Secukinumab for patients failing previous tumour necrosis factor-α inhibitor therapy: results of a randomized open-label study (SIGNATURE).

BACKGROUND: Efficacy data on therapies for patients with psoriasis who have failed tumour necrosis factor (TNF)-α inhibitor therapy is limited. OBJECTIVES: To determine the effectiveness and tolerability of secukinumab, an interleukin (IL)-17A inhibitor, in patients with moderate/severe chronic plaque psoriasis with documented efficacy failure of TNF-α inhibitor therapy (SIGNATURE study). METHODS: This was a randomized, open-label, noncomparator study in 53 dermatology centres in the U.K. and Republic of Ireland. Patients were randomized 1 : 1 to receive secukinumab 300 mg or 150 mg subcutaneously every week for 4 weeks, then 4-weekly thereafter. Patients were stratified by their prior efficacy failure with TNF-α inhibitors. Only patients who started and stayed on the same dose at each time point were included for efficacy assessments. RESULTS: In total, 233 patients were analysed. The primary end point was met, with a statistically significant improvement in response rates [75% reduction in Psoriasis Area and Severity Index (PASI 75)] from baseline to week 16 in both secukinumab 300 mg and 150 mg dose groups [77 of 118 patients (65·3%) and 51 of 115 patients (44·3%), respectively; P < 0·0001]. After 72 weeks, in patients starting and remaining on 300 mg, 77% (54 of 70) achieved PASI 75. Improvements in Dermatology Life Quality Index from baseline to week 16 occurred and were maintained up to 72 weeks. The safety profile was generally consistent with previous secukinumab studies, although a higher incidence of some adverse events (e.g. candida infections) was observed. CONCLUSIONS: This study provides evidence of efficacy and safety of secukinumab for treatment of patients with psoriasis who failed prior TNF-α inhibitor therapy. This study represents a 'real-world' population, providing reassurance that secukinumab is a treatment option in this difficult-to-treat population. What's already known about this topic? Conventional systemic nonbiological and tumour necrosis factor (TNF)-α inhibitor therapies for plaque psoriasis have not fully met patients' needs. There is a lack of data to support the treatment pathways for patients with psoriasis who have inadequate responses to TNF-α inhibitor therapy. Secukinumab, a recombinant high-affinity fully human monoclonal anti-human interleukin-17A antibody of the IgG1/κ-class, has shown excellent safety and efficacy in the treatment of moderate-to-severe psoriasis. What does this study add? This is the first study evaluating treatment with biologics after prior efficacy failure of TNF-α inhibitor therapy as defined by the U.K. National Institute for Health and Care Excellence criteria. Secukinumab is an effective treatment in this difficult-to-treat patient population. This study provides important practical information for clinicians managing psoriasis. Adverse events were consistent with the phase III programme for secukinumab, although some adverse events, e.g. candida, were increased.

Stability of Therapeutic Enzymes: Challenges and Recent Advances.

Enzymes are biocatalysts that have found profound applications in the current biotherapeutic industry and play a crucial role in diagnosis, prevention, and biochemical analysis of major diseases. However, stability, protein degradation and immunogenicity in the body present unique challenges that are faced upon sustained use of such enzymes. The present chapter is an attempt to dissect the state-of-the-art in relation to the challenges of development of therapeutic enzymes and the recent advances to address them. At the very outset, diseases where enzymes have found effective applications and the various causes of enzyme instability have been discussed. In recent times, polymer or nano- conjugated resistant delivery methods, as well as mutagenesis have led to manifold increase in enzyme stability against thermal denaturation, acidic gut environment, proteolysis and immunogenicity. Further, methods of analytical characterization of proteins have been highlighted and explored to shape future research directions.

Efficacy of ureolytic Enterobacter cloacae EMB19 mediated calcite precipitation in remediation of Zn (II).

In this study, urease mediated calcite precipitation technique was used for remediation of Zn (II). A urease positive Enterobacter cloacae EMB19 was used to produce calcite impregnated with Zn ions. In co-presence of Ca (II), Zn (II) concentrations of 10 and 20 mg L-1 were completely remediated by the bacterium from the media at 72 and 96 h of incubation, respectively. Co-precipitation of Ca (II) and Zn (II) to form calcite-Zn precipitate is one of the major mechanisms of Zn remediation in the present study. Role of urease in calcite-Zn precipitation was substantiated by using urease/carbonate and ammonium enriched cell free culture supernatant (CFS) obtained after sufficient microbial growth. Using CFS, 68% removal of initial 50 mg L-1 Zn (II) was detected. Energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction pattern (XRD) of the precipitate supports calcite mediated remediation of Zn. Remediation of multiple metals viz. Cd, Zn, Cu was also analyzed using CFS laden with urease. The preparation showed 40% Cd, 23% Zn, and 8% Cu reduction from the solution containing initial 25 mg L-1 of each metal. Overall, it can be concluded that, the E. cloacae mediated calcite precipitation technique could effectively be used for alleviation of Zn (II) and other heavy metals from the contaminated environment.

Harnessing the bio-mineralization ability of urease producing Serratia marcescens and Enterobacter cloacae EMB19 for remediation of heavy metal cadmium (II).

In the present study, urease positive Serratia marcescens (NCIM2919) and Enterobacter cloacae EMB19 (MTCC10649) were individually evaluated for remediation of cadmium (II) using ureolysis-induced calcium carbonate precipitation. Both the cultures were observed to efficiently remove cadmium from the media through co-precipitation of Cd (II) and Ca (II). S. marcescens and E. cloacae EMB19, respectively showed 96 and 98% removal of initial 5.0 mg L-1 soluble Cd (II) from the urea and CaCl2 laden media at 96 h of incubation period. At higher Cd (II) concentrations of 10 and 15 mg L-1, cadmium removal efficiency was much higher in case of E. cloacae EMB19 compared to S. marcescens. In-vitro cadmium (II) remediation study using urease containing cell-free culture supernatant of S. marcescens and E. cloacae EMB19 showed respective 98 and 53% removal of initial 50 mg L-1 Cd (II) from the reaction mixtures in co-presence of Ca (II). While in sole presence of Cd (II), only 16 and 8% removal of Cd (II) were detected for S. marcescens and E. cloacae EMB19, respectively. The elemental analysis of the co-precipitated mineral products using Energy Dispersive X-ray spectroscopy (EDX) clearly showed the prevalence of Ca and Cd ions. The morphology Cd-Ca composites formed with respect to both the cultures were observed to be of different shape and size as revealed through Scanning Electron Microscopy (SEM). Entire study hence comes out with a sustainable bioremediation option which could be effectively used to tackle Cd (II) or other heavy metal pollution.

Primary Sjogren's syndrome presenting as hypokalemic paralysis: A case series.

Primary Sjögren's syndrome (pSS) primarily involves exocrine glands, and renal tubular acidosis (RTA) is seen in one-third of the cases. RTA with hypokalemic paralysis as a presenting feature of pSS is described in few case reports in literature. We report 13 cases who presented as hypokalemic paralysis, and on evaluation were diagnosed to be pSS, as per the diagnostic criteria laid by the Sjögren's International Collaborative Clinical Alliance (2012). All patients were female, with a mean age at presentation being 33.1 ± 8.22 years (range, 25-48 years). Eleven patients had a complete distal RTA and two patients had incomplete distal RTA at the time of presentation. 62% (8/13) of patients had no signs and symptoms of exocrine gland involvement. All the cases were managed with oral alkali therapy, and six patients received additional immunomodulating agents. No improvement in renal tubular dysfunction (in the form of a reduction in the alkali dose) after immunomodulating therapy was observed over a mean follow-up of 2.8 years. Renal tubular dysfunction can be the presenting manifestation of pSS. It is important to consider the possible presence of this disorder in adults with otherwise unexplained distal RTA or hypokalemia.

2-Pyrrolidone synthesis from γ-aminobutyric acid produced by Lactobacillus brevis under solid-state fermentation utilizing toxic deoiled cottonseed cake.

There is an increasing demand of γ-aminobutyric acid (GABA) as drug and food additive, as well as feedstock to produce 2-pyrrolidone, a precursor for the synthesis of nylon 4. 2-Pyrrolidone is a petrochemical and depleting reserve which raises concern for its bio-based production. The study herein describes bio-based economical GABA production from Lactobacillus brevis by solid-state fermentation (SSF) using toxic deoiled cottonseed cake (CSC) as substrate. In general, the use of cottonseed cake remains restricted due to the presence of toxic gossypols. Thus, simultaneous detoxification observed during fermentation also widens the scope of utilization of this residual seedcake for feed use vis-a-vis production of other value added chemicals. The SSF conditions were optimized for maximum GABA production, viz., 19.7 mg/g, CSC of GABA was obtained at 6th day of fermentation with 70 % degradation of gossypols simultaneously. The potential of this bio-based GABA as a platform chemical is demonstrated in the synthesis of 2-pyrrolidone. Thus, a simple and cost-effective strategy for utilizing toxic biomass has been developed as an alternate to chemical synthetic route.

Structural elucidation and molecular characterization of Marinobacter sp. α-amylase.

Halophiles have been perceived as potential source of novel enzymes in recent years. The interest emanates from their ability to catalyze efficiently under high salt and organic solvents. Marinobacter sp. EMB8 α-amylase was found to be active and stable in salt and organic solvents. A study was carried out using circular dichroism (CD), fluorescence spectroscopy, and bioinformatics analysis of similar protein sequence to ascertain molecular basis of salt and solvent adaptability of α-amylase. Structural changes recorded in the presence of varying amounts of NaCl exhibited an increase in negative ellipticity as a function of salt, confirming that salt stabilizes the protein and increases the secondary structure, making it catalytically functional. The data of intrinsic and extrinsic fluorescence (using 1-anilinonaphthalene 8-sulfonate [ANS] as probe) further confirmed the role of salt. The α-amylase was active in the presence of nonpolar solvents, namely, hexane and decane, but inactivated by ethanol. The decrease in the activity was correlated with the loss of tertiary structure in the presence of ethanol. Guanidine hydrochloride and pH denaturation indicated the molten globule state at pH 4.0. Partial N-terminal amino acid sequence of the purified α-amylase revealed the relatedness to Pseudoalteromonas sp. α-amylase. "FVHLFEW" was found as the N-terminal signature sequence. Bioinformatics analysis was done using M. algicola α-amylase protein having the same N-terminal signature sequence. The three-dimensional structure of Marinobacter α-amylase was deduced using the I-TASSER server, which reflected the enrichment of acidic amino acids on the surface, imparting the stability in the presence of salt. Our study clearly indicate that salt is necessary for maintaining the secondary and tertiary structure of halophilic protein, which is a necessary prerequisite for catalysis.

Identification of performance indicators across a network of clinical cancer programs.

BACKGROUND: Cancer quality indicators have previously been described for a single tumour site or a single treatment modality, or according to distinct data sources. Our objective was to identify cancer quality indicators across all treatment modalities specific to breast, prostate, colorectal, and lung cancer. METHODS: Candidate indicators for each tumour site were extracted from the relevant literature and rated in a modified Delphi approach by multidisciplinary groups of expert clinicians from 3 clinical cancer programs. All rating rounds were conducted by e-mail, except for one that was conducted as a face-to-face expert panel meeting, thus modifying the original Delphi technique. Four high-level indicators were chosen for immediate data collection. A list of confounding variables was also constructed in a separate literature review. RESULTS: A total of 156 candidate indicators were identified for breast cancer, 68 for colorectal cancer, 40 for lung cancer, and 43 for prostate cancer. Iterative rounds of ratings led to a final list of 20 evidence- and consensus-based indicators each for colorectal and lung cancer, and 19 each for breast and prostate cancer. Approximately 30 clinicians participated in the selection of the breast, lung, and prostate indicators; approximately 50 clinicians participated in the selection of the colorectal indicators. CONCLUSIONS: The modified Delphi approach that incorporates an in-person meeting of expert clinicians is an effective and efficient method for performance indicator selection and offers the added benefit of optimal clinician engagement. The finalized indicator lists for each tumour site, together with salient confounding variables, can be directly adopted (or adapted) for deployment within a performance improvement program.

Persistence of Corynebacterium diphtheriae in Delhi & National Capital Region (NCR).

Despite the introduction of mass immunization, diphtheria continues to play a major role as a potentially lethal infectious disease in many countries. Delay in the specific therapy of diphtheria may result in death and, therefore, accurate diagnosis of diphtheria is imperative. This study was carried out at National Centre for Disease Control (NCDC), Delhi, India, on samples of suspected diphtheria cases referred from various government hospitals of Delhi and neighbouring areas during 2012-2014. Primary identification of Corynebacterium diphtheriae was done by standard culture, staining and biochemical tests followed by toxigenicity testing by Elek's test on samples positive for C. diphtheriae. The results showed persistence of toxigenic C. diphtheriae in our community indicating the possibility of inadequate immunization coverage.

Immobilization of halophilic Bacillus sp. EMB9 protease on functionalized silica nanoparticles and application in whey protein hydrolysis.

The present work targets the fabrication of an active, stable, reusable enzyme preparation using functionalized silica nanoparticles as an effective enzyme support for crude halophilic Bacillus sp. EMB9 protease. The immobilization efficiency under optimized conditions was 60%. Characterization of the immobilized preparation revealed marked increase in pH and thermal stability. It retained 80% of its original activity at 70 °C while t 1/2 at 50 °C showed a five-fold enhancement over that for the free protease. Kinetic constants K m and V max were indicative of a higher reaction velocity along with decreased affinity for substrate. The preparation could be efficiently reused up to 6 times and successfully hydrolysed whey proteins with high degree of hydrolysis. Immobilization of a crude halophilic protease on a nanobased scaffold makes the process cost effective and simple.

Effect of organic solvents on the structure and activity of moderately halophilic Bacillus sp. EMB9 protease.

Halophilic enzymes have been manifested for their stability and catalytic abilities under harsh operational conditions. These have been documented to withstand denaturation in presence of high temperature, pH, presence of organic solvents and chaotropic agents. The present study aims at understanding the stability and activity of a halophilic Bacillus sp. EMB9 protease in organic solvents. The protease was uniquely stable in polar solvents. A clear correlation was evident between the protease function and conformational transitions, validated by CD and fluorescence spectral studies. The study affirms that preservation of protein structure, possibly due to charge screening of the protein surface by Ca(2+) and Na(+) ions provides stability against organic solvents and averts denaturation. Salt was also found to exert a protective effect on dialyzed protease against chaotropism of solvents. Presence of 1 % (w/v) NaCl restored the activity in the dialyzed protease and prevented denaturation in methanol, toluene and n-decane. The work will have further implication on discerning protein folding in saline as well as non-aqueous environments.

Efficient proteolysis and application of an alkaline protease from halophilic Bacillus sp. EMB9.

A salt-stable alkaline protease from moderately halophilic Bacillus sp. EMB9, isolated from the western coast of India, is described. This protease was capable of efficiently removing silver from used/waste X-Ray films, as well as hydrolyzing defatted soy flour with 31% degree of hydrolysis (DH). Production of the protease was optimized by using response surface methodology. Ca(2+) and NaCl were the most critical factors in enhancing the yield. Under optimized culture conditions, a maximum of 369 U protease/mL was obtained, which is quite comparable to the yields of commercial proteases. The elevated production level coupled with ability to efficiently hydrolyze protein-laden soy flour and complete recovery of silver from used X-Ray films makes it a prospective industrial enzyme.

Genetic analysis of precore/core and partial pol genes in an unprecedented outbreak of fulminant hepatitis B in India.

We investigated an unprecedented outbreak of fulminant hepatitis B virus (HBV) that occurred in Modasa, Gujarat (India) in 2009. Genomic analysis of all fulminant hepatic failure cases confirmed exclusive predominance of subgenotype D1. A1762T, G1764A basal core promoter (BCP) mutations, insertion of isoleucine after nt 1843, stop codon mutation G1896A, G1862T transversion plus seven other mutations in the core gene caused inhibition of HBeAg expression implicating them as circulating precore/BCP mutant virus. Two rare mutations at amino acids 89 (Ile→Ala) and 119 (Leu→Ser) in addition to other mutations in the polymerase (pol) gene may have caused some alteration in either of four pol gene domains to affect encapsidation of pregenomic RNA to enhance pathogenicity. Sequence similarity among patients' sequences suggested an involvement of a single hepatitis B mutant strain/source to corroborate the finding of gross and continued usage of HBV mutant-contaminated syringes/needles by a physician which resulted in this unprecedented outbreak of fulminant hepatitis B. The fulminant exacerbation of the disease might be attributed to mutations in the BCP/precore/core and pol genes that may have occurred due to selection pressure during rapid spread/mutation of the virus.

rmpM gene as a genetic marker for human bacterial meningitis.

Meningitis is a bacterial, viral or fungal infection of the protective membrane meninges covering the brain and spinal cord. Viral and other forms of meningitis are mild and get cured within one or two week without any treatment. Whereas, bacterial meningitis can prove lethal if not being diagnosed or treated in time. Meningitis is a contagious infection and can spread from one person to another through coughing, sneezing or close contact. Usually the disease is diagnosed from cerebrospinal fluid (CSF) of the patients using culture, PCR, immunological and biochemical tests. All these methods suffer from one or more limitations. Our lab has developed a quick PCR based detection of Neisseria meningitidis (bacterial meningitis) directly from the patient CSF samples using specific primers of virulent rmpM gene. The overall analysis completes in 80 min for confirmation of the disease. Amplicon of 308 bp of rmpM gene does not show homology with other organisms and can be used as a genetic marker for human bacterial meningitis caused by Neisseria meningitidis.

speB gene as a specific genetic marker for early detection of rheumatic heart disease in human.

Streptococcus pyrogenic exotoxin B gene (speB) is chromosomally encoded pyrogenic and cardiotoxic virulence factor of S. pyogenes. Exotoxin B is produced only in a secreted form, as a 40 KD proprotein, which is subsequently processed to 28 KD in the mature form. Streptococcus pyogenes infection in human, causes initially pharyngitis due to inhalation of aerosols emitted by infected persons, develops rheumatic fever which leads to the rheumatic heart disease (damage of heart valves). The available detection methods are bacterial culture, ß-hemolysis, bacitracin sensitivity, hippurate test, phadebact test, CRP (C-reactive protein), ESR and PCR. All these methods are either expensive or non-confirmatory and have some limitations. Available PCR methods take more time and require other test to confirm the disease. Our PCR based detection of Streptococcus pyogenes in human using specific primers of speB gene completes overall analysis in 80 min which is the minimum time reported so far for the confirmation of the disease. Amplicon of 423bp of speB gene can be used as a specific genetic marker as it does not show homology with other organisms for early detection of rheumatic heart disease. Our method is specific virulence gene based which is quick, economical and more sensitive as compared with other methods.

Amplified fragment length polymorphism reveals specific epigenetic distinctions between Mycobacterium avium subspecies paratuberculosis isolates of various isolation types.

Amplified fragment length polymorphism (AFLP) was employed as a genetic analysis tool for the study of the genetic relatedness of Mycobacterium avium subsp. paratuberculosis isolates harvested from bovine fecal samples and from bovine or human tissues. This analysis revealed genetic differences between these two isolate types that were confirmed through cluster analysis. Dendrogram analysis separated these two isolate types based on the isolation scheme (tissue-associated versus fecal M. avium subsp. paratuberculosis isolates). Further sequence analysis of unique genetic regions from each isolation type revealed no genetic sequence differences. However, Clustal DNA alignments identified AFLP restriction enzyme sites that were undigested in the tissue-associated isolates. AFLP analysis also disclosed that the same AFLP restriction sites were digested in all of the fecal isolates. Sequence analysis further revealed a consensus sequence upstream of the undigested restriction sites for possible methyltransferase recognition in the tissue-associated M. avium subsp. paratuberculosis isolates.

sof gene as a specific genetic marker for detection of Streptococcus pyogenes causing pharyngitis and rheumatic heart disease.

Streptococcus pyogenes is a human pathogen causing invasive and non—invasive diseases, as well as severe sequels, such as rheumatic fever. Rheumatic heart disease is a sequel of rheumatic fever results from an untreated strep throat causing damage of the heart valves. The usual detection methods of strep throat are culture, virulent test, antibiotic sensitivity, CRP, ESR and PCR. These methods are expensive, time consuming and have some limitations. All reported PCR methods are based on either 16S rRNA or specific gene based along with other methods to confirm the disease in more than 1h. Here, we have developed a PCR based diagnosis of streptococcus pyogenes using specific primers of virulent sof gene (serum opacity factor) of S. pyogenes. Our method is an improvement of the existing methods and the overall analysis completes in 1 h which is the least time reported so far for the confirmation of the disease. Amplicon of 228 bp of sof gene does not show homology with other organisms and can be used as genetic marker for S. pyogenes.

Stability of haloalkaliphilic Geomicrobium sp. protease modulated by salt.

A novel protease from the halophilic bacterium Geomicrobium sp. EMB2 (MTCC 10310) is described. The activity of the protease was modulated by salt, and it exhibited remarkable stability in organic solvents, at alkaline pH, and in other denaturing conditions. The structural changes under various denaturing conditions were analyzed by measurements of intrinsic fluorescence and circular dichroism spectroscopy. Circular dichroism showed that the secondary structure of the protease was predominantly α-helical but unfolded in salt-free medium. The structure is regained by inclusion of NaCl in the range of 2-5%. The presence of NaCl exerted a protective effect against thermal, organic solvent, and guanidine hydrochloride denaturation by preventing unfolding.

Spontaneous inflammatory arthritis in HLA-B27 transgenic mice lacking beta 2-microglobulin: a model of human spondyloarthropathies.

Human class I major histocompatibility complex allele HLA-B27 is associated with a group of human diseases called "spondyloarthropathies." Studies on transgenic rats expressing HLA-B27 and human beta 2-microglobulin have confirmed the role of HLA-B27 in disease pathogenesis. Here we report spontaneous inflammatory arthritis in HLA-B27 transgenic mice lacking beta 2-microglobulin (B27+ beta 2m-/-). In the absence of beta 2-microglobulin, B27+ beta 2m-/- animals do not express the HLA-B27 transgene on the cell surface and have a very low level of CD8+ T cells. Most of the B27+ beta 2m-/- male mice showed nail changes, hair loss, and swelling in paws, which leads to ankylosis. The symptoms occur only after the B27+ beta 2m-/- mice are transferred from the specific pathogen-free mouse colony. These results suggest that aberrant assembly, transport, and expression of the HLA-B27 molecule may predispose an individual for development of the disease when exposed to an appropriate environmental trigger.