Dynamical Phase Transitions Reveal Amyloid-like States on Protein Folding Landscapes

Developing an understanding of protein misfolding processes presents a crucial challenge for unlocking the mysteries of human disease. In this article, we present our observations of β-sheet-rich misfolded states on a number of protein dynamical landscapes investigated through molecular dynamics simulation and Markov state models. We employ a nonequilibrium statistical mechanical theory to identify the glassy states in a protein’s dynamics, and we discuss the nonnative, β-sheet-rich states that play a distinct role in the slowest dynamics within seven protein folding systems. We highlight the fundamental similarity between these states and the amyloid structures responsible for many neurodegenerative diseases, and we discuss potential consequences for mechanisms of protein aggregation and intermolecular amyloid formation.     

Isolation of cold stress-responsive genes from Lepidium latifolium by suppressive subtraction hybridization

Suppression subtraction hybridization (SSH) libraries were constructed from RNA isolated from leaves of control and cold stress-induced Lepidium latifolium, a cold-tolerant plant species from high altitudes for isolation of cold-responsive genes. A total of 500 clones were obtained from the cold stress library. Dot blot expression analysis identified 157 clones that were upregulated and 75 that were downregulated during cold stress. These clones selected on the basis of their expression patterns on dot blot were sequenced. As much as 27 and 17 genes were identified from the forward and reverse libraries, respectively. The genes identified revealed homology with genes involved in diverse processes such as gene regulation/signaling, photosynthesis, DNA damage repair protein, pathogenesis-related protein, senescence-associated proteins and proteins with unknown functions.     

Polarizable Atomic Multipole X-Ray Refinement: Hydration Geometry and Application to Macromolecules

We recently developed a polarizable atomic multipole refinement method assisted by the AMOEBA force field for macromolecular crystallography. Compared to standard refinement procedures, the method uses a more rigorous treatment of x-ray scattering and electrostatics that can significantly improve the resultant information contained in an atomic model. We applied this method to high-resolution lysozyme and trypsin data sets, and validated its utility for precisely describing biomolecular electron density, as indicated by a 0.4-0.6% decrease in the R- and R_free-values, and a corresponding decrease in the relative energy of 0.4-0.8 Kcal/mol/residue. The re-refinements illustrate the ability of force-field electrostatics to orient water networks and catalytically relevant hydrogens, which can be used to make predictions regarding active site function, activity, and protein-ligand interaction energies. Re-refinement of a DNA crystal structure generates the zigzag spine pattern of hydrogen bonding in the minor groove without manual intervention. The polarizable atomic multipole electrostatics model implemented in the AMOEBA force field is applicable and informative for crystal structures solved at any resolution.     

DnaSAM: Software to perform neutrality testing for large datasets with complex null models

Patterns of DNA sequence polymorphisms can be used to understand the processes of demography and adaptation within natural populations. High-throughput generation of DNA sequence data has historically been the bottleneck with respect to data processing and experimental inference. Advances in marker technologies have largely solved this problem. Currently, the limiting step is computational, with most molecular population genetic software allowing a gene-by-gene analysis through a graphical user interface. An easy-to-use analysis program that allows both high-throughput processing of multiple sequence alignments along with the flexibility to simulate data under complex demographic scenarios is currently lacking. We introduce a new program, named DnaSAM, which allows high-throughput estimation of DNA sequence diversity and neutrality statistics from experimental data along with the ability to test those statistics via Monte Carlo coalescent simulations. These simulations are conducted using the ms program, which is able to incorporate several genetic parameters (e.g. recombination) and demographic scenarios (e.g. population bottlenecks). The output is a set of diversity and neutrality statistics with associated probability values under a user-specified null model that are stored in easy to manipulate text file.     

Changes in cholesterol levels in the plasma membrane modulate cell signaling and regulate cell adhesion and migration on fibronectin

The number and distribution of lipid molecules, including cholesterol in particular, in the plasma membrane, may play a key role in regulating several physiological processes in cells. We investigated the role of membrane cholesterol in regulating cell shape, adhesion and motility. The acute depletion of cholesterol from the plasma membrane of cells that were well spread and motile on fibronectin caused the rounding of these cells and decreased their adhesion to and motility on fibronectin. These modifications were less pronounced in cells plated on laminin, vitronectin or plastic, indicating that cholesterol-mediated changes in adhesion and motility are more specific for adhesion mediated by fibronectin-specific integrins, such as alpha5beta1. These changes were accompanied by remodeling of the actin cytoskeleton, the spatial reorganization of paxillin in the membrane, and changes to the dynamics of alpha5 integrin and paxillin-rich focal adhesions. Levels of tyrosine phosphorylation at position 576/577 of FAK and Erk1/Erk2 MAP-kinase activity levels were both lower in cholesterol-depleted than in control cells. These levels normalized only on fibronectin when cholesterol was reincorporated into the cell membrane. Thus, membrane cholesterol content has a specific effect on certain signaling pathways specifically involved in regulating cell motility on fibronectin and organization of the actin cytoskeleton.     

Biometry based ageing of nestling Indian Spotted Owlets ( Athene brama brama)

Biometric analysis helps in sex differentiation, understanding development and for studies of avian biology such as foraging ecology, evolutionary ecology, and survivorship. We suggest that biometry can also be a reliable, practical and inexpensive tool to determine the age of nestlings in the field by non-invasive methods. As an example we studied the biometry of wing, culmen, talon, tarsus and body mass of nestling southern Indian Spotted Owlets (Athene brama brama). Based on the growth pattern analysis using logistic growth model, discriminant analysis and CHAID (Chi-squared Automatic Interaction Detection) based decision tree, we show that biometry of nestling Spotted Owlets is an easy, reliable and inexpensive method to determine nestling age and to assess growth rate and relative nutritional status. These biometric parameters also allow us to predict their ability to initiate first flight from the nest site. This method is described here for the first time and we postulate that such charts can be devised for other avian species as well, so as to assist conservation biologists and bird rescuers.     

Sensitive and specific fluorescent probes for functional analysis of the three major types of mammalian ABC transporters

An underlying mechanism for multi drug resistance (MDR) is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins. ABC transporters also determine the general fate and effect of pharmaceutical agents in the body. The three major types of ABC transporters are MDR1 (P-gp, P-glycoprotein, ABCB1), MRP1/2 (ABCC1/2) and BCRP/MXR (ABCG2) proteins. Flow cytometry (FCM) allows determination of the functional expression levels of ABC transporters in live cells, but most dyes used as indicators (rhodamine 123, DiOC(2)(3), calcein-AM) have limited applicability as they do not detect all three major types of ABC transporters. Dyes with broad coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus may yield a significant percentage of false negative results. We describe two novel fluorescent probes that are substrates for all three common types of ABC transporters and can serve as indicators of MDR in flow cytometry assays using live cells. The probes exhibit fast internalization, favorable uptake/efflux kinetics and high sensitivity of MDR detection, as established by multidrug resistance activity factor (MAF) values and Kolmogorov-Smirnov statistical analysis. Used in combination with general or specific inhibitors of ABC transporters, both dyes readily identify functional efflux and are capable of detecting small levels of efflux as well as defining the type of multidrug resistance. The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of ABC transporter activity, and ready implementation on widely available instruments.     

Peptide derived from the lipid binding domain of Group IB human pancreatic phospholipase A2 possesses antibacterial activity

Group 1B human pancreatic secretory phospholipase A₂ (hp-sPLA₂), a digestive enzyme synthesized by pancreatic acinar cells and present in pancreatic juice, do not have antibacterial activity towards Escherichia coli. Our earlier results suggest that the N-terminal first ten amino acid residues of hp-sPLA₂ constitute major portion of the membrane binding domain of full-length enzyme and is responsible for the precise orientation of enzyme on the membrane surface by inserting into the lipid bilayers (Pande et al. (2006) Biochemistry, 45,12436–12447). In this study we report the antibacterial properties of a peptide (AVWQFRKMIK-CONH₂; N10 peptide), which corresponds to the N-terminal first ten amino acid residues of hp-sPLA₂, against E. coli. Full-length hp-sPLA₂, which contains this peptide sequence as N-terminal α-helix, did not showed detectable antibacterial activity. Presence of physiological concentration of salt or preincubation of N10 peptide with soluble anionic polymer inhibits the antibacterial activity indicating the importance of electrostatic interaction in binding of peptide to bacterial membrane. Addition of peptide resulted in destabilization of outer as well as inner cytoplasmic membrane of E. coli suggesting bacterial membranes to be the main target of action. N10 peptide exhibits strong synergism with lysozyme and potentiates the antibacterial activity of lysozyme. The peptide was inactive against human erythrocyte. Our result shows for the first time that a peptide fragment of hp-sPLA₂ possesses antibacterial activity towards E. coli and at subinhibitory concentration and can potentiate the antibacterial activity of membrane active enzyme. These observations suggest that N10 peptide may play an important role in the antimicrobial activity of pancreatic juice.     

The use of real-time PCR and species-specific primers for the identification and monitoring of Paecilomyces lilacinus

The Paecilomyces lilacinus is the most widely tested fungus for the control of root-knot and cyst nematodes. The fungus has also been implicated in a number of human and animal infections, difficulties in diagnosis often result in misdiagnosis or delays in identification leading to a delay in treatment. Here, we report the development of species-specific primers for the identification of P. lilacinus based on sequence information from the ITS gene, and their use in identifying P. lilacinus isolates, including clinical isolates of the fungus. The primer set generated a single PCR fragment of 130 bp in length that was specific to P. lilacinus and was also used to detect the presence of P. lilacinus from soil, roots and nematode eggs. Real-time PCR primers and a TaqMan probe were also developed and provided quantitative data on the population size of the fungus in two field sites. PCR, bait and culture methods were combined to investigate the presence and abundance of the fungus from two field sites in the United Kingdom where potato cyst nematode populations were naturally declining, and results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.     

Phylloplane bacteria increase seedling emergence, growth and yield of field-grown groundnut (Arachis hypogaea L.)

AIM: To isolate and characterize groundnut-associated bacterial isolates for growth promotion of groundnut in field. METHODS AND RESULTS: Three hundred and ninety-three groundnut-associated bacteria, representing the geocarposphere, phylloplane and rhizosphere, and endophytes were applied as seed treatment in greenhouse. Maximum increase in plant biomass (up to 26%) was observed following treatment with a rhizosphere isolate identified as Bacillus firmis GRS 123, and two phylloplane isolates Bacillus megaterium GPS 55 and Pseudomonas aeruginosa GPS 21. There was no correlation between the production of L-tryptophan-derived auxins and growth promotion by the test isolates. Actively growing cells and peat formulations of GRS 123 and GPS 55, and actively growing cells of GPS 21, significantly increased the plant growth and pod yield (up to 19%) in field. Rifampicin-resistant mutants of GRS 123 and GPS 21 colonized the ecto- and endorhizospheres of groundnut, respectively, up to 100 days after sowing (DAS), whereas GPS 55 was recovered from both the habitats at 100 DAS. CONCLUSION: Seed bacterization with phylloplane isolates promoted groundnut growth indicating the possibility of isolating rhizosphere beneficial bacteria from different habitats. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of phylloplane bacteria as effective plant growth-promoting rhizobacteria (PGPR) broadens the spectrum of PGPR available for field application.