostic ability is sometimes limited by the shorter low back pain episodes with which chiropractic patients often present. It may be a better predictor in patients whose episode duration is at least 2 weeks. Studies examining outcomes of stratified care in chiropractic patients are needed.

Characterization of Linum usitatissimum L. oil obtained from different extraction technique and in vitro antioxidant potential of supercritical fluid extract.

AIM: Present investigation was aimed to characterize the fixed oil of Linum usitatissimum L. using five different extraction methods: Supercritical fluid extraction (SFE), ultrasound-assistance, soxhlet extraction, solvent extraction, and three phase partitioning method. MATERIALS AND METHODS: The SFE conditions (temperature, pressure, and volume of CO2) were optimized prior for better yield. The extracted oils were analyzed and compared for their physiochemical parameters, high performance thin layer chromatography (HPTLC), gas chromatography-mass spectrometry (GC-MS), and Fourier-transformed infrared spectroscopy (FT-IR) fingerprinting. Antioxidant activity was also determined using 1,1-diphenyl-2-picrylhydrazyl and superoxide scavenging method. RESULT: The main fatty acids were α-linolenic acid, linoleic acid, palmitic acid, and stearic acid as obtained by GC-MS. HPTLC analysis revealed the presence of similar major components in chromatograms. Similarly, the pattern of peaks, as obtained in FT-IR and GC-MS spectra of same oils by different extraction methods, were superimposable. CONCLUSION: Analysis reported that the fixed oil of L. usitatissimum L. is a good source of n-3 fatty acid with the significant antioxidant activity of oil obtained from SFE extraction method.

Omalizumab therapy for asthma patients with poor adherence to inhaled corticosteroid therapy.

BACKGROUND: Omalizumab, an anti-IgE monoclonal antibody, is administered by injection once or twice monthly in offices and clinics. It offers a potential alternative intervention for patients with allergic asthma that is not well controlled because of recalcitrant poor adherence to inhaled corticosteroid therapy. OBJECTIVE: To assess the effect of omalizumab therapy by measuring airway responsiveness to adenosine, a marker of allergic airway inflammation, and resource use. METHODS: Patients (N = 17) aged 6 to 26 years (mean age, 16.4 years) with poorly controlled persistent allergic asthma, less than 50% adherence to inhaled corticosteroid therapy, a forced expiratory volume in 1 second (FEV1) of 60% predicted or higher, and adenosine provocation concentration that caused a decrease in FEV1 of 20% (PC20) of 60 mg/mL or less were randomized to receive 4 months of omalizumab or placebo in a double-blind, crossover trial with a 3- to 4-month washout between treatments. Patients were instructed to continue taking inhaled corticosteroids throughout the study. The PC20 was measured before and after each period. RESULTS: Fifteen patients completed the study. The mean baseline PC20 was 14.1 mg/mL (95% CI, 10.8-18.4 mg/mL). The fold change PC20 was 0.9 (95% CI, 0.5-1.7) during placebo and 3.1 (95% CI, 1.6-6.2) during omalizumab treatment; the estimated ratio was 3.4 (95% CI, 1.2-9.3; P = .02). Six patients required one or more short courses of oral corticosteroids for asthma exacerbations during placebo, but none required this intervention during omalizumab. During the study, the median prescription refills for inhaled corticosteroids was 0.15 (95% CI, 0.00-0.33) canisters per month. CONCLUSION: Omalizumab therapy is an alternative for patients with more severe poorly controlled asthma in whom adherence does not improve with conventional interventions. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00133042.

The use of individually wrapped presterilized small orthopaedic implants increase operating time: a prospective experimental study.

OBJECTIVE: There have been concerns about the potential increases in operating time associated with the use of individually wrapped presterilized small orthopaedic implants compared with our traditional method of screw banks. We set out to quantify this theory. DESIGN: Prospective experimental study. SETTING: Theatre. PARTICIPANTS: Orthopaedic Surgical Trainees and Theatre Scrub team. MAIN OUTCOME MEASURE: The time taken to complete the operation. RESULTS: The use of prepacked and sterilized implants added 2 min 56 s to the use of a bank with a full complement of normal screws that required tapping and 3 min 58 s if self-tapping screws were used (P < 0.001). CONCLUSION: Using individually wrapped presterilized small orthopaedic implants increases operating time.

Analytical and clinical performance characteristics of the Simplexa BK virus quantitative PCR assay for the diagnosis of polyomavirus-associated nephropathy in renal transplant recipients using plasma and urine specimens.

BACKGROUND: Polyomavirus-associated nephropathy is a significant cause of kidney rejection in renal transplant recipients. Quantification of BK viral load in plasma and urine can predict the development of polyomavirus-associated nephropathy, though each assay requires careful evaluation of analytical and clinical performance characteristics for optimal use. OBJECTIVES: This study evaluated the analytical and clinical performance characteristics of the Simplexa BK virus quantitative PCR assay. STUDY DESIGN: Analytical validation was performed using commercial standards, BK virus stock culture, and patient specimens. Clinical performance was evaluated using biopsy-proven BK nephropathy as the gold standard. RESULTS: The Simplexa BK virus quantitative PCR assay was linear over a range of 2.7-10.4 log(10) copies/mL. Limit of detection was 2.7-2.8 log(10) copies/mL in plasma and urine samples. Sensitivities were 100% and 100% and specificities were 84% and 86% for plasma and urine samples, respectively, when compared to a reference BK assay. Clinical cutoff values of 4.0 log(10) copies/mL (plasma) and 7.5 log(10) copies/mL (urine) yielded 100% sensitivity and specificities of 87.5% and 85%, respectively, for biopsy-proven polyomavirus nephropathy. CONCLUSIONS: The Simplexa BK virus quantitative PCR assay has high sensitivity and acceptable analytical characteristics for clinical use. The clinical cutoff values presented here provide a rational approach to the monitoring and treatment of renal transplant recipients for polyomavirus-associated nephropathy.