Biomarkers in renal cell carcinoma and their targeted therapies: a review.

Renal cell carcinoma (RCC) is one of the most life-threatening urinary malignancies displaying poor response to radiotherapy and chemotherapy. Although in the recent past there have been tremendous advancements in using targeted therapies for RCC, despite that it remains the most lethal urogenital cancer with a 5-year survival rate of roughly 76%. Timely diagnosis is still the key to prevent the progression of RCC into metastatic stages as well as to treat it. But due to the lack of definitive and specific diagnostic biomarkers for RCC and its asymptomatic nature in its early stages, it becomes very difficult to diagnose it. Reliable and distinct molecular markers can not only refine the diagnosis but also classifies the tumors into thier sub-types which can escort subsequent management and possible treatment for patients. Potential biomarkers can permit a greater degree of stratification of patients affected by RCC and help tailor novel targeted therapies. The review summarizes the most promising epigenetic [DNA methylation, microRNA (miRNA; miR), and long noncoding RNA (lncRNA)] and protein biomarkers that have been known to be specifically involved in diagnosis, cancer progression, and metastasis of RCC, thereby highlighting their utilization as non-invasive molecular markers in RCC. Also, the rationale and development of novel molecular targeted drugs and immunotherapy drugs [such as tyrosine kinase inhibitors and immune checkpoint inhibitors (ICIs)] as potential RCC therapeutics along with the proposed implication of these biomarkers in predicting response to targeted therapies will be discussed.

Molecular characterization and in silico analysis of oxalate decarboxylase of Pseudomonas sp. OXDC12.

Oxalate decarboxylase (OxDC) is a Mn-dependent hexameric enzyme that is highly important in management of calcium oxalate mediated nephrolithiasis. The present study reported the production and purification of OxDC from Pseudomonas sp. OXDC12 up to 45.3-fold with an overall yield of 7%. The purified OxDC displayed a single band of approximately 40 kDa on SDS-PAGE and 240 kDa on Native-PAGE suggesting it to be a hexameric enzyme. The purified OxDC displayed an optimum activity at 26 °C and pH 4.5 in the presence of substrate sodium oxalate (30 mg/mL) with a Km and Vmax value of 43.9 mM and 8.9 µmol/min, respectively and an activation energy of 52.49 kJ/mol. The enzyme activity was significantly enhanced by adding o-phenylenediamine to the reaction mixture. OxDC exhibited a very low 17 haemolytic activity which suggested a relatively safer therapeutic aspect of the tested OxDC. The structure prediction studies of the OxDC revealed a tertiary structure with α/ß chains that formed the ß barrel structure, typical of all cupin domains. The Ramachandran plot produced by PROCHECK shows that 90.5% of the residues are in the most favoured region and hence the OxDC model produced was a good one. Docking studies revealed the binding of the metal ions and ligands to cluster of three histidine residues in the N terminal domain that formed the active site pocket of the enzyme. It was suggested that the histidine coordinated Mn2+ ion was critical for substrate recognition and binding and was also directly involved in OxDC catalyses.highlightsOxalate decarboxylase (OxDC) was successfully purified from Pseudomonas sp. OXDC12 up-to 45.3-fold.The Km and Vmax values of the purified OxDC were calculated as 43.9 mM and 8.9 µmol/min, respectively.Genre analysis and structure prediction studies revealed the presence of ß barrel structure typical of all cupin domains. The model exhibited a bi-cupin domain that forms the dimer of the homo-hexameric OxDC.Docking experiments revealed that the cluster of three HIS residues in the N terminal domain of the tested enzyme formed the active site pocket for binding of Mn as well as the ligands.Communicated by Ramaswamy H. Sarma.

Screening, characterization and anti-cancer application of purified intracellular MGL.

L-methionine-γ-lyase (MGL) producing bacterial isolates were screened from soil samples that further characterized as 'Klebsiella oxytoca BLM-1' by biochemical and 16S rDNA sequencing. Intracellular MGL obtained from K. oxytoca BLM-1 by sonication was purified by Octyl-Sepharose and Sephadex G-200 column chromatography. MALDI-TOF-MS analysis of protein band (Mr ~ 63 kDa) confirmed the PLP-dependence and structural similarity with MGL enzyme. Purified MGL (1.1 µg) exhibited the maximum activity in potassium phosphate buffer (80 mM; with L-met 20 mM pH 7.0) at 37 °C. That further enhanced in the presence of NaCl (2 mM), Tween-80 (1.0 %; v/v) and EDTA (5 mM). Km and Vmax for purified MGL by using L-met as substrate was found to be 5.32 mM and 0.386 U/mL/min. The purified MGL showed PLP dependence and the half-life was 365.59 min. The MGL was effective against breast cancer (MCF7), gastric adenocarcinoma and human glioblastoma (U87MG) cancer cell lines with IC50 values of purified MGL 0.041 U/mL, 0.008 U/mL and 0.009 U/mL, respectively. The U87MG, greatly affected by MGL treatment, when cultured in DMEM medium (10 mL) with PLP, homocysteine and 10 % FCS as compared to control/untransformed mouse spleen cells.

An Insight in Developing Carrier-Free Immobilized Enzymes.

Enzymes play vital roles in all organisms. The enzymatic process is progressively at its peak, mainly for producing biochemical products with a higher value. The immobilization of enzymes can sometimes tremendously improve the outcome of biocatalytic processes, making the product(s) relatively pure and economical. Carrier-free immobilized enzymes can increase the yield of the product and the stability of the enzyme in biocatalysis. Immobilized enzymes are easier to purify. Due to these varied advantages, researchers are tempted to explore carrier-free methods used for the immobilization of enzymes. In this review article, we have discussed various aspects of enzyme immobilization, approaches followed to design a process used for immobilization of an enzyme and the advantages and disadvantages of various common processes used for enzyme immobilization.

Combination of classical and statistical approaches to enhance the fermentation conditions and increase the yield of Lipopeptide(s) by Pseudomonas sp. OXDC12: its partial purification and determining antifungal property.

Around 200 different lipopeptides (LPs) have been identified to date, most of which are produced via Bacillus and Pseudomonas species. The clinical nature of the lipopeptide (LP) has led to a big surge in its research. They show antimicrobial and antitumor activities due to which mass-scale production and purification of LPs are beneficial. Response surface methodology (RSM) approach has emerged as an alternative in the field of computational biology for optimizing the reaction parameters using statistical models. In the present study, Pseudomonas sp. strain OXDC12 was used for production and partial purification of LPs using Thin Layer Chromatography (TLC). The main goal of the study was to increase the overall yield of LPs by optimizing the different variables in the fermentation broth. This was achieved using a combination of one factor at a time (OFAT) and response surface methodology (RSM) approaches. OFAT technique was used to optimize the necessary parameters and was followed by the creation of statistical models (RSM) to optimize the remaining variables. Maximum mycelial growth inhibition (%) against the fungus Mucor sp. was 61.3% for LP. Overall, the combination of both OFAT and RSM helped in increasing the LPs yield by 3 folds from 367mg/L to 1169mg/L.

Purification, characterization and cytotoxic properties of a bacterial RNase.

An RNase produced by Bacillus safensis RB-5 was purified up to 22.32-fold by successive techniques of salting out, DEAE-anion exchange and gel permeation (Sephadex G-100) chromatography techniques with a yield of 2.27%. The purified RNase possessed a single band in SDS-PAGE (Mr ~ 60 kDa). The purified RNase showed optimal activity at temperature of 37 °C and pH 7.5 in the presence of substrate (Yeast RNA) and Mg2+ ions. The RNase activity was strongly inhibited by Hg2+ and mildly by Fe2+, Ba2+ and Zn2+ ions. Its half-life was found to be 8 h at 37 °C. The RNase kinetics study showed Km and Vmax value of 0.3 mM and 9.2 µmol/mg/min, respectively. The purified RNase also showed cytotoxic and antiproliferative activities towards a few transformed cell lines. The purified RNase (IC50 0.035 U/mL) effectively inhibited RD and Hep-2C cells proliferation & migration, while sparing HEK 293 cells. The purified RNase was cytotoxic as well as effective degrader of the RNA of transformed RD cells at low concentration. Moreover, the purified RNase of B. safensis RB-5 was found to possess a little hemolytic activity towards human RBCs.

Utility of Silane-Modified Magnesium-Based Magnetic Nanoparticles for Efficient Immobilization of Bacillus thermoamylovorans Lipase.

Enzymes and protein's immobilization on magnetic nano supports is emerging as a promising candidate in the food, medical field, and areas of environmental studies. This work presents a study on purified Bacillus thermoamylovorans lipase (BTL) by utilizing tetraethoxysilane (TEOS)-modified magnesium nano ferrite (MgNF) of 20 nm size. Its structural and morphological studies were investigated by powder X-ray diffractometry, high-resolution transmission electron microscopy, etc. Binding of BTL with MgNF was supported by using Fourier transform infrared spectroscopy. Magnetic behavior was examined by the vibrational sample magnetometer and Mössbauer spectrometer graphs. The enzymatic activity of BTL before and after immobilization was studied at different temperatures and reaction time. As per the Lineweaver-Burk plot, immobilized lipase has more biological affinity for fatty acids in comparison to the free lipase, and Kmax values of immobilized and free BTL were computed as 6.6 and 7.5 mM respectively, with excellent reusability(> 50%) even till 13 consecutive assay runs. Graphical Abstract.

Lipase catalysis in organic solvents: advantages and applications.

Lipases are industrial biocatalysts, which are involved in several novel reactions, occurring in aqueous medium as well as non-aqueous medium. Furthermore, they are well-known for their remarkable ability to carry out a wide variety of chemo-, regio- and enantio-selective transformations. Lipases have been gained attention worldwide by organic chemists due to their general ease of handling, broad substrate tolerance, high stability towards temperatures and solvents and convenient commercial availability. Most of the synthetic reactions on industrial scale are carried out in organic solvents because of the easy solubility of non-polar compounds. The effect of organic system on their stability and activity may determine the biocatalysis pace. Because of worldwide use of lipases, there is a need to understand the mechanisms behind the lipase-catalyzed reactions in organic solvents. The unique interfacial activation of lipases has always fascinated enzymologists and recently, biophysicists and crystallographers have made progress in understanding the structure-function relationships of these enzymes. The present review describes the advantages of lipase-catalyzed reactions in organic solvents and various effects of organic solvents on their activity.

Comparative analysis of zinc finger proteins involved in plant disease resistance.

A meta-analysis was performed to understand the role of zinc finger domains in proteins of resistance (R) genes cloned from different crops. We analyzed protein sequences of seventy R genes of various crops in which twenty six proteins were found to have zinc finger domains along with nucleotide binding sites - leucine rice repeats (NBS-LRR) domains. We identified thirty four zinc finger domains in the R proteins of nine crops and were grouped into 19 types of zinc fingers. The size of individual zinc finger domain within the R genes varied from 11 to 84 amino acids, whereas the size of proteins containing these domains varied from 263 to 1305 amino acids. The biophysical analysis revealed that molecular weight of Pi54 zinc finger was lowest whereas the highest one was found in rice Pib zinc finger named as Transposes Transcription Factor (TTF). The instability (R(2) =0.95) and the aliphatic (R(2) =0.94) indices profile of zinc finger domains follows the polynomial distribution pattern. The pairwise identity analysis showed that the Lin11, Isl-1 & Mec-3 (LIM) zinc finger domain of rice blast resistance protein pi21 have 12.3% similarity with the nuclear transcription factor, X-box binding-like 1 (NFX) type zinc finger domain of Pi54 protein. For the first time, we reported that Pi54 (Pi-k(h)-Tetep), a rice blast resistance (R) protein have a small zinc finger domain of NFX type located on the C-terminal in between NBS and LRR domains of the R-protein. Compositional analysis depicted by the helical wheel diagram revealed the presence of a hydrophobic region within this domain which might help in exposing the LRR region for a possible R-Avr interaction. This domain is unique among all other cloned plant disease resistance genes and might play an important role in broad-spectrum nature of rice blast resistance gene Pi54.

In vitro antibacterial and antimalarial activity of dehydrophenylalanine-containing undecapeptides alone and in combination with drugs.

A set of three cationic undecapeptides, analogous to the previously reported peptide VS2 (KWΔFWKΔFVKΔFVK), was created by alanine substitution in order to probe the effect of hydrophobicity on peptide activity. The activities of these peptides were determined against Escherichia coli, Staphylococcus aureus and the malaria parasite Plasmodium falciparum. VA1, the closest analogue of VS2, showed five-fold augmented activity [minimum inhibitory concentration (MIC)=10 µM] against the Gram-positive bacterium S. aureus. The designed analogues were non-haemolytic and non-cytotoxic at their MICs and clinically relevant concentrations. By alanine substitution, it was also possible to probe the critical role of tryptophan residues in determining peptide potency. Circular dichroism studies of the peptides in a membrane-mimetic system showed a correlation between peptide helicity and antimicrobial activity. The peptides were also tested in combination with sublethal concentrations of antibiotic drugs (rifampicin and kanamycin) and the antimalarial drug chloroquine. In combination with these drugs, the effect of the peptides was synergistic or additive. These results provide insight into basic design principles for generating new clinically relevant lead peptides. It also provides an alternative strategy where a peptide and a non-peptide drug can be used in combination to battle increasingly drug-resistant microbes.

The single functional blast resistance gene Pi54 activates a complex defence mechanism in rice.

The Pi54 gene (Pi-k(h)) confers a high degree of resistance to diverse strains of the fungus Magnaporthe oryzae. In order to understand the genome-wide co-expression of genes in the transgenic rice plant Taipei 309 (TP) containing the Pi54 gene, microarray analysis was performed at 72 h post-inoculation of the M. oryzae strain PLP-1. A total of 1154 differentially expressing genes were identified in TP-Pi54 plants. Of these, 587 were up-regulated, whereas 567 genes were found to be down-regulated. 107 genes were found that were exclusively up-regulated and 58 genes that were down- regulated in the case of TP-Pi54. Various defence response genes, such as callose, laccase, PAL, and peroxidase, and genes related to transcription factors like NAC6, Dof zinc finger, MAD box, bZIP, and WRKY were found to be up-regulated in the transgenic line. The enzymatic activities of six plant defence response enzymes, such as peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, ß-glucosidase, ß-1,3-glucanase, and chitinase, were found to be significantly high in TP-Pi54 at different stages of inoculation by M. oryzae. The total phenol content also increased significantly in resistant transgenic plants after pathogen inoculation. This study suggests the activation of defence response and transcription factor-related genes and a higher expression of key enzymes involved in the defence response pathway in the rice line TP-Pi54, thus leading to incompatible host-pathogen interaction.

Bioresolution of benzyl glycidyl ether using whole cells of Bacillus alcalophilus.

The incubation of whole Bacillus alcalophilus cells grown on a mineral supplemented medium (MSM) containing 1% (w/v) sucrose as carbon source, 1.2% (w/v) tryptone as nitrogen source at pH 6.5 and temperature 30 °C in 24 h kinetically resolved benzyl glycidyl ether (1 mg/ml) to provide (S)-benzyl glycidyl ether with 30% ee and (R)-3-benzyloxypropane-1,2-diol with 40% ee.

Enzymatic Synthesis of Isopropyl Acetate by Immobilized Bacillus cereus Lipase in Organic Medium.

Selective production of fragrance fatty acid ester from isopropanol and acetic acid has been achieved using silica-immobilized lipase of Bacillus cereus MTCC 8372. A purified thermoalkalophilic extracellular lipase was immobilized by adsorption onto the silica. The effects of various parameters like molar ratio of substrates (isopropanol and acetic acid; 25 to 100 mM), concentration of biocatalyst (25-125 mg/mL), reaction time, reaction temperature, organic solvents, molecular sieves, and initial water activity were studied for optimal ester synthesis. Under optimized conditions, 66.0 mM of isopropyl acetate was produced when isopropanol and acetic acid were used at 100 mM: 75 mM in 9 h at 55°C in n-heptane under continuous shaking (160 rpm) using bound lipase (25 mg). Addition of molecular sieves (3 Å × 1.5 mm) resulted in a marked increase in ester synthesis (73.0 mM). Ester synthesis was enhanced by water activity associated with pre-equilibrated saturated salt solution of LiCl. The immobilized lipase retained more than 50% of its activity after the 6th cycle of reuse.

Synthesis of isopropyl ferulate using silica-immobilized lipase in an organic medium.

Immobilization of lipases has proved to be a useful technique for improving an enzyme's activity in organic solvents. In the present study, the performance of a silica-immobilized lipase was evaluated for the synthesis of isopropyl ferulate in DMSO. The biocatalyst was cross-linked onto the matrix with 1% glutaraldehyde. The effects of various parameters, molar ratio of ferulic acid to isopropyl alcohol (25 mM : 100 mM), concentration of biocatalyst (2.5-20 mg/mL), molecular sieves (25-250 mg/mL), and various salt ions, were studied consecutively as a function of percent esterification. Immobilized lipase at 25 mg/mL showed maximum esterification (~84%) of ferulic acid and isopropanol at a molar ratio of 25 mM : 100 mM, respectively, in DMSO at 45°C in 3 h under shaking (150 rpm). To overcome the inhibitory effect of water (a byproduct) if any, in the reaction mixture, molecular sieves (3 Å × 1.5 mm; 100 mg/mL) were added to the reaction mixture to promote the forward reaction. Salt ions like Ca(2+), Cd(2+), and Fe(2+) enhanced the activity of immobilized biocatalyst while a few ions like Co(2+), Zn(2+), Mg(2+), Mn(2+), Al(3+), and Na(+) had mild inhibitory effect. Approximately, one third of total decrease in the esterification efficacy was observed after the 5th repetitive cycle of esterification.

Methods for inhibition of residual lipase activity in colorimetric assay: a comparative study.

A comparative study of various treatments for inhibition of the residual activity of a lipase (obtained from Bacillus coagulans MTCC-6375) in a colorimetric assay using p-nitrophenyl palmitate (pNPP) was made. Direct chilling of contents of reaction mixture or addition of chilled mixture of ethanol : acetone (1:1) decreased the residual lipase activity by 94.0 and 95.0% respectively, as compared to lipase incubated at 45 degrees C for 20 min (control). Amongst various ionic and non-ionic detergents, Triton X-100 (0.07%, v/v) and sodium lauryl sarcosine or SLS (0.25%, w/v) partially, and SDS (0.05%, w/v) completely blocked the residual lipase activity of B. coagulans lipase in colorimetric assay. Addition of a serine protease inhibitor, PMSF (15 mM) or EDTA (200 mM) inhibited residual lipase activity by 99.5 and 100%, respectively. However, addition of reducing agents viz., 2-mercaptoethanol and dithiothreitol caused decomposition of chromogenic substrate (pNPP) thus rendering the colorimetric method unfit for lipase assay. EDTA (200 mM) and SDS (0.05%, w/v) were also highly effective in inhibiting the residual activities of lipases of Pseudomonas aeruginosa MTCC-4713, P. cepacia and commercial grade lipolytic preparations such as lipozyme, lipolase and porcine pancreatic lipase. However, PMSF (15 mM) completely inhibited the residual activity of lipase of P. aeruginosa.