Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase.

Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. The mutation of multiple conserved residues reduces substrate inhibition, suggesting that substrate inhibition is an evolutionary well conserved feature in AK. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity.

A Preemptive Living Donor Renal Transplant in an HIV Positive Patient from an HIV Positive Donor.

With recent advances in the treatment of human immunodeficiency virus (HIV), renal transplantation is no longer considered a contraindication in properly selected HIV-positive patients. Several studies have demonstrated comparable patient and graft outcomes between HIV-negative and HIV-positive renal transplant recipients. Most of the information on outcomes of HIV-positive to HIV-positive transplantation is based on data from deceased donors. There are only a handful of case reports about living donor renal transplantation in an HIV-positive patient from an HIV-positive donor. Furthermore, there is no report in the world of preemptive living donor renal transplantation in this setting. Here, we report a case of successful preemptive renal transplantation in an HIV-positive recipient from an HIV-positive living donor performed at our center.

An Unusual Presentation of Multiple Myeloma.

Multiple myeloma commonly presents as anemia, renal failure, bone pain, and infections. Presentation with epistaxis is extremely rare, and hence myeloma as the etiologic factor is seldom considered. We report the case of a patient who initially presented with recurrent epistaxis and then with myasthenia. It was only when he developed acute kidney injury 4 months after the initial presentation with epistaxis that a diagnosis of multiple myeloma was made.

Coronary imaging of cardiac allograft vasculopathy predicts current and future deterioration of left ventricular function in patients with orthotopic heart transplantation.

BACKGROUND: Intravascular ultrasound (IVUS) and optical coherence tomography (OCT) improve sensitivity of cardiac allograft vasculopathy (CAV) detection compared to invasive coronary angiography (ICA), but their ability to predict clinical events is unknown. We determined whether severe CAV detected with ICA, IVUS, or OCT correlates with graft function. METHODS: Comparison of specific vessel parameters between IVUS and OCT on 20 patients attending for angiography 12-24 months post-orthotopic heart transplant. Serial left ventricular ejection fraction (EF) was recorded prospectively. RESULTS: Analyzing 55 coronary arteries, OCT and IVUS correlated well for vessel CAV characteristics. A mean intimal thickness (MIT)OCT  > .25 mm had a sensitivity of 86.7% and specificity of 74.3% at detecting Stanford grade 4 CAV. Those with angiographically evident CAV had significant reduction in graft EF over 7.3 years follow-up (median ΔEF -2% vs +1.5%, P = .03). Patients with MITOCT  > .25 mm in at least one vessel had a lower median EF at time of surveillance (57% vs 62%, P = .014). Two MACEs were noted. CONCLUSION: Imaging with OCT correlates well with IVUS for CAV detection. Combined angiography and OCT to screen for CAV within 12-24 months of transplant predicts concurrent and future deterioration in graft function.

Retrospective Diagnosis of Nail-patella Syndrome.

A 37 years old female presented with asymptomatic nephrotic range proteinuria due to focal segmental glomerulosclerosis (FSGS). She was treated with steroids and mycophenolate mofetil to which there was no response and progressed to advanced chronic kidney disease. When her brother who was being evaluated as a potential donor, for renal transplant, was found to have proteinuria and a genetic study for the steroid-resistant nephrotic syndrome was done. This revealed mutation in the LMX1B gene. It is then that a diagnosis of nail-patella syndrome (NPS) was made. She underwent a successful renal transplant with her father as a donor and is doing well.

Evidence of the Effectiveness of Pictorial Health Warnings on Cigarette Packaging in Nepal.

BACKGROUND: Tobacco Control Act of 2010 mandates government to implement at least 75% pictorial health warnings (PHWs) on tobacco packaging that was enforced in 2013. The purpose of the study was to assess the effectiveness of PHWs and its impact to the policy change. METHODS: A cross-sectional study was conducted in 9 cities between September 2014 and March 2015. Direct interviews were made among 2250 randomly selected individuals. The effectiveness of PHWs were measured as perceived: i) scariness; ii) quit motivation iii) convincing youth not to start smoking; iv) encouraging ex-smokers to remain as quitters; v) building public awareness. Logistic regression analysis was used to determine the factors associated with the effectiveness of PHWs. RESULTS: Of the 2250 participants, 29.8% (670) were current smokers, 8.6% (193) were ex-smokers and 97.6% believed that smoking was addictive. PHWs made 83% of the participants scared. Participants believed that PHWs would be effective in motivating smokers to quit (80.2%), in convincing youth not to start smoking (86.8%), in encouraging ex-smokers to remain as quitters (89.1%) and in building public awareness on the dangers of smoking (94%). PHWs made 58% of the current smokers intended to quit smoking and reduced their daily intake of cigarettes from 11 to 5 on average.  Current smokers preferred to purchase loose cigarettes rather than a pack. The covariates significantly associated with the effectiveness of PHWs were current smokers, ex-smokers and addiction. CONCLUSION: PHWs were found important to motivate smokers to quit smoking, to reduce consumption of cigarettes and to prevent relapse in ex-smokers. Evidence from the study had triggered policy changes which included enlargement of the size of PHW to 90% and the release of a notification to ban selling of loose cigarettes. Thus, the warning messages with pictures are required to be improved and rotated.

Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype.

The relationship between sequence variation and phenotype is poorly understood. Here, we use metabolomic analysis to elucidate the molecular mechanism underlying the filamentous phenotype of E. coli strains that carry destabilizing mutations in dihydrofolate reductase (DHFR). We find that partial loss of DHFR activity causes reversible filamentation despite SOS response indicative of DNA damage, in contrast to thymineless death (TLD) achieved by complete inhibition of DHFR activity by high concentrations of antibiotic trimethoprim. This phenotype is triggered by a disproportionate drop in intracellular dTTP, which could not be explained by drop in dTMP based on the Michaelis-Menten-like in vitro activity curve of thymidylate kinase (Tmk), a downstream enzyme that phosphorylates dTMP to dTDP. Instead, we show that a highly cooperative (Hill coefficient 2.5) in vivo activity of Tmk is the cause of suboptimal dTTP levels. dTMP supplementation rescues filamentation and restores in vivo Tmk kinetics to Michaelis-Menten. Overall, this study highlights the important role of cellular environment in sculpting enzymatic kinetics with system-level implications for bacterial phenotype.

Management of arrhythmias in pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a potentially devastating clinical condition with a poor long-term prognosis. Cardiac arrhythmias are frequent in PH, and pulmonary hypertensives are particularly susceptible to the adverse haemodynamic effects of heart rhythm disorders. However, arrhythmia management in PH patients can be more challenging than in the general population due to the particular physiological idiosyncrasies associated with the condition. Here, we summarise and appraise the data pertaining to multimodality treatment of cardiac arrhythmias in PH to help refine the management strategy for this vulnerable patient group. RESULTS: The majority of our understanding of the safety and effectiveness of different arrhythmia treatments in PH is based on observational and retrospective data. Rhythm control is the overall goal, and for atrial and ventricular tachyarrhythmias, referral for catheter ablation, ideally using electroanatomical mapping technology in specialist centres, is the preferable means of achieving this. Contradictory viewpoints are expounded regarding the safety of beta blocker use in PH, though in three small prospective clinical trials and at least six animal models they appear to be well-tolerated. Nevertheless, amiodarone remains the preferred pharmacological treatment. Direct current cardioversion can be carried out effectively to terminate tachyarrhythmias in both the emergency and elective setting, though mechanistic studies demonstrate a higher recurrence rate in PH patients. Individual reports and series suggest that device implantation may be technically challenging and associated with a higher complication rate due to anatomical distortion and chamber enlargement. Modulation of sympathetic input to the heart appears to reduce arrhythmia vulnerability in canine models of PH, and its clinical application in humans is a worthwhile area of further study. CONCLUSION: Prompt restoration of sinus rhythm improves outcomes in PH, and at present, the most reliable and safest strategy for long-term rhythm control is amiodarone and, where possible, ablation. Reinforcement of the evidence base with randomised prospective trials is necessary. This would be particularly beneficial to clarify the role of atrial fibrillation ablation and the safety and efficacy of beta-blockers. In addition, a more comprehensive assessment of the vulnerability of PH patients to potentially fatal brady- and ventricular tachyarrhythmias may help guide recommendations for provision of primary prevention device therapy.

Early use of tocilizumab in the prevention of adult respiratory failure in SARS-CoV-2 infections and the utilization of interleukin-6 levels in the management.

Respiratory failure in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection appears related to cytokine release syndrome that often results in mechanical ventilation (MV). We investigated the role of tocilizumab (TCZ) on interleukin-6 (IL-6) trends and MV in patients with SARS-CoV-2. In this longitudinal observational study, 112 patients were evaluated from 1 February to 31 May 2020. TCZ was administered followed by methylprednisolone to patients with >3L oxygen requirement and pneumonia severity index score ≤130 with computed tomography scan changes. IL-6, C-reactive protein (CRP), ferritin, lactate dehydrogenase (LDH), D-dimer, and procalcitonin were monitored on days 0, 3, and 6 of therapy. Statistical analyses were performed with significance ≤0.05. Eighty out of 112 SARS-CoV-2-positive patients (45 males, 56.96%; 34 females, 43.04%) were included in this study. Seven patients expired (8.75%) and nine patients required MV (11.25%). Median IL-6 levels pre-administration of TCZ was 342.50 (78.25-666.25) pg/mL compared with post-administration on day 3 (563; 162-783) pg/mL (P < .00001). On day 6, the median dropped to 545 (333.50-678.50) pg/mL compared with day 3 (P = .709). CRP, ferritin, LDH, and D-dimer levels were reduced after TCZ therapy. Early use of TCZ may reduce the need for MV and decrease CRP, ferritin, LDH, and D-dimer levels. The sequential use of methylprednisolone for 72 hours seems to potentiate the effect and prolong the suppression of the cytokine storm. IL-6 levels may be helpful as a prognostic tool.

Novel arterioplasty of severe left pulmonary artery stenosis using native main pulmonary artery.

There are multiple approaches described for the repair of stenosed branch pulmonary arteries. Regardless of the technique used, restenosis is common. We describe a case of severe left pulmonary artery stenosis repaired with a novel technique using the native main pulmonary artery which was transected and turned down to create a direct anastomosis with the left pulmonary artery. This tension-free tissue to tissue anastomosis resulted in hemodynamically gratifying results with the potential for growth.

Crystalline Nephropathy due to APRT Deficiency: A Preventable Cause of Renal and Renal Allograft Failure.

Adenine phosphororibosyl transferase (APRT) deficiency, a rare inborn error of metabolism is inherited as an autosomal recessive trait. It presents with 2,8-dihydroxyadenine (2,8-DHA) crystal nephropathy and recurrent nephrolithiasis and often progresses to end stage renal disease (ESRD). After transplant, it can recur in the allograft. If APRT deficiency is recognized early, renal failure can be prevented, arrested or reversed in native kidney and in allograft by treatment with allopurinol, which inhibits xanthine oxidase and reduces 2,8-DHA formation. We report two cases of APRT deficiency from our center. DNA sequencing of APRT gene performed in one of the cases revealed a pathogenic variant in Exon1 of APRT gene (c.3G>C; p.Met1). This variant affects the translation initiation codon and results in a start loss. The variant has previously been reported in two cases with APRT deficiency.

One stage combined repair of intracardiac defects with vascular ring employing sternotomy approach.

We present an unusual case of atrial septal defect and ventricular septal defect with a vascular ring formed by a right-sided aortic arch with an aberrant left subclavian artery that gave rise to a patent ductus arteriosus connecting to the main pulmonary artery. We performed a single-stage repair of the intracardiac defects and division of vascular ring with a sternotomy instead of the traditionally practiced dual approach. This included division and reimplantation of the aberrant left subclavian artery to the left carotid artery after transection. This approach has not been described so far.

Transcatheter closure of elongated and pulmonary hypertensive patent arterial duct in infants using Amplatzer vascular plug II.

OBJECTIVE: To evaluate the feasibility, efficacy, and safety of Amplatzer vascular plug II in large and elongated ducts in infants. INTRODUCTION: Patent arterial duct device closure is technically challenging in infants with large and elongated ducts because Amplatzer duct occluder and Amplatzer duct occluder II have high chances of causing aortic coarctation and left pulmonary artery stenosis, respectively. The Amplatzer vascular plug II being soft with no retention discs on either sides helps in mitigating these problems. METHOD: This is a prospective, observational study involving infants with clinical, echocardiographic and angiographic evidence of large left to right shunt. All the children underwent duct closure using Amplatzer vascular plug II. RESULTS: Eighteen infants qualified for the study. Mean age and weight were 8.63 ± 3.84 months and 6.3 ± 1.7 kg, respectively. The angiographic mean duct diameter at the pulmonary artery end was 4.66 ± 0.92 mm, and the mean duct length was 9.4 ± 2.48 mm. The size of Amplatzer vascular plug II used varied from 6 mm to 10 mm. Technical success was achieved in 16/18 cases. One patient had device embolisation, and in the other, the device was found to be unstable. The ratio of Amplatzer vascular plug II size to the duct diameter was 1.65 ± 0.27, while the ratio of ductal length to device length was 1.48 ± 0.46 in those with successful outcome. CONCLUSIONS: Amplatzer vascular plug II is a safe and effective option in appropriately selected infants with elongated ducts. Diameter and length of Amplatzer vascular plug II vis-a-vis those of the ductus are important determinants of the successful outcome.

Substrate inhibition imposes fitness penalty at high protein stability.

Proteins are only moderately stable. It has long been debated whether this narrow range of stabilities is solely a result of neutral drift toward lower stability or purifying selection against excess stability-for which no experimental evidence was found so far-is also at work. Here, we show that mutations outside the active site in the essential Escherichia coli enzyme adenylate kinase (Adk) result in a stability-dependent increase in substrate inhibition by AMP, thereby impairing overall enzyme activity at high stability. Such inhibition caused substantial fitness defects not only in the presence of excess substrate but also under physiological conditions. In the latter case, substrate inhibition caused differential accumulation of AMP in the stationary phase for the inhibition-prone mutants. Furthermore, we show that changes in flux through Adk could accurately describe the variation in fitness effects. Taken together, these data suggest that selection against substrate inhibition and hence excess stability may be an important factor determining stability observed for modern-day Adk.

Long-term follow-up of an adult with left pulmonary artery stenting and absent right pulmonary artery: a case report.

BACKGROUND: Unilateral absence of pulmonary artery is a rare congenital abnormality with varied clinical presentations. We present a unique case of congenital absence of right pulmonary artery (RPA) with left pulmonary artery (LPA) origin stenosis without any intracardiac or extracardiac lesion and its long-term follow-up. CASE SUMMARY: A 68-year-old woman underwent successful LPA stenting 23 years back for absent RPA and LPA origin stenosis. She was recently evaluated for breathlessness, almost two decades after the procedure. On evaluation, she was found to have severe pulmonary hypertension (Group 5) without any distortion of the LPA and with normally functioning stent. DISCUSSION: This report discusses the long-term outcome of stenting in the setting of severe stenosis of a single pulmonary artery. Over the years, the patient went on to develop severe segmental pulmonary hypertension (Group 5).

Accessibility of the Shine-Dalgarno Sequence Dictates N-Terminal Codon Bias in E. coli.

Despite considerable efforts, no physical mechanism has been shown to explain N-terminal codon bias in prokaryotic genomes. Using a systematic study of synonymous substitutions in two endogenous E. coli genes, we show that interactions between the coding region and the upstream Shine-Dalgarno (SD) sequence modulate the efficiency of translation initiation, affecting both intracellular mRNA and protein levels due to the inherent coupling of transcription and translation in E. coli. We further demonstrate that far-downstream mutations can also modulate mRNA levels by occluding the SD sequence through the formation of non-equilibrium secondary structures. By contrast, a non-endogenous RNA polymerase that decouples transcription and translation largely alleviates the effects of synonymous substitutions on mRNA levels. Finally, a complementary statistical analysis of the E. coli genome specifically implicates avoidance of intra-molecular base pairing with the SD sequence. Our results provide general physical insights into the coding-level features that optimize protein expression in prokaryotes.

Intraoperative customized double-patch device with twin sutures for multiple muscular septal defects.

OBJECTIVES: Closure of multiple muscular ventricular septal defects (VSDs) remains a challenge because of anatomical complexity. METHODS: We mapped all the VSDs using en face reconstruction of the right ventricular septal surface through echocardiography and then performed an 'Intraoperative Customized Double-Patch Device' technique to surgically close them in 39 patients (male:female = 25:14). The median age of the patients was 6 months (2 months-10 years), and mean weight was 5.98 ± 4.21 kg. A patch of polytetrafluoroethylene was placed on the left ventricular side of the defect and another on the right ventricular side, and they were anchored to each other using 2 polypropylene sutures. Residual shunts were evaluated using intraoperative echocardiography and measurements of right atrial-pulmonary arterial saturation were taken in all patients. RESULTS: The distribution of muscular VSDs was as follows: anterior muscular 12, posterior muscular 18, mid-muscular 11 and apical 9. The associated lesions included perimembranous VSD (n = 28), tetralogy of Fallot (n = 6), double-outlet right ventricle (n = 2) and supramitral membrane (n = 2). Mean clamp time and bypass time were 93 ± 19 min and 147 ± 26 min, respectively. Mean hospital stay was 11 ± 3.39 days with no in-hospital mortality. Five patients with significant residual shunts needed concomitant PA banding. All patients remained in New York Heart Association Class I. There was either no residual shunt (n = 3) or trivial shunt (n = 2) among the banded patients. All patients remained symptom-free and continued to thrive well at the most recent follow-up (3.48 ± 1.51 years). CONCLUSIONS: Muscular VSDs can be mapped through en face reconstruction and closed using intraoperative customized double-patch device technique in a variety of situations with satisfactory immediate and short-term results.

Dual pathology causing severe pulmonary hypertension following surgical repair of total anomalous pulmonary venous connection: Successful outcome following serial transcatheter interventions.

Surgical repair of total anomalous pulmonary venous connection (TAPVC) can be complicated by the development of pulmonary venous stenosis later on. In addition, the vertical vein, if left unligated, can remain patent and lead to hemodynamically significant left to right shunting. We report an infant who required transcatheter correction of both these problems after surgical repair of TAPVC.

Trade-offs between microbial growth phases lead to frequency-dependent and non-transitive selection.

Mutations in a microbial population can increase the frequency of a genotype not only by increasing its exponential growth rate, but also by decreasing its lag time or adjusting the yield (resource efficiency). The contribution of multiple life-history traits to selection is a critical question for evolutionary biology as we seek to predict the evolutionary fates of mutations. Here we use a model of microbial growth to show that there are two distinct components of selection corresponding to the growth and lag phases, while the yield modulates their relative importance. The model predicts rich population dynamics when there are trade-offs between phases: multiple strains can coexist or exhibit bistability due to frequency-dependent selection, and strains can engage in rock-paper-scissors interactions due to non-transitive selection. We characterize the environmental conditions and patterns of traits necessary to realize these phenomena, which we show to be readily accessible to experiments. Our results provide a theoretical framework for analysing high-throughput measurements of microbial growth traits, especially interpreting the pleiotropy and correlations between traits across mutants. This work also highlights the need for more comprehensive measurements of selection in simple microbial systems, where the concept of an ordinary fitness landscape breaks down.

Optimization of lag phase shapes the evolution of a bacterial enzyme.

Mutations provide the variation that drives evolution, yet their effects on fitness remain poorly understood. Here we explore how mutations in the essential enzyme adenylate kinase (Adk) of Escherichia coli affect multiple phases of population growth. We introduce a biophysical fitness landscape for these phases, showing how they depend on molecular and cellular properties of Adk. We find that Adk catalytic capacity in the cell (the product of activity and abundance) is the major determinant of mutational fitness effects. We show that bacterial lag times are at a well-defined optimum with respect to Adk's catalytic capacity, while exponential growth rates are only weakly affected by variation in Adk. Direct pairwise competitions between strains show how environmental conditions modulate the outcome of a competition where growth rates and lag times have a tradeoff, shedding light on the multidimensional nature of fitness and its importance in the evolutionary optimization of enzymes.