Isolation and Characterisation of a Recombinant Antibody Fragment That Binds NCAM1-Expressing Intervertebral Disc Cells

Degeneration of the intervertebral discs (IVD) is a leading cause of neck and low back pain. Degeneration begins in the central nucleus pulposus region, leading to loss of IVD osmotic properties. Regeneration approaches include administration of matrix-mimicking scaffolds, cells and/or therapeutic factors. Cell-targeting strategies are likely to improve delivery due to the low cell numbers in the IVD. Single-chain antibody fragments (scFvs) that bind IVD cells were isolated for potential delivery of therapeutics to degenerated IVD. The most cell-distal domain of neural cell adhesion molecule 1 (NCAM1) was cloned and expressed in Escherichia coli. Phage display technology was used to isolate a human scFv against the recombinant domain by panning a scFv library on the immobilised protein. The isolated scFv bound cultured rat astrocytes, as well as bovine nucleus pulposus and annulus fibrosus cells in immunocytochemical studies. The scFv also labelled cells in bovine spinal cord and six-month and two-year old bovine IVD sections by immunohistochemistry. Antibody fragments can provide cell-binding moieties at improved cost, time, yield and functionalisation potential over whole antibodies. The described scFv has potential application in delivery of therapeutics to NCAM1-expressing cells in degenerated IVD.     

A Prospective Epidemiological Study of Acute Mountain Sickness in Nepalese Pilgrims Ascending to High Altitude (4380 m)

Background

Each year, thousands of pilgrims travel to the Janai Purnima festival in Gosainkunda, Nepal (4380 m), ascending rapidly and often without the aid of pharmaceutical prophylaxis.

Methods

During the 2012 Janai Purnima festival, 538 subjects were recruited in Dhunche (1950 m) before ascending to Gosainkunda. Through interviews, subjects provided demographic information, ratings of AMS symptoms (Lake Louise Scores; LLS), ascent profiles, and strategies for prophylaxis.

Results

In the 491 subjects (91% follow-up rate) who were assessed upon arrival at Gosainkunda, the incidence of AMS was 34.0%. AMS was more common in females than in males (RR = 1.57; 95% CI = 1.23, 2.00), and the AMS incidence was greater in subjects >35 years compared to subjects ≤35 years (RR = 1.63; 95% CI = 1.36, 1.95). There was a greater incidence of AMS in subjects who chose to use garlic as a prophylactic compared to those who did not (RR = 1.69; 95% CI = 1.26, 2.28). Although the LLS of brothers had a moderate correlation (intraclass correlation = 0.40, p = 0.023), sibling AMS status was a weak predictor of AMS.

Conclusions

The incidence of AMS upon reaching 4380 m was 34% in a large population of Nepalese pilgrims. Sex, age, and ascent rate were significant factors in the development of AMS, and traditional Nepalese remedies were ineffective in the prevention of AMS.     

Prediction of the Recognition Sites on 16S and 23S rRNAs from E.coli for the Formation of 16S-23S rRNA Complex

Abstract

Interactions between RNA molecules have been postulated to play an important role in the assembly of ribosomes. Using the sequence analysis and the search of continuous complementary regions on 16S rRNA and 23S rRNA, the recognition sites involved in the formation of ribosome of E.coli are postulated. The number of postulated sites was narrowed down by taking available experimental data. The suggestive evidence for correct postulation is obtained from sequence comparison studies of 16S and 23S rRNAs from various species. The sites 891–899 and 1195–1203 on 16S rRNA along with the corresponding complementary sites 1904–1912 and 760–768 on 23S rRNA are predicted to be the most probable candidates for the sites of recognition between 16S and 23S rRNAs. The possibility of the involvement of the additional site 630–638 on 16S rRNA with its complementary site 2031–2039 on 23S rRNA cannot be ruled out.     

Agrobacterium-mediated transformation of promising oil-bearing marine algae Parachlorella kessleri

Parachlorella kessleri is a unicellular alga which grows in fresh as well as marine water and is commercially important as biomass/lipid feedstock and in bioremediation. The present study describes the successful transformation of marine P. kessleri with the help of Agrobacterium tumefaciens. Transformed marine P. kessleri was able to tolerate more than 10 mg l^?1 hygromycin concentration. Co-cultivation conditions were modulated to allow the simultaneous growth of both marine P. kessleri and A. tumefaciens. For co-cultivation, P. kessleri was shifted from Walne’s to tris acetate phosphate medium to reduce the antibiotic requirement during selection. In the present study, the transfer of T-DNA was successful without using acetosyringone. Biochemical and genetic analyses were performed for expression of transgenes by GUS assay and PCR in transformants. Establishment of this protocol would be useful in further genetic modification of oil-bearing Parachlorella species.     

Center Frequency and Bandwidth Reconfigurable Spoof Surface Plasmonic Metamaterial Band-Pass Filter

Plasmonics - In this study, we report a design concept to obtain center frequency and bandwidth reconfigurable spoof surface plasmon polaritons (SSPP) band-pass filter using T-shaped spoof SPP...     

Hydrolysis of soluble starch using Bacillus licheniformis alpha-amylase immobilized on superporous CELBEADS

In the present work, indigenously prepared rigid superporous (pore size of approximately 3 microm) cross-linked cellulose matrix (CELBEADS) has been used as a support for the immobilization of Bacillus licheniformis alpha-amylase (BLA). Optimum pH and temperature, and Michaelis-Menten constants were determined for both free and immobilized BLA. Immobilized BLA was observed to produce a different saccharide profile than free BLA at any value of dextrose equivalent. It was observed that pH, temperature, and initial starch concentration has a significant effect on the saccharide profile of starch hydrolysate produced using immobilized BLA in the batch mode, whereas the ratio of concentration of enzyme units to initial starch concentration has no influence on the same. Hence immobilized BLA can be used as an additional tool for production of maltodextrins with different saccharide profiles. Immobilized BLA has better thermostability than free BLA. Immobilized BLA was found to retain full activity even after eight batches of hydrolysis, each of 8h duration at 55 degrees C and 90 mg/mL initial starch concentration. A semiempirical model has been used for the prediction of saccharide composition of starch hydrolysate with respect to time.     

Exploring the therapeutic potential of staphylococcal phage formulations: Current challenges and applications in phage therapy

Unconstrained consumption of antibiotics throughout the expanse of the 21st century has resulted in increased antimicrobial resistance (AMR) among bacterial pathogens, a transpiring predicament affecting the public healthcare sector. The upsurge of multidrug-resistant pathogens, including Staphylococcus aureus, synchronously with the breakdown of the conventional antibiotic pipeline has led to the exploration of alternate strategies. Phage therapy applications have thus gained immense prominence among the scientific community to conquer this notorious pathogen associated with wide-ranging clinical manifestations, especially in immunosuppressed individuals. In this direction, a plethora of phage formulations like topical solutions, medicated dressings impregnated with phages, liposomal entrapments, etc., have been considered as an effective and upcoming strategy. Owing to the synergistic effect of phages with other antibacterial agents, they can be easily exploited for biomedical application. This review primarily focuses on the therapeutic implications of S. aureus phages in the biotechnological and medical arena. Through this review article, we have also discussed the current status and the incurring challenges in phage therapy.     

Detection of Shiga-like toxin on the outer membranes of Shigella species and Escherichia coli K-12

Abstract Outer membranes of Shigella species and E. coli K-12 carrying large invasive plasmids and isogenic non-invasive strains without plasmids were analyzed by SDS-PAGE. The immunoblotting analysis of the outer membrane proteins of these bacteria was performed with monoclonal antibody (mAb) made against A and B subunits of Shiga-like toxin (SLT). The SLT was detected in the outer membranes of S. dysenteriae 1 IDBM11, S. sonnei PNS20, S. flexneri M90T, S. dysenteriae 60R, and E. coli K-12 strain AB2463. The two other E. coli K-12 strains, C600 and 933J were included as controls for low and high toxin producers respectively. The outer membrane protein band of molecular weight 70 kDa was common to all bacterial strains studied. The most prominent band of 70 kDa protein was seen to be present in the high toxin producing plasmidless strain of S. dysenteriae 60R and the lysogenic strain of E. coli 933J. The invasive strains of S. dysenteriae 1 and S. flexneri M90T which carry the large invasive plasmids showed the least prominent band of 70 kDa protein.
The immunoblotting analysis of Shiga-toxin partially purified from the S. dysenteriae 60R strain revealed the absence of 70 kDa band on SDS-PAGE, instead the two dissociated subunits were seen. Furthermore, periplasmic Shiga-toxin proteins also showed the complete dissociation into A and B subunits. However, under the same denaturing conditions, the 70 kDa protein band cross-reacting with mAb against A and B subunits was still present in the outer membranes of all different strains.     

Essential role for both CD80 and CD86 costimulation, but not CD40 interactions, in allergen-induced Th2 cytokine production from asthmatic bronchial tissue: role for alphabeta, but not gammadelta, T cells

CD80 and CD86 interact with CD28 and deliver costimulatory signals required for T cell activation. We demonstrate that ex vivo allergen stimulation of bronchial biopsy tissue from mild atopic asthmatic, but not atopic nonasthmatic, subjects induced production of IL-5, IL-4, and IL-13. Explants from both study groups did not produce IFN-gamma, but secreted the chemokine RANTES without any overt stimulation. In addition to allergen, stimulation of asthmatic explants with mAbs to CD3 and TCR-alphabeta but not TCR-gammadelta induced IL-5 secretion. Allergen-induced IL-5 and IL-13 production by the asthmatic tissue was inhibited by anti-CD80 and, to a lesser extent, by anti-CD86 mAbs. In contrast, the production of these cytokines by PBMCs was not affected by mAbs to CD80, was inhibited by anti-CD86, and was strongly attenuated in the presence of both Abs. FACS analysis revealed that stimulated asthmatic bronchial tissue was comprised of CD4+ T cells that expressed surface CD28 (75. 3%) but little CTLA-4 (4.0%). Neutralizing mAbs to CD40 ligand had no effect on the cytokine levels produced by asthmatic tissue or PBMCs. Collectively, these findings suggest that allergen-specific alphabeta T cells are resident in asthmatic bronchial tissue and demonstrate that costimulation by both CD80 and CD86 is essential for allergen-induced cytokine production. In contrast, CD86 appears to be the principal costimulatory molecule required in PBMC responses. Attenuation of type 2 alphabeta T cell responses in the bronchial mucosa by blocking these costimulatory molecules may be of therapeutic potential in asthma.