Exploring the binding preferences/specificity in the active site of human cathepsin E

Aspartic proteinases are produced in the human body by a variety of cells. Some of these proteins, examples of which are pepsin, gastricsin, and renin, are secreted and exert their effects in the extracellular spaces. Cathepsin D and cathepsin E on the other hand are intracellular enzymes. The least characterized of the human aspartic proteinases is cathepsin E. Presented here are results of studies designed to characterize the binding specificities in the active site of human cathepsin E with comparison to othermechanistically similar enzymes. A peptide series based on Lys-Pro-Ala-Lys-Phe*Nph-Arg-Leu was generatedto elucidate the specificity in the individual binding pockets with systematic substitutions in the P_5? P₂ and P₂′-P₃′ based on charge, hydrophobicity, and hydrogen bonding. Also, to explore the S₂ binding preferences, asecond series of peptides based on Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu was generated with systematic replacements in the P₂ position. Kinetic parameters were determined forboth sets of peptides. The results were correlated to a rule-based structural model of human cathepsin E, constructed on the known three-dimensional structures of several highly homologous aspartic proteinases; porcine pepsin, bovine chymosin, yeast proteinase A, human cathepsin D, andmouse and human renin. Important specificity-determining interactions were found in the S₃ (Glu13) and S₂ (Thr-222, Gln-287, Leu-289, Ile-300)subsites. © 1995 Wiley-Liss, Inc.     

Costs of Dengue Control Activities and Hospitalizations in the Public Health Sector during an Epidemic Year in Urban Sri Lanka

Background

Reported as a public health problem since the 1960s in Sri Lanka, dengue has become a high priority disease for public health authorities. The Ministry of Health is responsible for controlling dengue and other disease outbreaks and associated health care. The involvement of large numbers of public health staff in dengue control activities year-round and the provision of free medical care to dengue patients at secondary care hospitals place a formidable financial burden on the public health sector.

Methods

We estimated the public sector costs of dengue control activities and the direct costs of hospitalizations in Colombo, the most heavily urbanized district in Sri Lanka, during the epidemic year of 2012 from the Ministry of Health’s perspective. The financial costs borne by public health agencies and hospitals are collected using cost extraction tools designed specifically for the study and analysed retrospectively using a combination of activity-based and gross costing approaches.

Results

The total cost of dengue control and reported hospitalizations was estimated at US$3.45 million (US$1.50 per capita) in Colombo district in 2012. Personnel costs accounted for the largest shares of the total costs of dengue control activities (79%) and hospitalizations (46%). The results indicated a per capita cost of US$0.42 for dengue control activities. The average costs per hospitalization ranged between US$216–609 for pediatric cases and between US$196–866 for adult cases according to disease severity and treatment setting.

Conclusions

This analysis is a first attempt to assess the economic burden of dengue response in the public health sector in Sri Lanka. Country-specific evidence is needed for setting public health priorities and deciding about the deployment of existing or new technologies. Our results suggest that dengue poses a major economic burden on the public health sector in Sri Lanka.     

Laboratory-Enhanced Dengue Sentinel Surveillance in Colombo District,Sri Lanka: 2012-2014

Introduction

Dengue has emerged as a significant public health problem in Sri Lanka. Historically surveillance was passive, with mandatory dengue notifications based on clinical diagnosis with only limited laboratory confirmation. To obtain more accurate data on the disease burden of dengue, we set up a laboratory-based enhanced sentinel surveillance system in Colombo District. Here we describe the study design and report our findings of enhanced surveillance in the years 2012–2014.

Methods

Three outpatient clinics and three government hospitals in Colombo District that covered most of the Colombo metropolitan area were selected for the sentinel surveillance system. Up to 60 patients per week presenting with an undifferentiated fever were enrolled. Acute blood samples from each patient were tested by dengue specific PCR, NS1 ELISA and IgM ELISA. A sub-set of samples was sent to Duke-NUS Singapore for quality assurance, virus isolation and serotyping. Trained medical research assistants used a standardized case report form to record clinical and epidemiological data. Clinical diagnoses by the clinicians-in-charge were recorded for hospitalized cases.

Results

Of 3,127 febrile cases, 43.6% were PCR and/or NS1 positive for dengue. A high proportion of lab confirmed dengue was observed from inpatients (IPD) (53.9%) compared to outpatient (clinics in hospitals and general practice) (7.6%). Dengue hemorrhagic fever (DHF) was diagnosed in 11% of patients at the time of first contact, and the median day of illness at time of presentation to the sentinel sites was 4. Dengue serotype 1 was responsible for 85% of the cases and serotype 4 for 15%. The sensitivity and specificity of the clinicians’ presumptive diagnosis of dengue was 84% and 34%, respectively.

Conclusion

DENV-1, and to a lesser degree DENV-4, infection were responsible for a high proportion of febrile illnesses in Colombo in the years 2012 to 2014. Clinicians’ diagnoses were associated with high sensitivity, but laboratory confirmation is required to enhance specificity.     

Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence

Statistical analysis of 12 unstable and 32 stable proteins revealed that there are certain dipeptides, the occurrence of which is significantly different in the unstable proteins compared with those in the stable ones. Based on the impact of these dipeptides on the unstable proteins over the stable ones, a weight value of instability is assigned to each of the dipeptides. For a given protein the summation of these weight values normalized to the length of its sequence helps to distinguish between unstable and stable proteins. Results suggest that the in vivo instability of proteins is possibly determined by the order of certain amino acids in its sequence. An attempt is made to correlate metabolic stability of proteins with features of their primary sequence where weight values of instability for a protein of known sequence could thus be used as an index for predicting its stability characteristics.     

Benzyl prolinate derivatives as novel selective KCC2 blockers

The discovery and optimization of a novel class of selective submicromolar KCC2 blockers is described. Details of synthesis and SAR are given together with ADME properties of selected compounds. A methylsulfone residue on the R¹ phenyl group improved the overall general profile of these prolinate derivatives.     

Foliar sclereids in Memecylon and Lijndenia (Melastomataceae) from Borneo, Java, Malaya and Sumatra

Foliar sclereids are investigated in 41 Memecylon species and in Lijndenia laurina Zoll. & Mor. (= Memecylon oligoneurum Bl.). The predominance of filiform sclereids in the Memecylon species is remarkable. Lijndenia laurina has ramiform sclereids.     

Mitochondrial Damage-Associated Molecular Patterns (MTDs) Are Released during Hepatic Ischemia Reperfusion and Induce Inflammatory Responses

Ischemia / reperfusion injury (IRI) during the course of liver transplantation enhances the immunogenicity of allografts and thus impacts overall graft outcome. This sterile inflammatory insult is known to activate innate immunity and propagate organ damage through the recognition of damage-associate molecular pattern (DAMP) molecules. The purpose of the present study was to investigate the role of mitochondrial DAMPs (MTDs) in the pathogenesis of hepatic IRI. Using in vitro models we observed that levels of MTDs were significantly higher in both transplantation-associated and warm IR, and that co-culture of MTDs with human and rat hepatocytes significantly increased cell death. MTDs were also released in an in vivo rat model of hepatic IRI and associated with increased secretion of inflammatory cytokines (TNF-α, IL-6, and IL-10) and increased liver injury compared to the sham group. Our results suggest that hepatic IR results in a significant increase of MTDs both in vitro and in vivo suggesting that MTDs may serve as a novel marker in hepatic IRI. Co-culture of MTDs with hepatocytes showed a decrease in cell viability in a concentration dependent manner, which indicates that MTDs is a toxic mediator participating in the pathogenesis of liver IR injury.     

Deprotonation of hydrochloride salts of amino acid esters and peptide esters using commercial zinc dust.

The deprotonation of hydrochloride salts of ethyl and methyl esters of amino acids and peptides is accomplished using activated zinc dust. The reaction is neat and quantitative. Thus, the free amino acid esters and peptide esters have been isolated in good yield and purity.     

Fitness traits of insecticide resistant and susceptible strains of tea mosquito bug Helopeltis theivora Waterhouse (Heteroptera: Miridae)

We compared the biological traits of insecticide resistant and susceptible field populations of tea mosquito bug Helopeltis theivora Waterhouse. The insecticide resistant population of the conventional Tea Estate “Chuapara” of the Dooars, Jalpaiguri, differed significantly from the susceptible strain of the organic Tea Estate “Makibari” of Darjeeling. Both these tea plantation areas are located in the northern part of West Bengal, India. Adverse changes in biological and developmental traits were observed mainly in: (i) reduction in oviposition period; (ii) fecundity; and (iii) prolongation of nymphal and total developmental period. However, all other parameters such as pre- and post oviposition periods, egg incubation period, hatchability and adult longevity were not significantly different. These results clearly demonstrated that only certain fitness components in the resistant strains appear to be adaptively changed and lowered.