The binding of analogs of porphyrins and chlorins with elongated side chains to albumin

In previous studies, we demonstrated that elongation of side chains of several sensitizers endowed them with higher affinity for artificial and natural membranes and caused their deeper localization in membranes. In the present study, we employed eight hematoporphyrin and protoporphyrin analogs and four groups containing three chlorin analogs each, all synthesized with variable numbers of methylenes in their alkyl carboxylic chains. We show that these tetrapyrroles’ affinity for bovine serum albumin (BSA) and their localization in the binding site are also modulated by chain lengths. The binding constants of the hematoporphyrins and protoporphyrins to BSA increased as the number of methylenes was increased. The binding of the chlorins depended on the substitution at the meso position opposite to the chains. The quenching of the sensitizers’ florescence by external iodide ions decreased as the side chains became longer, indicating to deeper insertion of the molecules into the BSA binding pocket. To corroborate this conclusion, we studied the efficiency of photodamage caused to tryptophan in BSA upon illumination of the bound sensitizers. The efficiency was found to depend on the side-chain lengths of the photosensitizer. We conclude that the protein site that hosts these sensitizers accommodates different analogs at positions that differ slightly from each other. These differences are manifested in the ease of access of iodide from the external aqueous phase, and in the proximity of the photosensitizers to the tryptophan. In the course of this study, we developed the kinetic equations that have to be employed when the sensitizer itself is being destroyed.     

Plasma kinetics and matrix residues of deoxynivalenol (DON) and zearalenone (ZEN) are altered in endotoxaemic pigs independent of LPS entry site

This study aimed to investigate a potential modulatory effect of E. coli lipopolysaccharide (LPS) on the kinetics of deoxynivalenol (DON) and zearalenone (ZEN) after pre- or post-hepatic LPS administration to unravel the putative role of the liver. Fifteen barrows were fed a diet containing mycotoxin-contaminated maize (4.59 mg DON/kg feed, 0.22 mg ZEN/kg feed) for 29 days and equipped with pre-hepatic catheters (portal vein, “po”) and post-hepatic catheters (jugular vein, “ju”), facilitating simultaneous infusion of LPS (“LPS group”, 7.5 μg/kg body weight) or 0.9% sterile NaCl solution (control, “CON group”, equivolumar to LPS group) and blood sampling. This resulted in three infusion groups, depending on infusion site: CON_ju-CON_po, CON_ju-LPS_po, and LPS_ju-CON_po. On day 29, pigs were fed their morning ration (700 g/pig) (?15 min), and blood samples were collected at regular intervals relative to infusion start. At 195 min, pigs were sacrificed and bile, urine, liquor, and liver samples collected. DON concentrations in jugular and portal blood decreased in both LPS-infused groups, whereas the ZEN concentrations increased, regardless of the treatment site. In liver tissue, a decrease of both toxin concentrations was observed in endotoxaemic pigs as well as a drop in hepatic conjugation, regardless of LPS entry site. In contrast to our hypothesis, DON and ZEN were not differently altered depending on the LPS-entry site. Neither the absorption nor the accumulation of DON and ZEN in different tissues differed significantly between animals which were infused with LPS via either the jugular or portal vein.     

Two interferons alpha influence each other during their interaction with the extracellular domain of human type interferon receptor subunit 2

The interaction between two human interferons alpha (IFN-alphas) and the extracellular (EC) domain of human type I IFN receptor subunit 2 (IFNAR2) was analyzed. Previous experiments using Daudi cells showed that IFN-alpha21b and some IFN-alpha hybrids (made from IFN-alpha2c and 21b) competed poorly for the IFN-alpha2b binding site. This study examined the causes of the poor competition between these IFN-alphas. IFN-alpha2c and the IFN hybrid CM3 {IFN-alpha21b(1-75)(81-95)/IFN-alpha2c(76-80) (96-166), Y86K} were selected for this study based on their cell binding and biological properties. Competitive binding ELISA, native electrophoresis followed by Western blot, electrospray ionization mass spectrometry (ESI-MS), surface plasmon resonance biosensor (SPR) analysis, as well as neutralization of antiproliferative activities on Daudi cells in the presence of soluble IFNAR2-EC show evidence that each of the described IFN-alpha subtypes affected the binding of the other IFN-alpha to IFNAR2-EC by affecting the stability of the complex, i.e., dissociation of the complex. Moreover, native electrophoresis with different IFNAR2-EC mutants showed that IFN-alpha2c and CM3 utilize different amino acids in the binding domain of IFNAR2-EC. In addition to that, analytical ultracentrifugation (AUC) revealed differences in the oligomeric state of the two studied interferons. Our results demonstrated that two individual IFN-alphas interact differentially with IFNAR2-EC and influence each other during this interaction. This study contributes to the understanding of the mutual interaction between multiple IFN-alpha subtypes during the competition for binding to the receptor.     

Projections of the Current and Future Disease Burden of Hepatitis C Virus Infection in Malaysia

Background

The prevalence of hepatitis C virus (HCV) infection in Malaysia has been estimated at 2.5% of the adult population. Our objective, satisfying one of the directives of the WHO Framework for Global Action on Viral Hepatitis, was to forecast the HCV disease burden in Malaysia using modelling methods.

Methods

An age-structured multi-state Markov model was developed to simulate the natural history of HCV infection. We tested three historical incidence scenarios that would give rise to the estimated prevalence in 2009, and calculated the incidence of cirrhosis, end-stage liver disease, and death, and disability-adjusted life-years (DALYs) under each scenario, to the year 2039. In the baseline scenario, current antiviral treatment levels were extended from 2014 to the end of the simulation period. To estimate the disease burden averted under current sustained virological response rates and treatment levels, the baseline scenario was compared to a counterfactual scenario in which no past or future treatment is assumed.

Results

In the baseline scenario, the projected disease burden for the year 2039 is 94,900 DALYs/year (95% credible interval (CrI): 77,100 to 124,500), with 2,002 (95% CrI: 1340 to 3040) and 540 (95% CrI: 251 to 1,030) individuals predicted to develop decompensated cirrhosis and hepatocellular carcinoma, respectively, in that year. Although current treatment practice is estimated to avert a cumulative total of 2,200 deaths from DC or HCC, a cumulative total of 63,900 HCV-related deaths is projected by 2039.

Conclusions

The HCV-related disease burden is already high and is forecast to rise steeply over the coming decades under current levels of antiviral treatment. Increased governmental resources to improve HCV screening and treatment rates and to reduce transmission are essential to address the high projected HCV disease burden in Malaysia.     

Surface plasmon resonance biosensor for direct detection of antibody against Epstein-Barr virus

This paper describes the direct label-free detection of antibodies against the Epstein-Barr virus (anti-EBNA) using a surface plasmon resonance (SPR) biosensor. The antibody detection was performed using the immunoreaction between anti-EBNA and a respective synthetic peptide (EBNA-1), which was conjugated with bovine serum albumin (BSA-EBNA) and immobilized on the sensor surface. Three immobilization chemistries for the attachment of BSA-EBNA were investigated to optimize ligand density and minimize loss of EBNA-1 immunoreactivity. The developed SPR biosensor functionalized with the optimal immobilization method was calibrated and characterized in terms of detection limit, reproducibility, regenerability and storability. It was demonstrated that the sensor is capable of detecting concentrations of anti-EBNA as low as 0.2 ng/ml (approximately 1 pM) both in buffer and 1% human serum and can be stored and regenerated for repeated use.     

Octyl gallate has potent anti-inflammasome activity by directly binding to NLRP3 LRR domain

The NOD-, LRR-, and Pyrin domain-containing protein 3 (NLRP3) inflammasome plays key roles in regulating inflammation. Numerous studies show that the abnormal activation of NLRP3 associates with the initiation and progression of various diseases. Hence, the NLRP3 inflammasome may be a promising therapeutic target for these diseases. Octyl gallate (OG) is a small molecule with antioxidant, antimicrobial, antifungal, and anti-inflammatory activities; however, the mechanism underlying its anti-inflammatory activity is still unclear. Here, we developed a screening system for NLRP3-inflammasome inhibitors. A total of 3287 small molecules were screened for inhibitors of nigericin-induced NLRP3 oligomerization. OG was identified as a novel inhibitor. We show that OG directly targets the LRR domain of NLRP3 and thereby blocks the inflammatory cascade of the NLRP3 inflammasome. This contrasts with the mode-of-action of other direct NLRP3 inhibitors, which all bind to the NACHT domain of NLRP3. Interestingly, OG also inhibits the priming step by downregulating the Raf-MEK1/2-ERK1/2 axis. Thus, OG inhibits the NLRP3 inflammasome by two distinct mechanisms. Importantly, OG injection ameliorated the inflammation in mouse models of foot gout and sepsis. Our study identifies OG as a potential therapeutic agent for NLRP3-associated diseases.     

Identification of a Novel Afipia Species Isolated from an Indian Flying Fox

An old world fruit bat Pteropus giganteus, held in captivity and suffering from necrosis of its wing digits, failed to respond to antibiotic therapy and succumbed to the infection. Samples submitted to the National Centre for Foreign Animal Disease were tested for viral infection. Vero E6 cells exhibited minor but unique cytopathic effects on second blind passage, and full CPE by passage four. Utilizing an unbiased random amplification technique from cell culture supernatant, we identified a bacterium belonging to the Bradyrhizobiaceae. Purification of cell culture supernatant on TY media revealed a slow growing bacterial isolate. In this study using electron microscopy, 16S rRNA gene analysis and whole genome sequencing, we identify a novel bacterial species associated with the site of infection belonging to the genus Afipia. This genus of bacteria is very diverse, with only a limited number of species characterized. Afipia felis, previously described as the etiological agent to cause cat scratch disease, and Afipia septicemium, most recently shown to cause disease in humans, highlight the potential for members of this genus to form a branch of opportunistic pathogens within the Bradyrhizobiaceae. Increased utilization of next generation sequencing and genomics will aid in classifying additional members of this intriguing bacterial genera.