Transorbital Sonographic Evaluation of Normal Optic Nerve Sheath Diameter in Healthy Volunteers in Bangladesh

Introduction

Measurement of optic nerve sheath diameter (ONSD) by ultrasound is increasingly used as a marker to detect raised intracranial pressure (ICP). ONSD varies with age and there is no clear consensus between studies for an upper limit of normal. Knowledge of normal ONSD in a healthy population is essential to interpret this measurement.

Methods

In a prospective observational study, ONSD was measured using a 15 MHz ultrasound probe in healthy volunteers in Chittagong, Bangladesh. The aims were to determine the normal range of ONSD in healthy Bangladeshi adults and children, compare measurements in males and females, horizontal and vertical beam orientations and left and right eyes in the same individual and to determine whether ONSD varies with head circumference independent of age.

Results

136 subjects were enrolled, 12.5% of whom were age 16 or under. Median ONSD was 4.41 mm with 95% of subjects in the range 4.25–4.75 mm. ONSD was bimodally distributed. There was no relationship between ONSD and age (≥4 years), gender, head circumference, and no difference in left vs right eye or horizontal vs vertical beam.

Conclusions

Ultrasonographic ONSD in Bangladeshi healthy volunteers has a narrow bimodal distribution independent of age (≥4 years), gender and head circumference. ONSD >4.75 mm in this population should be considered abnormal.     

MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach

Propagation of signals from G protein-coupled receptors (GPCRs) in cells is primarily mediated by protein-protein interactions. MAS is a GPCR that was initially discovered as an oncogene and is now known to play an important role in cardiovascular physiology. Current literature suggests that MAS interacts with common heterotrimeric G-proteins, but MAS interaction with proteins which might mediate G protein-independent or atypical signaling is unknown. In this study we hypothesized that MAS C-terminal tail (Ct) is a major determinant of receptor-scaffold protein interactions mediating MAS signaling. Mass-spectrometry based proteomic analysis was used to comprehensively identify the proteins that interact with MAS Ct comprising the PDZ-binding motif (PDZ-BM). We identified both PDZ and non-PDZ proteins from human embryonic kidney cell line, mouse atrial cardiomyocyte cell line and human heart tissue to interact specifically with MAS Ct. For the first time our study provides a panel of PDZ and other proteins that potentially interact with MAS with high significance. A ‘cardiac-specific finger print’ of MAS interacting PDZ proteins was identified which includes DLG1, MAGI1 and SNTA. Cell based experiments with wild-type and mutant MAS lacking the PDZ-BM validated MAS interaction with PDZ proteins DLG1 and TJP2. Bioinformatics analysis suggested well-known multi-protein scaffold complexes involved in nitric oxide signaling (NOS), cell-cell signaling of neuromuscular junctions, synapses and epithelial cells. Majority of these protein hits were predicted to be part of disease categories comprising cancers and malignant tumors. We propose a ‘MAS-signalosome’ model to stimulate further research in understanding the molecular mechanism of MAS function. Identifying hierarchy of interactions of ‘signalosome’ components with MAS will be a necessary step in future to fully understand the physiological and pathological functions of this enigmatic receptor.     

Methylene amine substituted arylindenopyrimidines as potent adenosine A2A/A1 antagonists

A novel series of arylindenopyrimidines were identified as A_2A and A₁ receptor antagonists. The series was optimized for in vitro activity by substituting the 8- and 9-positions with methylene amine substituents. The compounds show excellent activity in mouse models of Parkinson’s disease when dosed orally.     

Role of Glu312 in binding and positioning of the substrate for the hydride transfer reaction in choline oxidase

Choline oxidase catalyzes the oxidation of choline to glycine betaine, a compatible solute that accumulates in pathogenic bacteria and plants so they can withstand osmotic and temperature stresses. The crystal structure of choline oxidase was determined and refined to a resolution of 1.86 A with data collected at 100 K using synchrotron X-ray radiation. The structure reveals a covalent linkage between His99 Nepsilon2 and FAD C8M atoms, and a 123 A3 solvent-excluded cavity adjacent to the re face of the flavin. A hypothetical model for choline docked into the cavity suggests that several aromatic residues and Glu312 may orient the cationic substrate for efficient catalysis. The role of the negative charge on Glu312 was investigated by engineering variant enzymes in which Glu312 was replaced with alanine, glutamine, or aspartate. The Glu312Ala enzyme was inactive. The Glu312Gln enzyme exhibited a Kd value for choline at least 500 times larger than that of the wild-type enzyme. The Glu312Asp enzyme had a kcat/KO2 value similar to that of the wild-type enzyme but kcat and kcat/Km values that were 230 and 35 times lower, respectively, than in the wild-type enzyme. These data are consistent with the spatial location of the negative charge on residue 312 being important for the oxidation of the alcohol substrate. Solvent viscosity and substrate kinetic isotope effects suggest the presence of an internal equilibrium in the Glu312Asp enzyme prior to the hydride transfer reaction. Altogether, the crystallographic and mechanistic data suggest that Glu312 is important for binding and positioning of the substrate in the active site of choline oxidase.     

Therapeutic potential of HIV-1 entry inhibitor peptidomimetics

Human immunodeficiency virus 1 (HIV-1) infection remains a public health concern globally. Although great strides in the management of HIV-1 have been achieved, current highly active antiretroviral therapy is limited by multidrug resistance, prolonged use-related effects, and inability to purge the HIV-1 latent pool. Even though novel therapeutic options with HIV-1 broadly neutralizing antibodies (bNAbs) are being explored, the scalability of bNAbs is limited by economic cost of production and obligatory requirement for parenteral administration. However, these limitations can be addressed by antibody mimetics/peptidomimetics of HIV-1 bNAbs. In this review we discuss the limitations of HIV-1 bNAbs as HIV-1 entry inhibitors and explore the potential therapeutic use of antibody mimetics/peptidomimetics of HIV-1 entry inhibitors as an alternative for HIV-1 bNAbs. We highlight the reduced cost of production, high specificity, and oral bioavailability of peptidomimetics compared to bNAbs to demonstrate their suitability as candidates for novel HIV-1 therapy and conclude with some perspectives on future research toward HIV-1 novel drug discovery.     

Temperature as a predictive tool for plantar triaxial loading

Diabetic foot ulcers are caused by moderate repetitive plantar stresses in the presence of peripheral neuropathy. In severe cases, the development of these foot ulcers can lead to lower extremity amputations. Plantar pressure measurements have been considered a capable predictor of ulceration sites in the past, but some investigations have pointed out inconsistencies when solely relying on this method. The other component of ground reaction forces/stresses, shear, has been understudied due to a lack of adequate equipment. Recent articles reported the potential clinical significance of shear in diabetic ulcer etiology. With the lack of adequate tools, plantar temperature has been used as an alternative method for determining plantar triaxial loading and/or shear. However, this method has not been previously validated. The purpose of this study was to analyze the potential association between exercise-induced plantar temperature increase and plantar stresses. Thirteen healthy individuals walked on a treadmill for 10 minutes at 3.2 km/h. Pre and post-exercise temperature profiles were obtained with a thermal camera. Plantar triaxial stresses were quantified with a custom-built stress plate. A statistically significant correlation was observed between peak shear stress (PSS) and temperature increase (r=0.78), but not between peak resultant stress (PRS) and temperature increase (r=0.46). Plantar temperature increase could predict the location of PSS and PRS in 23% and 39% of the subjects, respectively. Only a moderate linear relationship was established between triaxial plantar stresses and walking-induced temperature increase. Future research will investigate the value of nonlinear models in predicting plantar loading through foot temperature.     

Quality improvement programme to achieve acceptable colonoscopy completion rates: prospective before and after study

Problem A large audit of colonoscopy in the United Kingdom showed that the unadjusted completion rate was 57% when stringent criteria for identifying the caecum were applied. The caecum should be reached 90% of the time. Little information is available on what units or operators need to do to improve to acceptable levels.Design Quality improvement programme using two completed cycles of audit.Setting Endoscopy department in a university linked general hospital in northeast England.Key measures for improvement Colonoscopy completion rate.Strategy for change Two audit cycles were completed between 1999 and 2002. Changes to practice were based on results of audit and took into account the opinions of relevant staff. Lack of time for each colonoscopy, poor bowel preparation, especially in frail patients, and a mismatch between number of colonoscopies done and completion rate for individual operators were responsible for failed colonoscopies. Appropriate changes were made.Effects of change The initial crude colonoscopy completion rate was 60%, improving to 71% after the first round of audit and 88% after the second round, which approximates to the agreed audit standard of 90%. The final adjusted completion rate was 94%.Lessons learnt Achievement of the national targets in a UK general hospital is possible by lengthening appointments, admitting frail patients for bowel preparation to one ward, and allocating colonoscopies to the most successful operators.     

Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase

The FAD-dependent choline oxidase has a flavin cofactor covalently attached to the protein via histidine 99 through an 8α-N(3)-histidyl linkage. The enzyme catalyzes the four-electron oxidation of choline to glycine betaine, forming betaine aldehyde as an enzyme-bound intermediate. The variant form of choline oxidase in which the histidine residue has been replaced with asparagine was used to investigate the contribution of the 8α-N(3)-histidyl linkage of FAD to the protein toward the reaction catalyzed by the enzyme. Decreases of 10-fold and 30-fold in the k_cat/K_m and k_cat values were observed as compared with wild-type choline oxidase at pH 10 and 25 °C, with no significant effect on k_cat/K_O using choline as substrate. Both the k_cat/K_m and k_cat values increased with increasing pH to limiting values at high pH consistent with the participation of an unprotonated group in the reductive half-reaction and the overall turnover of the enzyme. The pH independence of both ^D(k_cat/K_m) and ^Dk_cat, with average values of 9.2 ± 3.3 and 7.4 ± 0.5, respectively, is consistent with absence of external forward and reverse commitments to catalysis, and the chemical step of CH bond cleavage being rate-limiting for both the reductive half-reaction and the overall enzyme turnover. The temperature dependence of the ^Dk_red values suggests disruption of the preorganization in the asparagine variant enzyme. Altogether, the data presented in this study are consistent with the FAD-histidyl covalent linkage being important for the optimal positioning of the hydride ion donor and acceptor in the tunneling reaction catalyzed by choline oxidase.A number of enzymes, including dehydrogenases (1–3), monooxygenases (4–7), halogenases (8–11), and oxidases (7, 12, 13), employ flavin cofactors (FAD or FMN) for their catalytic processes. About a tenth of all flavoproteins have been shown to contain a covalently attached cofactor, which may be linked at the C8M position via histidyl, tyrosyl, or cysteinyl side chains or at the C6M position via a cysteinyl side chain (14). Glucooligosaccharide oxidase (15, 16), hexose oxidase (17), and berberine bridge enzyme (18, 19) are examples of flavoproteins (FAD as cofactor) with both linkages present in one flavin molecule. The covalent linkages in flavin-dependent enzymes have been shown to stabilize protein structure (20–22), prevent loss of loosely bound flavin cofactors (23), modulate the redox potential of the flavin microenvironment (20, 23–27), facilitate electron transfer reactions (28), and contribute to substrate binding as in the case of the cysteinyl linkage (20). However, no study has implicated a mechanistic role of the flavin covalent linkages in enzymatic reactions in which a hydride ion is transferred by quantum mechanical tunneling.The discovery of quantum mechanical tunneling in enzymatic reactions, in which hydrogen atoms, protons, and hydride ions are transferred, has attracted considerable interest in enzyme studies geared toward understanding the mechanisms underlying the several orders of magnitudes in the rate enhancements of protein-catalyzed reactions compared with non-enzymatic ones. Tunneling mechanisms have been shown in a wide array of cofactor-dependent enzymes, including flavoenzymes. Examples of flavoenzymes in which the tunneling mechanisms have been demonstrated include morphinone reductase (29, 30), pentaerythritol tetranitrate reductase (29), glucose oxidase (31–33), and choline oxidase (34). Mechanistic data on Class 2 dihydroorotate dehydrogenases, also with a flavin cofactor (FMN) covalently linked to the protein moiety (35, 36), could only propose a mechanism that is either stepwise or concerted with significant quantum mechanical tunneling for the hydride transfer from C6 and the deprotonation at C5 in the oxidation of dihydroorotate to orotate (37). This leaves choline oxidase as the only characterized enzyme with a covalently attached flavin cofactor (12, 38), where the oxidation of its substrate occurs unequivocally by quantum mechanical tunneling.Choline oxidase from Arthrobacter globiformis catalyzes the two-step FAD-dependent oxidation of the primary alcohol substrate choline to glycine betaine with betaine aldehyde, which is predominantly bound to the enzyme and forms a gem-diol species, as intermediate (Scheme 1). Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism against stress conditions, thus making genetic engineering of relevant plants of economic interest (39–45), and the biosynthetic pathway for the osmolyte is a potential drug target in human microbial infections of clinical interest (46–48). The first oxidation step catalyzed by choline oxidase involves the transfer of a hydride ion from a deprotonated choline to the protein-bound flavin followed by reaction of the anionic flavin hydroquinone with molecular oxygen to regenerate the oxidized FAD (for a recent review see Ref. 50). The gem-diol choline, i.e. hydrated betaine aldehyde, is the substrate for the second oxidation step (49), suggesting that the reaction may follow a similar mechanism. The isoalloxazine ring of the flavin cofactor, which is buried within the protein, is physically constrained through a covalent linkage via the C(8) methyl of the flavin and the N(3) atom of the histidine side chain at position 99 (Fig. 1) (12). Also contributing to the physical constrain are the proximity of Ile-103 to the pyrimidine ring and the interactions of the backbone atoms of residues His-99 through Ile-103 with the isoalloxazine ring. The rigid positioning of the isoalloxazine ring could only permit a solvent-excluded cavity of ∼125 ų adjacent to the re face of the FAD to accommodate a 93-ų choline molecule in the substrate binding domain (12). Mechanistic data thus far obtained on choline oxidase, coupled with the crystal structure of the wild-type enzyme resolved to 1.86 Å, are consistent with a quantum tunneling mechanism for the hydride ion transfer occurring within a highly preorganized enzyme-substrate complex (Scheme 2) (12, 34, 50). Exploitation of the tunneling mechanism requires minimal independent movement of the hydride ion donor and acceptor, with the only dynamic motions permitted being the ones that promote the hydride transfer reaction.Open in a separate windowSCHEME 1.Two-step, four-electron oxidation of choline catalyzed by choline oxidase.Open in a separate windowFIGURE 1.x-ray crystal structure of the active site of wild-type choline oxidase resolved to 1.86 Å (PDB 2jbv). Note the significant distortion of the flavin ring at the C(4a) atom, which is due to the presence of a C(4a) adduct (69).Open in a separate windowSCHEME 2.The hydride ion transfer reaction from the α-carbon of the activated choline alkoxide species to the N(5) atom of the isoalloxazine ring of the enzyme-bound flavin in choline oxidase.In the present study, the contribution of the physically constrained flavin isoalloxazine ring to the reaction catalyzed by choline oxidase has been investigated in a variant enzyme in which the histidine residue at position 99 was replaced with an asparagine. The results suggest that, although not being required per se, the covalent linkage in choline oxidase contributes to the hydride tunneling reaction by either preventing independent movement or contributing to the optimal positioning of the flavin acting as hydride ion acceptor with respect to the alkoxide species acting as a donor. However, the covalent linkage is not required for the reaction.     

Rational design of femtomolar inhibitors of isoleucyl tRNA synthetase from a binding model for pseudomonic acid-A

This paper describes the design and characterization of novel inhibitors of IleRS, whose binding affinity approaches the tightest reported for noncovalent inhibition. Compounds were designed from a binding model for the natural product pseudomonic acid-A (PS-A) together with a detailed understanding of the reaction cycle of IleRS and characterization of the mode of binding of the reaction intermediate IleAMP. The interactions of the compounds with IleRS were characterized by inhibition of aminoacylation of tRNA or PP(i)/ATP exchange at supersaturating substrate concentration and by transient kinetics and calorimetry methods. A detailed understanding of the interaction of a comprehensive series of compounds with IleRS allowed the identification of key features and hence the design of exquisitely potent inhibitors. Predictions based on these results have been recently supported by a docking model based on the crystal structure of IleRS with PS-A [Silvian, L. F., Wang J. M., and Steitz T. A. (1999) Science 285 1074-1077].