CKAP5 stabilizes CENP-E at kinetochores by regulating microtubule-chromosome attachments.

Stabilization of microtubule plus end-directed kinesin CENP-E at the metaphase kinetochores is important for chromosome alignment, but its mechanism remains unclear. Here, we show that CKAP5, a conserved microtubule plus tip protein, regulates CENP-E at kinetochores in human cells. Depletion of CKAP5 impairs CENP-E localization at kinetochores at the metaphase plate and results in increased kinetochore-microtubule stability and attachment errors. Erroneous attachments are also supported by computational modeling. Analysis of CKAP5 knockout cancer cells of multiple tissue origins shows that CKAP5 is preferentially essential in aneuploid, chromosomally unstable cells, and the sensitivity to CKAP5 depletion is correlated to that of CENP-E depletion. CKAP5 depletion leads to reduction in CENP-E-BubR1 interaction and the interaction is rescued by TOG4-TOG5 domain of CKAP5. The same domain can rescue CKAP5 depletion-induced CENP-E removal from the kinetochores. Interestingly, CKAP5 depletion facilitates recruitment of PP1 to the kinetochores and furthermore, a PP1 target site-specific CENP-E phospho-mimicking mutant gets stabilized at kinetochores in the CKAP5-depleted cells. Together, the results support a model in which CKAP5 controls mitotic chromosome attachment errors by stabilizing CENP-E at kinetochores and by regulating stability of the kinetochore-attached microtubules.

Regulation of α-synuclein by chaperones in mammalian cells.

Neurodegeneration in patients with Parkinson's disease is correlated with the occurrence of Lewy bodies-intracellular inclusions that contain aggregates of the intrinsically disordered protein α-synuclein1. The aggregation propensity of α-synuclein in cells is modulated by specific factors that include post-translational modifications2,3, Abelson-kinase-mediated phosphorylation4,5 and interactions with intracellular machineries such as molecular chaperones, although the underlying mechanisms are unclear6-8. Here we systematically characterize the interaction of molecular chaperones with α-synuclein in vitro as well as in cells at the atomic level. We find that six highly divergent molecular chaperones commonly recognize a canonical motif in α-synuclein, consisting of the N terminus and a segment around Tyr39, and hinder the aggregation of α-synuclein. NMR experiments9 in cells show that the same transient interaction pattern is preserved inside living mammalian cells. Specific inhibition of the interactions between α-synuclein and the chaperone HSC70 and members of the HSP90 family, including HSP90ß, results in transient membrane binding and triggers a remarkable re-localization of α-synuclein to the mitochondria and concomitant formation of aggregates. Phosphorylation of α-synuclein at Tyr39 directly impairs the interaction of α-synuclein with chaperones, thus providing a functional explanation for the role of Abelson kinase in Parkinson's disease. Our results establish a master regulatory mechanism of α-synuclein function and aggregation in mammalian cells, extending the functional repertoire of molecular chaperones and highlighting new perspectives for therapeutic interventions for Parkinson's disease.

Stability of Cytoplasmic Membrane of Escherichia coli Bacteria in Aqueous and Ethanolic Environment.

The mechanism of action of any antibacterial agent or disinfectant depends largely on their interaction with the bacterial membrane. Herein, we use the SPICA (surface property fitting coarse graining) force-field and develop a coarse-grained (CG) model for the structure of the cytoplasmic membrane of Escherichia coli (E. coli) and its interaction with water and ethanol. We elucidate the impact of different concentrations of ethanol on the cytoplasmic membrane bilayers and vesicles of E. coli using the CG molecular dynamics (CG MD) simulations. Our modeling approach first focuses on the parametrization of the required force-field for POPG lipid and its interaction with water, ethanol, and POPE lipid. Subsequently, the structural stability of the E. coli bacterial membrane in the presence of high and low concentrations of ethanol is delineated. Both flat bilayers as well as vesicles of E. coli membrane were considered for the CG MD. Our results reveal that, at low ethanol concentrations (<30 mol %), the size of the E. coli vesicles increases with discernible deformations in their shapes. Because of ethanol-induced interdigitation, thinning of the E. coli vesicular membrane is also observed. However, at higher ethanol concentrations (>30 mol %), the integrity of the vesicles is lost because of deteriorating invasion of ethanol molecules into the vesicle bilayer and significant weakening of lipid-lipid interactions. At higher ethanol concentrations (40 and 70 mol %), both the multivesicle and single-vesicle bacterial membranes exhibit a similar rupturing pattern wherein the extraction of lipids from the membrane and formation of aggregates of the component lipids are observed. These aggregates consist of polar head groups of 3-5 POPE/POPG lipids with intertwined nonpolar tails.

Structural insights into α-synuclein monomer-fibril interactions.

Protein aggregation into amyloid fibrils is associated with multiple neurodegenerative diseases, including Parkinson's disease. Kinetic data and biophysical characterization have shown that the secondary nucleation pathway highly accelerates aggregation via the absorption of monomeric protein on the surface of amyloid fibrils. Here, we used NMR and electron paramagnetic resonance spectroscopy to investigate the interaction of monomeric α-synuclein (α-Syn) with its fibrillar form. We demonstrate that α-Syn monomers interact transiently via their positively charged N terminus with the negatively charged flexible C-terminal ends of the fibrils. These intermolecular interactions reduce intramolecular contacts in monomeric α-Syn, yielding further unfolding of the partially collapsed intrinsically disordered states of α-Syn along with a possible increase in the local concentration of soluble α-Syn and alignment of individual monomers on the fibril surface. Our data indicate that intramolecular unfolding critically contributes to the aggregation kinetics of α-Syn during secondary nucleation.

Leveraging QM/MM and Molecular Dynamics Simulations to Decipher the Reaction Mechanism of the Cas9 HNH Domain to Investigate Off-Target Effects.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology is an RNA-guided targeted genome-editing tool using Cas family proteins. Two magnesium-dependent nuclease domains of the Cas9 enzyme, termed HNH and RuvC, are responsible for cleaving the target DNA (t-DNA) and nontarget DNA strands, respectively. The HNH domain is believed to determine the DNA cleavage activity of both endonuclease domains and is sensitive to complementary RNA-DNA base pairing. However, the underlying molecular mechanisms of CRISPR-Cas9, by which it rebukes or accepts mismatches, are poorly understood. Thus, investigation of the structure and dynamics of the catalytic state of Cas9 with either matched or mismatched t-DNA can provide insights into improving its specificity by reducing off-target cleavages. Here, we focus on a recently discovered catalytic-active form of the Streptococcus pyogenes Cas9 (SpCas9) and employ classical molecular dynamics and coupled quantum mechanics/molecular mechanics simulations to study two possible mechanisms of t-DNA cleavage reaction catalyzed by the HNH domain. Moreover, by designing a mismatched t-DNA structure called MM5 (C to G at the fifth position from the protospacer adjacent motif region), the impact of single-guide RNA (sgRNA) and t-DNA complementarity on the catalysis process was investigated. Based on these simulations, our calculated binding affinities, minimum energy paths, and analysis of catalytically important residues provide atomic-level details of the differences between matched and mismatched cleavage reactions. In addition, several residues exhibit significant differences in their catalytic roles for the two studied systems, including K253, K263, R820, K896, and K913.

N-acetyl-L-tryptophan (NAT) provides protection to intestinal epithelial cells (IEC-6) against radiation-induced apoptosis via modulation of oxidative stress and mitochondrial membrane integrity.

BACKGROUND: Ionizing radiation generates oxidative stress in biological systems via inducing free radicals. Gastro-intestinal system has been known for its high radiosensitivity. Therefore, to develop an effective radiation countermeasure for gastrointestinal system, N-acetyl L-tryptophan was evaluated for its radioprotective efficacy using intestinal epithelial cells-6 (IEC-6) cells as the experimental model. METHODS AND RESULTS: Cellular metabolic and lysosomal activity of L-NAT and L-NAT treated irradiated IEC-6 cells were assessed by MTT and NRU staining, respectively. ROS and mitochondrial superoxide levels along with mitochondrial disruption were detected using specific fluorescent probes.  Endogenous antioxidants (CAT, SOD, GST, GPx) activities were determined using calorimetric assay.  Apoptosis and DNA damage were assessed using flow cytometery and Comet assay, respectively. Results of the study were demonstrated that L-NAT pre-treatment (- 1 h) to irradiated IEC-6 cells significantly contribute to ensuring 84.36% to 87.68% (p < 0.0001) survival at 0.1 µg/mL concentration against LD50 radiation dose (LD50; 20 Gy). Similar level of radioprotection was observed with a clonogenic assay against γ radiation (LD50; 5 Gy). L-NAT was found to provide radioprotection by neutralizing radiation-induced oxidative stress, enhancing antioxidant enzymes (CAT, SOD, GST, and GPx), and protecting DNA from radiation-induced damage. Further, significant restoration of mitochondrial membrane integrity along with apoptosis inhibition was observed with irradiated IEC-6 cells upon L-NAT pretreatment.

Structural adaptations in the bovine serum albumin protein in archetypal deep eutectic solvent reline and its aqueous mixtures.

The global concern over the environmental impact and challenges associated with the use of conventional solvents in biotransformation processes have pushed the search for alternative solvents. Recently, deep eutectic solvents (DESs) have appeared as a promising replacement with better biocompatibility and have been postulated to hold great potential in protein engineering and crystallization processes. In this context, herein, we have investigated the effect of reline (a choline chloride : urea mixture in 1 : 2 proportion) DES in its pure and hydrated forms on the structural stability and conformation of the bovine serum albumin (BSA) protein using all-atom molecular dynamics simulations. We observe a substantial overall expansion of the BSA structure with a simultaneous increment in the solvent accessible surface area, signifying the influence of reline on the BSA tertiary structure. These induced structural perturbations are quite pronounced in reline-water mixtures. Concomitantly, a notable reline concentration-dependent disruption of the BSA secondary structure through the melting of α-helices, mainly driven by H-bonding interactions, is observed. In the presence of pure reline, significant rigidity in the protein backbone is also observed. Thus, despite the expansion, the BSA tertiary structure in pure reline is found to be most close to the native protein structure and remains in a partially folded state at all the studied reline concentrations. In pure reline, BSA-urea hydrogen bonding is more prevalent than BSA-[Ch]+. We also observe that in aqueous reline systems, the BSA-water hydrogen bonds are mostly compensated by BSA-urea hydrogen bonds. The aqueous re-equilibration of these partially denatured protein conformations showed a significant recovery of secondary and tertiary structures, where the recovery is most profound for the BSA conformation extracted from pure reline.

Coordinated Actions of Cas9 HNH and RuvC Nuclease Domains Are Regulated by the Bridge Helix and the Target DNA Sequence.

CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection in bacteria and archaea against intruding genomic materials. Cas9, a type-II CRISPR effector protein, is widely used for gene editing applications since a single guide RNA can direct Cas9 to cleave specific genomic targets. The conformational changes associated with RNA/DNA binding are being modulated to develop Cas9 variants with reduced off-target cleavage. Previously, we showed that proline substitutions in the arginine-rich bridge helix (BH) of Streptococcus pyogenes Cas9 (SpyCas9-L64P-K65P, SpyCas92Pro) improve target DNA cleavage selectivity. In this study, we establish that kinetic analysis of the cleavage of supercoiled plasmid substrates provides a facile means to analyze the use of two parallel routes for DNA linearization by SpyCas9: (i) nicking by HNH followed by RuvC cleavage (the TS (target strand) pathway) and (ii) nicking by RuvC followed by HNH cleavage (the NTS (nontarget strand) pathway). BH substitutions and DNA mismatches alter the individual rate constants, resulting in changes in the relative use of the two pathways and the production of nicked and linear species within a given pathway. The results reveal coordinated actions between HNH and RuvC to linearize DNA, which is modulated by the integrity of the BH and the position of the mismatch in the substrate, with each condition producing distinct conformational energy landscapes as observed by molecular dynamics simulations. Overall, our results indicate that BH interactions with RNA/DNA enable target DNA discrimination through the differential use of the parallel sequential pathways driven by HNH/RuvC coordination.

Biochemical and functional characterization of the SMC holocomplex from Mycobacterium smegmatis.

Multi-subunit SMC complexes are required to perform essential functions, such as chromosome compaction, segregation and DNA repair, from bacteria to humans. Prokaryotic SMC proteins form complexes with two non-SMC subunits, ScpA and ScpB, to condense the chromosome. The mutants of both scpa and scpb genes in Bacillus subtilis have been shown to display characteristic phenotypes such as growth defects and increased frequency of anucleate cells. Here, we studied the function of the Smc-ScpAB complex from Mycobacterium smegmatis. We observed no significant growth difference between the scpb null mutant and wild-type M. smegmatis under both standard and stress conditions. Furthermore, we characterized the Smc-ScpAB holocomplex from M. smegmatis. The MsSMC consists of the dimerization hinge and ATPase head domains connected by long coiled-coils. The MsSMC interacts with two non-SMC proteins, ScpA and ScpB, and the resulting holocomplex binds to different DNA substrates independent of ATP. The Smc-ScpAB complex showed DNA-stimulated ATPase activity in the presence of ssDNA. A cytological profiling assay revealed that upon overexpression the Smc-ScpAB ternary complex compacts the decondensed nucleoid of rifampicin-treated wild-type and null mukb mutant of Escherichia coli in vivo. Together, our study suggests that M. smegmatis has a functional Smc-ScpAB complex capable of DNA binding and condensation. Based on our observations, we speculate that the presence of alternative SMCs such as MksB or other SMC homologues might have rescued the scpb mutant phenotype in M. smegmatis.

Calcium- and calmodulin-regulated microtubule-associated proteins as signal-integration hubs at the plasma membrane-cytoskeleton nexus.

Plant growth and development are a genetically predetermined series of events but can change dramatically in response to environmental stimuli, involving perpetual pattern formation and reprogramming of development. The rate of growth is determined by cell division and subsequent cell expansion, which are restricted and controlled by the cell wall-plasma membrane-cytoskeleton continuum, and are coordinated by intricate networks that facilitate intra- and intercellular communication. An essential role in cellular signaling is played by calcium ions, which act as universal second messengers that transduce, integrate, and multiply incoming signals during numerous plant growth processes, in part by regulation of the microtubule cytoskeleton. In this review, we highlight recent advances in the understanding of calcium-mediated regulation of microtubule-associated proteins, their function at the microtubule cytoskeleton, and their potential role as hubs in crosstalk with other signaling pathways.

Microtubule-associated protein IQ67 DOMAIN5 regulates morphogenesis of leaf pavement cells in Arabidopsis thaliana.

Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation, and cell morphology. Their organization and dynamics are co-ordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization, and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced anisotropic expansion. Lastly, we demonstrate IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays and provide first evidence for important roles for calcium in regulation of PC morphogenesis. Our work identifies IQD5 as a novel player in PC shape regulation and, for the first time, links calcium signaling to developmental processes that regulate anisotropic growth in PCs.

Mitochondrial Localization of Highly Fluorescent and Photostable BODIPY-Based Ruthenium(II), Rhodium(III), and Iridium(III) Metal Complexes.

A new N,O-based BODIPY ligand was synthesized and further utilized to develop highly fluorescent and photostable Ru(II), Rh(III), and Ir(III) metal complexes. The complexes were fully characterized by different analytical techniques including single-crystal XRD studies. The photostabilities and live cell imaging capabilities of the complexes were investigated via confocal microscopy. The complexes localized specifically in the mitochondria of live cells and showed negligible cytotoxicities at a concentration used for imaging purposes. They also exhibited high photostabilities, with fluorescence intensities remaining above 50% after 1800 scans.

DMSO induced dehydration of heterogeneous lipid bilayers and its impact on their structures.

Recently, we have reported that higher concentrations of dimethyl sulfoxide (DMSO) exhibit an enhancement in the structural ordering of the homogeneous N-palmitoyl-sphingomyelin (PSM) bilayer, whereas the presence of DMSO at lower concentrations leads to minor destabilization of the PSM bilayer structure. In this study, we aim to understand how these two modes of action of DMSO diversify for heterogeneous bilayers by employing atomistic molecular dynamic simulations. A binary bilayer system comprising PSM and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and a ternary bilayer system consisting of cholesterol along with PSM and POPC are the two heterogeneous biomimetic bilayers studied herein. We have simulated both the mixed lipid bilayer systems at 323 K, which is above the main phase transition temperature of the PSM lipid. This study reveals that DMSO exerts contrasting effects on the structure and stability of mixed bilayer systems, depending on its concentration. At 5 mol% of DMSO, the binary bilayer system shows slight disordering of lipid tails in conjunction with an appreciable increase in the area per lipid (APL), whereas for the ternary bilayer system, the orientational ordering of the lipid tails does not alter much; however, a slight expansion in the APL is observed. On the other hand, at 20 mol% of DMSO, an appreciable increase in the ordering of lipid tails for both the mixed bilayer systems occurs, depicting an enhancement in the structural stability of the bilayers. Furthermore, the H-bond analysis reveals that water-lipid H-bonding interaction decreases with increasing concentration of DMSO. We also observe contraction of the water-lipid interfacial region, pointing out DMSO induced dehydration at the lipid head-group region, and the dehydration effect is prominent for 20 mol% of DMSO. Furthermore, the computed free energies suggest that the free energy required for the transfer of a DMSO molecule from the lipid head-group region to the lipid head-tail interface is higher for the cholesterol containing ternary bilayer.

Design and Construction of Aroyl-Hydrazone Derivatives: Synthesis, Crystal Structure, Molecular Docking and Their Biological Activities.

Here, we report the synthesis and characterization of four new aroyl-hydrazone derivatives L1 -L4 , and their structural as well as biological activities have been explored. In addition to docking with bovine serum albumin (BSA) and duplex DNA, the experimental results demonstrate the effective binding of L1 -L4 with BSA protein and calf thymus DNA (ct-DNA) which is in agreement with the docking results. Further biological activities of L1 -L4 have been examined through molecular docking with different proteins which are involved in the propagation of viral or cancer diseases. L1 shows best binding affinity with influenza A virus polymerase PB2 subunit (2VY7) with binding energy -11.42 kcal/mol and inhibition constant 4.23 nm, whereas L2 strongly bind with the hepatitis C virus NS5B polymerase (2WCX) with binding energy -10.47 kcal/mol and inhibition constant 21.06 nm. Ligand L3 binds strongly with TGF-beta receptor 1 (3FAA) and L4 with cancer-related EphA2 protein kinases (1MQB) with binding energy -10.61 kcal/mol, -10.02 kcal/mol and inhibition constant 16.67 nm and 45.41 nm, respectively. The binding energies of L1 -L4 are comparable with binding energies of their proven inhibitors. L1 , L3 and L4 can be considered as both 3FAA and 1MQB dual targeting anticancer agents, while L1 and L3 are both 2VY7 and 2WCX dual targeting antiviral agents. On the other side, L2 and L4 target only one virus related target (2WCX). Furthermore, the geometry optimizations of L1 -L4 were performed via density functional theory (DFT). Moreover, all four ligands (L1 -L4 ) were characterized by NMR, FT-IR, ESI-MS, elemental analysis and their molecular structures were validated by single crystal X-ray diffraction studies.

Modulation of the extent of structural heterogeneity in α-synuclein fibrils by the small molecule thioflavin T.

The transition of intrinsically disordered, monomeric α-synuclein into ß-sheet-rich oligomers and fibrils is associated with multiple neurodegenerative diseases. Fibrillar aggregates possessing distinct structures that differ in toxicity have been observed in different pathological phenotypes. Understanding the mechanism of the formation of various fibril polymorphs with differing cytotoxic effects is essential for determining how the aggregation reaction could be modulated to favor nontoxic fibrils over toxic fibrils. In this study, two morphologically different α-synuclein fibrils, one helical and the other ribbon-like, are shown to form together. Surprisingly, a widely used small molecule for probing aggregation reactions, thioflavin T (ThT), was found to tune the structural heterogeneity found in the fibrils. The ribbon-like fibrils formed in the presence of ThT were found to have a longer structural core than the helical fibrils formed in the absence of ThT. The ribbon-like fibrils are also more toxic to cells. By facilitating the formation of ribbon-like fibrils over helical fibrils, ThT reduced the extent of fibril polymorphism. This study highlights the role of a small molecule such as ThT in selectively favoring the formation of a specific type of fibril by binding to aggregates formed early on one of multiple pathways, thereby altering the structural core and external morphology of the fibrils formed.

15N transverse relaxation measurements for the characterization of µs-ms dynamics are deteriorated by the deuterium isotope effect on 15N resulting from solvent exchange.

15N R2 relaxation measurements are key for the elucidation of the dynamics of both folded and intrinsically disordered proteins (IDPs). Here we show, on the example of the intrinsically disordered protein α-synuclein and the folded domain PDZ2, that at physiological pH and near physiological temperatures amide-water exchange can severely skew Hahn-echo based 15N R2 relaxation measurements as well as low frequency data points in CPMG relaxation dispersion experiments. The nature thereof is the solvent exchange with deuterium in the sample buffer, which modulates the 15N chemical shift tensor via the deuterium isotope effect, adding to the apparent relaxation decay which leads to systematic errors in the relaxation data. This results in an artificial increase of the measured apparent 15N R2 rate constants-which should not be mistaken with protein inherent chemical exchange contributions, Rex, to 15N R2. For measurements of 15N R2 rate constants of IDPs and folded proteins at physiological temperatures and pH, we recommend therefore the use of a very low D2O molar fraction in the sample buffer, as low as 1%, or the use of an external D2O reference along with a modified 15N R2 Hahn-echo based experiment. This combination allows for the measurement of Rex contributions to 15N R2 originating from conformational exchange in a time window from µs to ms.

Impact of amphiphilic molecules on the structure and stability of homogeneous sphingomyelin bilayer: Insights from atomistic simulations.

Modulation of lipid membrane properties due to the permeation of amphiphiles is an important biological process pertaining to many applications in the field of pharmaceutics, toxicology, and biotechnology. Sphingolipids are both structural and functional lipids that constitute an important component of mechanically stable and chemically resistant outer leaflets of plasma membranes. Here, we present an atomistic molecular dynamics simulation study to appreciate the concentration-dependent effects of small amphiphilic molecules, such as ethanol, acetone, and dimethyl sulfoxide (DMSO), on the structure and stability of a fully hydrated homogeneous N-palmitoyl-sphingomyelin (PSM) bilayer. The study reveals an increase in the lateral expansion of the bilayer along with disordering of the hydrophobic lipid tails on increasing the concentration of ethanol. At higher concentrations of ethanol, rupturing of the bilayer is quite evident through the analysis of partial electron density profiles and lipid tail order parameters. For ethanol containing systems, permeation of water molecules in the hydrophobic part of the bilayer is allowed through local defects made due to the entry of ethanol molecules via ethanol-ethanol and ethanol-PSM hydrogen bonds. Moreover, the extent of PSM-PSM hydrogen bonding decreases with increasing ethanol concentration. On the other hand, acetone and DMSO exhibit minimal effects on the stability of the PSM bilayer at their lower concentrations, but at higher concentrations they tend to enhance the stability of the bilayer. The simulated potential of mean force (PMF) profiles for the translocation of the three solutes studied reveal that the free-energy of transfer of an ethanol molecule across the PSM lipid head region is lower than that for acetone and DMSO molecules. However, highest free-energy rise in the core hydrophobic part of the bilayer is observed for the DMSO molecule, whereas the ethanol and acetone PMF profiles show a lower barrier in the hydrophobic region of the bilayer.

A study on oncogenic role of leptin and leptin receptor in oral squamous cell.

Leptin been mainly produced by adipose tissue and cancer cells is the most studied adipokine, amongst the several cytokines. Leptin is an antiapoptotic molecule and inducer of cancer stem cells as well as activates cell proliferation. Its oncogenic, mitogenic, proinflammatory and proangiogenic actions lead to its vital roles in tumourigenesis. Two common functional DNA polymorphisms in the genes of leptin G2548A (LEP) and leptin receptor A668G (LEPR) affect the amount of circulating cytokine-type hormone leptin with risk for development of oral squamous cell carcinoma (OSCC). The present study investigated whether these LEP and LEPR gene polymorphisms are affecting risk for OSCC by comparing the genotypes of patients with controls. A total of 306 OSCC and 228 controls participated in this study. We have determined the frequency of LEP (G2548A) and LEPR (A668G) gene polymorphisms in OSCC cases and controls by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). The incidence of leptin gene G2548A homozygous mutant AA polymorphism was significantly increased in the OSCC patients (p = 0.002, odds ratio (OR) = 2.4, 95 % confidence interval (CI) = 1.37-4.22) when compared with controls, and leptin receptor A668G homozygous mutant GG polymorphism was significantly high in the OSCC patients as compared to controls (p = 0.000, OR = 3.8, 95 % CI = 1.98-7.62). The polymorphism of homozygous mutant allele A of leptin gene and G allele of leptin receptor may be associated with increased risk for OSCC. The observations showed regular increase of supporting role of leptin in OSCC. The present study showed an association of AA genotype and A allele of LEP G2548A as well as GG genotype and G allele of LEPR A668G polymorphisms with increased risk for OSCC in north Indian patients. Moreover, the combination of both the polymorphisms may be involved in susceptibility and progression of OSCC.

Mycorrhizal association and its relation with pteridophytes.

Mycorrhizal association is one of the earliest and diversely distributed symbiotic associations on the Earth. This association helped early terrestrial plants to colonize the land by improved supply of nutrients like phosphate, nitrogen and zinc. It also helped plants to tolerate unfavorable soil conditions with increased water retention capacity, resistance to drought and pathogens. In return, fungi benefitted with carbon as their food source from the plants. More than 80% of terrestrial plants including pteridophytes, gymnosperms and angiosperms are reported to form arbuscular mycorrhizal (AM) association. Plants with root systems appeared on land during the Devonian period and many of them like pteridophytes still exist today. Various molecular and fossil studies confirm that the plants belonging to Ordovician-Devonian are associated with fungi, which are very similar to genus Glomus. AM association is very common in pteridophytes and the growth of its sporophyte and gametophyte is directly affected in the presence of AM association. Pteridophytes as early land plants with root systems have a very significant place in the plant kingdom. They have evolved and adapted to fill various habitats and facilitated early terrestrialization of other land plants by providing suitable niche with the help of AM fungi. In spite of pteridophytes being a very important plant group in the land system, very few reports are available on fungal-pteridophyte association. The present review is an effort to gather information about AM association in pteridophytes that might help in unraveling the evolution and significance of plant and fungi association.