Kamaldeep Gill

Isolation and characterization of novel protein with anti-fungal and anti-inflammatory properties from Aloe vera leaf gel.

The Aloe protein of 14 kDa from the Aloe vera leaf gel was isolated by an ion exchange chromatography using DEAE-cellulose and CM-cellulose column. The purified Aloe protein exhibited a potent anti-fungal activity against Candida paraprilosis, Candida krusei and Candida albicans. In addition, the purified Aloe protein also showed an anti-inflammatory property against pure lipoxygenase and cyclooxygenase-2 with 84% and 73% inhibition, respectively, and was verified by binding with these proteins by real time method by the phenomenon of surface plasmon resonance. This Aloe protein is a novel protein possessing antifungal and anti-inflammatory properties and thus sets a platform to be used as a medicinal plant product.

Sirtuin1: A Promising Serum Protein Marker for Early Detection of Alzheimer’s Disease

Sirtuin (SIRT) pathway has a crucial role in Alzheimer’s disease (AD). The present study evaluated the alterations in serum sirtuin1 (SIRT1) concentration in healthy individuals (young and old) and patients with AD and mild cognitive impairment (MCI). Blood samples were collected from 40 AD and 9 MCI patients as cases and 22 young healthy adults and 22 healthy elderly individuals as controls. Serum SIRT1 was estimated by Surface Plasmon Resonance (SPR), Western Blot and Enzyme Linked Immunosorbent Assay (ELISA). A significant (p<0.0001) decline in SIRT1 concentration was observed in patients with AD (2.27±0.46 ng/µl) and MCI (3.64±0.15 ng/µl) compared to healthy elderly individuals (4.82±0.4 ng/µl). The serum SIRT1 concentration in healthy elderly was also significantly lower (p<0.0001) compared to young healthy controls (8.16±0.87 ng/µl). This study, first of its kind, has demonstrated, decline in serum concentration of SIRT1 in healthy individuals as they age. In patients with AD and MCI the decline was even more pronounced, which provides an opportunity to develop this protein as a predictive marker of AD in early stages with suitable cut off values.

A novel antimicrobial peptide derived from modified N-terminal domain of bovine lactoferrin: design, synthesis, activity against multidrug-resistant bacteria and Candida.

Lactoferrin (LF) is believed to contribute to the hosts defense against microbial infections. This work focuses on the antibacterial and antifungal activities of a designed peptide, L10 (WFRKQLKW) by modifying the first eight N-terminal residues of bovine LF by selective homologous substitution of amino acids on the basis of hydrophobicity, L10 has shown potent antibacterial and antifungal properties against clinically isolated extended spectrum beta lactamases (ESBL), producing gram-negative bacteria as well as Candida strains with minimal inhibitory concentrations (MIC) ranging from 1 to 8 μg/mL and 6.5 μg/mL, respectively. The peptide was found to be least hemolytic at a concentration of 800 μg/mL. Interaction with lipopolysaccharide (LPS) and lipid A (LA) suggests that the peptide targets the membrane of gram-negative bacteria. The membrane interactive nature of the peptide, both antibacterial and antifungal, was further confirmed by visual observations employing electron microscopy. Further analyses, by means of propidium iodide based flow cytometry, also supported the membrane permeabilization of Candida cells. The peptide was also found to possess anti-inflammatory properties, by virtue of its ability to inhibit cyclooxygenase-2 (COX-2). L10 therefore emerges as a potential therapeutic remedial solution for infections caused by ESBL positive, gram-negative bacteria and multidrug-resistant (MDR) fungal strains, on account of its multifunctional activities. This study may open up new approach to develop and design novel antimicrobials.

Antifungal and Antiproliferative Protein from Cicer arietinum: A Bioactive Compound against Emerging Pathogens

The emergence of epidemic fungal pathogenic resistance to current antifungal drugs has increased the interest in developing alternative antibiotics from natural sources. Cicer arietinum is well known for its medicinal properties. The aim of this work was to isolate antimicrobial proteins from Cicer arietinum. An antifungal protein, C-25, was isolated from Cicer arietinum and purified by gel filtration. C-25 protein was tested using agar diffusion method against human pathogenic fungi of ATCC strains and against clinical isolates of Candida krusei, Candida tropicalis, and Candida parapsilosis, and MIC values determined were varied from 1.56 to 12.5 μg/mL. The SEM study demonstrated that C-25 induces the bleb-like surface changes, irregular cell surface, and cell wall disruption of the fungi at different time intervals. Cytotoxic activity was studied on oral cancer cells and normal cells. It also inhibits the growth of fungal strains which are resistant to fluconazole. It reduced the cell proliferation of human oral carcinoma cells at the concentration of 37.5 μg/mL (IC50) and no toxic effect was found on normal human peripheral blood mononuclear cells even at higher concentration of 600 μg/mL. It can be concluded that C-25 can be considered as an effective antimycotic as well as antiproliferative agent against human oral cancer cells.

Quantification of p38αMAP kinase: a prognostic marker in HNSCC with respect to radiation therapy.

BACKGROUND Translation of early findings from basic research is aimed to benefit cancer therapeutics. We report the p38α level in serum of head and neck squamous cell carcinoma (HNSCC) patients indicating it as a prognostic marker and established its correlation with radiation therapy (RT). METHODS The case-controlled study was performed on 120 HNSCC patients from whom 81 patients and 45 controls were statistically analyzed. The p38α estimation was done at pre-RT, during-RT and post-RT using a real time Surface Plasmon Resonance (SPR) technology, ELISA and western blot. RESULTS HNSCC patients showed threefold increase in p38α level when compared to control (p value<0.0001). The estimated concentration of p38α in a temporal manner, before-RT, during-RT and post-RT was 0.61 ng/μl (95%CI: 0.53-0.69), 0.35 ng/μl (95%CI: 0.31-0.38) and 0.30 ng/μl (95%CI: 0.26-0.33), respectively. Among the 81 cases, 70 patients (86.42%) showed a declined p38α in response to RT as evaluated by SPR and were responding clinically (clinical tumor regression). CONCLUSIONS This study showed elevated p38α level at cancer diagnosis and a statistically significant decline during-RT and post-RT periods. Hence, it can emerge as a prognostic marker supporting the candidature of p38α as a suitable serum marker in HNSCC.

Structure Based Design and Synthesis of Peptide Inhibitor of Human LOX-12: In Vitro and In Vivo Analysis of a Novel Therapeutic Agent for Breast Cancer

Human breast cancer cell proliferation involves a complex interaction between growth factors, steroid hormones and peptide hormones. The interaction of growth factors, such as epidermal growth factor (EGF), with their receptors on breast cancer cells can lead to the hydrolysis of phospholipids and release of fatty acid such as arachidonic acid, which can be further metabolized by cyclooxygenase (COX) and lipoxygenase (LOX) pathways to produce prostaglandins. The high concentration of prostaglandins has been associated with chronic inflammatory diseases and several types of human cancers. This is due to the over expression COX, LOX and other inflammatory enzymes. Ten peptides were designed and synthesized by solid phase peptide synthesis and analyzed in vitro for enzyme inhibition. Out of these peptides, YWCS had shown significant inhibitory effects. The dissociation constant (KD) was determined by surface plasmon resonance (SPR) analysis and was found to be 3.39×10−8 M and 8.6×10−8 M for YWCS and baicalein (positive control), respectively. The kinetic constant Ki was 72.45×10−7 M as determined by kinetic assay. The peptide significantly reduced the cell viability of estrogen positive MCF-7 and estrogen negative MDA-MB-231 cell line with the half maximal concentration (IC50) of 75 µM and 400 µM, respectively. The peptide also induced 49.8% and 20.8% apoptosis in breast cancer cells MCF-7 and MDA-MB-231, respectively. The YWCS was also found to be least hemolytic at a concentration of 358 µM. In vivo studies had shown that the peptide significantly inhibits tumor growth in mice (p<0.017). This peptide can be used as a lead compound and complement for ongoing efforts to develop differentiation therapies for breast cancer.

The over expression of cathelicidin peptide LL37 in head and neck squamous cell carcinoma: the peptide marker for the prognosis of cancer.

The over expression of LL37, an antimicrobial peptide belonging~to the cathelicidin family has implication in the progression of human malignancy but the exact role is still not clear. This study aims to elucidate the correlation of LL37 with Head and Neck Squamous Cell Carcinoma (HNSCC) and the consequences of radiotherapy on it. The LL37 levels were quantified in serum samples of control and HNSCC patients at pre-RT, during-RT and post-RT using a real time Surface Plasmon Resonance technology and ELISA. The LL37 of 50 HNSCC patients was significantly (p < 0.0001) threefold higher than the 25 controls and declined with respect to radiation therapy (p < 0.0001) supporting its candidature as a prognostic marker in HNSCC.

Novel synthetic anti-fungal tripeptide effective against Candida krusei

Introduction: Candida species are the major fungal pathogens of humans. Among them, Candida krusei have emerged as a notable pathogen with a spectrum of clinical manifestations and is known to develop resistance against azoles mainly fluconazole. Anti-microbial peptides play important roles in the early mucosal defence against infection and are potent anti-fungal agents since they fight against fungal infection as well as have ability to regulate host immune defence system. The aim of the study was to synthesize a small anti fungal peptide. Materials and Methods: The series of tripeptides were synthesized and screened for antifungal activity against Candida strains according to CLSI guidelines. Toxicity effect of peptide was tested with human erythrocytes. The mode of action of peptide on fungus was resolved by scanning electron microscopy (SEM) studies Results: The tripeptide FAR showed a prominent anti fungal activity among the series. The minimum inhibitory concentration and minimum fungicidal concentration of tripeptide FAR was found to be 171.25 μg/ml and 685 μg/ml, respectively against Candida krusei . The therapeutic index was 2.9. The haemolytic experiment revealed that this peptide is non - toxic to human cells. The SEM studies showed disruption of cell wall and bleb-like surface changes and irregular cell surface. Conclusion: The peptide showed a significant antifungal activity against C. krusei. Thus, it can set a platform for the design of new effective therapeutic agents against C. krusei.

Prognostic Significance of Cyclooxygenase-2 and Response to Chemotherapy in Invasive Ductal Breast Carcinoma Patients by Real Time Surface Plasmon Resonance Analysis

Cyclooxygenase-2 (COX-2), an inducible enzyme, has been implicated in the progression and angiogenesis of breast cancer. The aim of the study is to quantify the concentration of COX-2 and its association with clinico-pathological parameters and response to treatment in patients with invasive ductal carcinoma receiving both neo-adjuvant and adjuvant chemotherapy. The level of COX-2 was estimated using a novel biosensor-based surface plasmon resonance technique in serum of 84 patients with breast cancer (48 patients of neo-adjuvant chemotherapy and 36 patients of adjuvant chemotherapy) and 40 age- and gender-matched normal individuals. A significant increase in COX-2 level was observed in patients compared with normal individuals (p>0.0001). The COX-2 level in serum was found to be significantly higher in patients with lymph node involvement (p<0.0061). 68% (33/48) of the patients receiving neo-adjuvant chemotherapy showed significantly (p<0.0025) reduced COX-2 levels. This study shows significant decrease of COX-2 level in patients with breast cancer treated with both neo-adjuvant and adjuvant chemotherapy. Estimation of COX-2 level in serum may serve as a tumor biomarker in patients with breast cancer.

The Rational Design of Specific Peptide Inhibitor against p38α MAPK at Allosteric-Site: A Therapeutic Modality for HNSCC

p38α is a significant target for drug designing against cancer. The overproduction of p38α MAPK promotes tumorigenesis in head and neck squamous cell carcinoma (HNSCC). The ATP binding and an allosteric site referred as DFG are the key sites of the p38α mitogen activated protein kinase (MAPK) exploited for the design of inhibitors. This study demonstrated design of peptide inhibitor on the basis of allosteric site using Glide molecular docking software and the biochemical analysis of the best modeled peptide. The best fitted tetrapeptide (FWCS) in the allosteric site inhibited the pure recombinant and serum p38α of HNSCC patients by 74 and 72%, respectively. The potency of the peptide was demonstrated by its IC50 (4.6 nM) and KD (3.41×10−10 M) values, determined by ELISA and by surface plasmon resonance (SPR) technology, respectively. The cell viability of oral cancer i.e. KB cell line was reduced in dose dependent manner by 60 and 97% by the treatment of peptide and the IC50 was 600 and 210 µM after 24 and 72 h incubation, respectively. Our result provides an insight for the development of a proficient small peptide as a promising anticancer agent targeting DFG site of p38α kinase.