Protein stabilization through phage display

RNase S consists of two proteolytic fragments of RNase A, residues 1-20 (S20) and residues 21-124 (S pro). A 15-mer peptide (S15p) with high affinity for S pro was selected from a phage display library. Peptide residues that are buried in the structure of the wild type complex are conserved in S15p though there are several changes at other positions. Isothermal titration calorimetry studies show that the affinity of S15p is comparable to that of the wild type peptide at 25 degrees C. However, the magnitudes of DeltaH(o) and DeltaC(p) are lower for S15p, suggesting that the thermal stability of the complex is enhanced. In agreement with this prediction, at pH 6, the T(m) of the S15p complex was found to be 10 degrees C higher than that of the wild type complex. This suggests that for proteins where fragment complementation systems exist, phage display can be used to find mutations that increase protein thermal stability.     

Virological Characteristics of Acute Hepatitis B in Eastern India: Critical Differences with Chronic Infection

Hepatitis B Virus (HBV) manifests high genetic variability and is classifiable into ten genotypes (A-J). HBV infection can lead to variable clinical outcomes, ranging from self-limiting acute hepatitis to active chronic hepatitis, cirrhosis and hepatocellular carcinoma. The present study characterizes HBV strains circulating among patients with acute (AHB) and chronic HBV infection (CHB). Among a total of 653 HBsAg positive cases, 40 manifested acute infection. After sequencing the surface(S), basal core promoter/pre-core(BCP/PC) and the X gene regions, phylogenetic tree was constructed using MEGA4 by neighbor-joining method. Statistical robustness was established with bootstrap analysis. Nucleotide diversity was determined by Shannon entropy per site using the Entropy program of the Los Alamos National Laboratories. Analyses of acute patients revealed that HBV/D2 is the major circulating sub-genotype and commonly associated with sexual promiscuity and the age group between15-30 years. Comparison of AHB and CHB patients revealed that HBeAg positivity, ALT levels and genotype D were significantly high in AHB, whereas CHB patients were predominantly male, had a high viral load, and were commonly associated with genotype C. The frequencies of mutations in the S, BCP/PC, and X gene were low in AHB as compared to CHB. Drug resistant mutations were not detectable in the polymerase gene of AHB. Average nucleotide diversity in AHB was considerably low as compared to CHB. Further, the highest average ΔH (average difference in entropy between chronic and acute infection) was observed in the BCP/PC region implying that this region was most vulnerable to mutations upon HBV persistence, especially in case of genotype C. Additionally, among all substitutions, the A1762T and G1764A BCP mutations were the strongest indicators of chronicity. In conclusion, the study exhibits a general portrait of HBV strains circulating among acute hepatitis B patients in Eastern India and their intricate differences with chronic patients which should be useful from the clinical point of view.     

Discovery of a novel class of biphenyl pyrazole sodium channel blockers for treatment of neuropathic pain

A series of novel biphenyl pyrazole dicarboxamides were identified as potential sodium channel blockers for treatment of neuropathic pain. Compound 20 had outstanding efficacy in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain.     

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.     

Design and synthesis of piperazine-indole p38α MAP kinase inhibitors with improved pharmacokinetic profiles

Derivatives of the 4-fluorobenzyl dimethylpiperazine-indole class of p38α MAP kinase inhibitors are described. Biological evaluation of these compounds focused on maintaining activity while improving pharmacokinetic (PK) properties. Improved properties were observed for structures bearing substitutions on the benzylic methylene.     

The chemokine receptor CXCR3 attenuates the control of chronic Mycobacterium tuberculosis infection in BALB/c mice

The chemokine receptor CXCR3 plays a significant role in regulating the migration of Th1 cells. Given the importance of Th1 immunity in the control of tuberculous infection, the results of the present study demonstrating that CXCR3-deficient BALB/c mice are more resistant to Mycobacterium tuberculosis, compared with wild-type mice, is surprising. This enhanced resistance manifests in the chronic but not the acute phase of infection. Remarkable differences in the cellular composition of the pulmonic granuloma of the CXCR3(-/-) and wild-type mice were found, the most striking being the increase in the number of CD4(+) T cells in the knockout strain. In the chronic phase of infection, the number of CD69-expressing CD4(+) T lymphocytes in the lungs of CXCR3(-/-) mice was higher than in wild-type mice. Additionally, at 1 mo postinfection, the number of IFN-gamma-producing CD4(+) T cells in the lungs and mediastinal lymph nodes of the CXCR3-deficient strain was elevated compared with wild-type mice. Pulmonic expression of IFN-gamma, IL-12, TNF-alpha, or NO synthase 2, the principal antimycobacterial factors, were equivalent in the two mouse strains. These results indicate that: 1) CXCR3 plays a role in modulating the cellular composition of tuberculous granuloma; 2) CXCR3 impairs antimycobacterial activity in chronic tuberculosis; and 3) in the absence of CXCR3, mice exhibit a heightened state of CD4(+) T lymphocyte activation in the chronic phase of infection that is associated with enhanced CD4(+) T cell priming. Therefore, CXCR3 can attenuate the host immune response to M. tuberculosis by adversely affecting T cell priming.     

RECOVIR: An application package to automatically identify some single stranded RNA viruses using capsid protein residues that uniquely distinguish among these viruses

Background  

Most single stranded RNA (ssRNA) viruses mutate rapidly to generate large number of strains having highly divergent capsid sequences. Accurate strain recognition in uncharacterized target capsid sequences is essential for epidemiology, diagnostics, and vaccine development. Strain recognition based on similarity scores between target sequences and sequences of homology matched reference strains is often time consuming and ambiguous. This is especially true if only partial target sequences are available or if different ssRNA virus families are jointly analyzed. In such cases, knowledge of residues that uniquely distinguish among known reference strains is critical for rapid and unambiguous strain identification. Conventional sequence comparisons are unable to identify such capsid residues due to high sequence divergence among the ssRNA virus reference strains. Consequently, automated general methods to reliably identify strains using strain distinguishing residues are not currently available.     

The C‐terminal calcium‐sensitive disordered motifs regulate isoform‐specific polymerization characteristics of calsequestrin

Calsequestrin (CASQ) exists as two distinct isoforms CASQ1 and CASQ2 in all vertebrates. Although the isoforms exhibit unique functional characteristic, the structural basis for the same is yet to be fully defined. Interestingly, the C‐terminal region of the two isoforms exhibit significant differences both in length and amino acid composition; forming Dn‐motif and DEXn‐motif in CASQ1 and CASQ2, respectively. Here, we investigated if the unique C‐terminal motifs possess Ca²⁺‐sensitivity and affect protein function. Sequence analysis shows that both the Dn‐ and DEXn‐motifs are intrinsically disordered regions (IDRs) of the protein, a feature that is conserved from fish to man. Using purified synthetic peptides, we show that these motifs undergo distinctive Ca²⁺‐mediated folding suggesting that these disordered motifs are Ca²⁺‐sensitivity. We generated chimeric proteins by swapping the C‐terminal portions between CASQ1 and CASQ2. Our studies show that the C‐terminal portions do not play significant role in protein folding. An interesting finding of the current study is that the switching of the C‐terminal portion completely reverses the polymerization kinetics. Collectively, these data suggest that these Ca²⁺‐sensitivity IDRs located at the back‐to‐back dimer interface influence isoform‐specific Ca²⁺‐dependent polymerization properties of CASQ. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 15–22, 2015.     

Gene expression changes reflect clinical response in a placebo-controlled randomized trial of abatacept in patients with diffuse cutaneous systemic sclerosis

IntroductionSystemic sclerosis is an autoimmune disease characterized by inflammation and fibrosis of the skin and internal organs. We sought to assess the clinical and molecular effects associated with response to intravenous abatacept in patients with diffuse cutaneous systemic.MethodsAdult diffuse cutaneous systemic sclerosis patients were randomized in a 2:1 double-blinded fashion to receive abatacept or placebo over 24 weeks. Primary outcomes were safety and the change in modified Rodnan Skin Score (mRSS) at week 24 compared with baseline. Improvers were defined as patients with a decrease in mRSS of ≥30 % post-treatment compared to baseline. Skin biopsies were obtained for differential gene expression and pathway enrichment analyses and intrinsic gene expression subset assignment.ResultsTen subjects were randomized to abatacept (n = 7) or placebo (n = 3). Disease duration from first non-Raynaud’s symptom was significantly longer (8.8 ± 3.8 years vs. 2.4 ± 1.6 years, p = 0.004) and median mRSS was higher (30 vs. 22, p = 0.05) in the placebo compared to abatacept group. Adverse events were similar in the two groups. Five out of seven patients (71 %) randomized to abatacept and one out of three patients (33 %) randomized to placebo experienced ≥30 % improvement in skin score. Subjects receiving abatacept showed a trend toward improvement in mRSS at week 24 (−8.6 ± 7.5, p = 0.0625) while those in the placebo group did not (−2.3 ± 15, p = 0.75). After adjusting for disease duration, mRSS significantly improved in the abatacept compared with the placebo group (abatacept vs. placebo mRSS decrease estimate −9.8, 95 % confidence interval −16.7 to −3.0, p = 0.0114). In the abatacept group, the patients in the inflammatory intrinsic subset showed a trend toward greater improvement in skin score at 24 weeks compared with the patients in the normal-like intrinsic subset (−13.5 ± 3.1 vs. −4.5 ± 6.4, p = 0.067). Abatacept resulted in decreased CD28 co-stimulatory gene expression in improvers consistent with its mechanism of action. Improvers mapped to the inflammatory intrinsic subset and showed decreased gene expression in inflammatory pathways, while non-improver and placebos showed stable or reverse gene expression over 24 weeks.ConclusionsClinical improvement following abatacept therapy was associated with modulation of inflammatory pathways in skin.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00442611","term_id":"NCT00442611"}}NCT00442611. Registered 1 March 2007.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0669-3) contains supplementary material, which is available to authorized users.