Predicting Neuropathy and Reactions in Leprosy at Diagnosis and Before Incident Events—Results from the INFIR Cohort Study

Background

Leprosy is a disease of skin and peripheral nerves. The process of nerve injury occurs gradually through the course of the disease as well as acutely in association with reactions. The INFIR (ILEP Nerve Function Impairment and Reactions) Cohort was established to identify clinically relevant neurological and immunological predictors for nerve injury and reactions.

Methodology/Principal Findings

The study, in two centres in India, recruited 188 new, previously untreated patients with multi-bacillary leprosy who had no recent nerve damage. These patients underwent a series of novel blood tests and nerve function testing including motor and sensory nerve conduction, warm and cold detection thresholds, vibrometry, dynamometry, monofilament sensory testing and voluntary muscle testing at diagnosis and at monthly follow up for the first year and every second month for the second year. During the 2 year follow up a total of 74 incident events were detected. Sub-clinical changes to nerve function at diagnosis and during follow-up predicted these new nerve events. Serological assays at baseline and immediately before an event were not predictive; however, change in TNF alpha before an event was a statistically significant predictor of that event.

Conclusions/Significance

These findings increase our understanding of the processes of nerve damage in leprosy showing that nerve function impairment is more widespread than previously appreciated. Any nerve involvement, including sub-clinical changes, is predictive of further nerve function impairment. These new factors could be used to identify patients at high risk of developing impairment and disability.     

Multiple Sequence Signals Direct Recognition and Degradation of Protein Substrates by the AAA+ Protease HslUV

Proteolysis is important for protein quality control and for the proper regulation of many intracellular processes in prokaryotes and eukaryotes. Discerning substrates from other cellular proteins is a key aspect of proteolytic function. The Escherichia coli HslUV protease is a member of a major family of ATP-dependent AAA+ degradation machines. HslU hexamers recognize and unfold native protein substrates and then translocate the polypeptide into the degradation chamber of the HslV peptidase. Although a wealth of structural information is available for this system, relatively little is known about mechanisms of substrate recognition. Here, we demonstrate that mutations in the unstructured N-terminal and C-terminal sequences of two model substrates alter HslUV recognition and degradation kinetics, including changes in V_max. By introducing N- or C-terminal sequences that serve as recognition sites for specific peptide-binding proteins, we show that blocking either terminus of the substrate interferes with HslUV degradation, with synergistic effects when both termini are obstructed. These results support a model in which one terminus of the substrate is tethered to the protease and the other terminus is engaged by the translocation/unfolding machinery in the HslU pore. Thus, degradation appears to consist of discrete steps, which involve the interaction of different terminal sequence signals in the substrate with different receptor sites in the HslUV protease.     

Assessment of the Correlation Between Two Defining Criteria for Bidirectional Isthmic Block in the Ablation of Typical Atrial Flutter

Background

A complete, bidirectional conduction block in the cavotricuspid isthmus (CTI) represents the end-point of the typical atrial flutter ablation. We investigated the correlation between two criteria for successful ablation, one based on the atrial bipolar electrogram morphology before and after complete CTI conduction block, compared to the standard criteria of differential pacing and reversal in the right atrial depolarization sequence during coronary sinus (CS) pacing.

Method

We conducted a retrospective study in 111 patients (81 males, average age 62±10 years) who underwent an atrial flutter ablation during September 2007 - July 2009 in the Cardiology - Rehabilitation Hospital, UMF Cluj-Napoca. We assessed the presence of a bidirectional block at the end of the procedure using the standard criteria. We then analyzed the morphology of the bipolar atrial electrograms adjacent to the ablation line, before and after CTI conduction block.

Results

A change from a qRs morphology to a rSr'' morphology when pacing from the coronary sinus and from a rsr'' morphology to a QRS morphology when pacing from the low-lateral right atrium was associated with a CTI conduction block. Sensitivity (Se), specificity(Sp), positive predictive value (PPV), negative predictive value (NPV) were 96%, 89%, 99% and 67% respectively.

Conclusion

Our study suggests that the analysis of the atrial bipolar electrogram next to the ablation line before and after CTI ablation may be used as a reliable criterion to validate CTI conduction block due to its high sensitivity, specificity and positive predictive value.     

Classification and regression tree and spatial analyses reveal geographic heterogeneity in genome wide linkage study of Indian visceral leishmaniasis

Background

Genome wide linkage studies (GWLS) have provided evidence for loci controlling visceral leishmaniasis on Chromosomes 1p22, 6q27, 22q12 in Sudan and 6q27, 9p21, 17q11-q21 in Brazil. Genome wide studies from the major focus of disease in India have not previously been reported.

Methods and Findings

We undertook a GWLS in India in which a primary ∼10 cM (515 microsatellites) scan was carried out in 58 multicase pedigrees (74 nuclear families; 176 affected, 353 total individuals) and replication sought in 79 pedigrees (102 nuclear families; 218 affected, 473 total individuals). The primary scan provided evidence (≥2 adjacent markers allele-sharing LOD≥0.59; nominal P≤0.05) for linkage on Chromosomes 2, 5, 6, 7, 8, 10, 11, 20 and X, with peaks at 6p25.3-p24.3 and 8p23.1-p21.3 contributed to largely by 31 Hindu families and at Xq21.1-q26.1 by 27 Muslim families. Refined mapping confirmed linkage across all primary scan families at 2q12.2-q14.1 and 11q13.2-q23.3, but only 11q13.2-q23.3 replicated (combined LOD = 1.59; P = 0.0034). Linkage at 6p25.3-p24.3 and 8p23.1-p21.3, and at Xq21.1-q26.1, was confirmed by refined mapping for primary Hindu and Muslim families, respectively, but only Xq21.1-q26.1 replicated across all Muslim families (combined LOD 1.49; P = 0.0045). STRUCTURE and SMARTPCA did not identify population genetic substructure related to religious group. Classification and regression tree, and spatial interpolation, analyses confirm geographical heterogeneity for linkages at 6p25.3-p24.3, 8p23.1-p21.3 and Xq21.1-q26.1, with specific clusters of families contributing LOD scores of 2.13 (P = 0.0009), 1.75 (P = 0.002) and 1.84 (P = 0.001), respectively.

Conclusions

GWLS has identified novel loci that show geographical heterogeneity in their influence on susceptibility to VL in India.     

Spectrophotometric and steady-state kinetic analysis of the biosynthetic arginine decarboxylase of Yersinia pestis utilizing arginine analogues as inhibitors and alternative substrates

The PLP-dependent, biosynthetic arginine decarboxylase (ADC) of Yersinia pestis was investigated using steady-state kinetics employing structural analogues of arginine as both alternative substrates and competitive inhibitors. The inhibitor analysis indicates that binding of the carboxyl and guanidinium groups of the substrate, l-arginine, provides essentially all of the free energy change realized upon substrate binding in the ground state. Furthermore, recognition of the guanidinium group is primarily responsible for substrate specificity. Comparison of the steady-state parameters for a series of alternative substrates that contained chemically modified guanidinium moieties provides evidence of a role for induced fit in ADC catalysis. ADC was also characterized by UV/vis and fluorescence spectrophotometry in the presence or absence of a number of arginine analogues. The enzyme complexes formed served as models for the adsorption complex and the external aldimine complex of the enzyme with the substrate.     

Use of scanning electron microscopy and energy dispersive X-ray spectroscopy to identify key fouling species during alternating tangential filtration

Alternating tangential flow filtration (ATF) has become one of the primary methods for cell retention and clarification in perfusion bioreactors. However, membrane fouling can cause product sieving losses that limit the performance of these systems. This study used scanning electron microscopy and energy dispersive X-ray spectroscopy to identify the nature and location of foulants on 0.2 μm polyethersulfone hollow fiber membranes after use in industrial Chinese hamster ovary cell perfusion bioreactors for monoclonal antibody production. Membrane fouling was dominated by proteinaceous material, primarily host cell proteins along with some monoclonal antibody. Fouling occurred primarily on the lumen surface with much less protein trapped within the depth of the fiber. Protein deposition was also most pronounced near the inlet/exit of the hollow fibers, which are the regions with the greatest flux (and transmembrane pressure) during the cyclical operation of the ATF. These results provide important insights into the underlying phenomena governing the fouling behavior of ATF systems for continuous bioprocessing.     

Inverting character of alpha-glucuronidase A from Aspergillus tubingensis

Alpha-glucuronidase A from Aspergillus tubingensis was found to be capable of liberating 4-O-methyl-D-glucuronic acid (MeGlcA) only from those beechwood glucuronoxylan fragments in which the acid is attached to the non-reducing terminal xylopyranosyl residue. Reduced aldotetrauronic acid, 4-O-methyl-D-glucuronosyl-alpha-1,2-D-xylopyranosyl-beta-1,4-xylopyranosyl-beta-1,4-xylitol, was found to be a suitable substrate to follow the stereochemical course of the hydrolytic reaction catalyzed by the purified enzyme. The configuration of the liberated MeGlcA was followed in a D(2)O reaction mixture by (1)H-NMR spectroscopy. It was unambiguously established that MeGlcA was released from the substrate as its beta-anomer from which the alpha-anomer was formed on mutarotation. This result represents the first experimental evidence for the inverting character of a microbial alpha-glucuronidase, a member of glycosyl hydrolase family 67 (EC 3.1.1.139).     

Overexpression and purification of recombinant eel calcitonin and its phylogenetic analysis

Calcitonin (CT), a peptide hormone that is widely used for the treatment of osteoporosis, Paget's disease, hypercalcemic shock and chronic pain in terminal cancer patients, is produced by the para-follicular cells of the thyroid gland in mammals and by the ultimobranchial gland of birds and fish. Fish calcitonin, like eel calcitonin (eCT), is more potent and longer lasting than human CT and is one of the many bioactive peptides that require C-terminal amidation for full biological activity. In this study we describe the over-expression and over-production of C-terminal amidated eCT in recombinant Streptomyces avermitilis. A phylogenetic analysis was performed with all the known CT amino acid sequences.     

A simple and inexpensive "cell dissociation sieve-tissue grinder" apparatus

A simple and inexpensive cell dissociation sieve-tissue grinder apparatus consisting essentially of stainless steel sieve (the one popularly used for sieving tea leaves) and a glass syringe plunger acting as pestle, is described for making single cell suspension.