Effect of temperature and nucleotide on the binding of BiP chaperone to a protein substrate.

BiP (immunoglobulin heavy-chain binding protein) is a Hsp70 monomeric ATPase motor that plays broad and crucial roles in maintaining proteostasis inside the cell. Structurally, BiP is formed by two domains, a nucleotide-binding domain (NBD) with ATPase activity connected by a flexible hydrophobic linker to the substrate-binding domain. While the ATPase and substrate binding activities of BiP are allosterically coupled, the latter is also dependent on nucleotide binding.

Temperature dependent mechanical unfolding and refolding of a protein studied by thermo-regulated optical tweezers.

Temperature is a useful system variable to gather kinetic and thermodynamic information from proteins. Usually, free energy and the associated entropic and enthalpic contributions are obtained by quantifying the conformational equilibrium based on melting experiments performed in bulk conditions. Such experiments are suitable only for those small single-domain proteins whose side reactions of irreversible aggregation are unlikely to occur.

Lactic acid from vaginal microbiota enhances cervicovaginal epithelial barrier integrity by promoting tight junction protein expression.

Background: Women with a cervicovaginal microbiota dominated by Lactobacillus spp. are at reduced risk of acquiring sexually transmitted infections including HIV, but the biological mechanisms involved remain poorly defined. Here, we performed metaproteomics on vaginal swab samples from young South African women (n = 113) and transcriptomics analysis of cervicovaginal epithelial cell cultures to examine the ability of lactic acid, a metabolite produced by cervicovaginal lactobacilli, to modulate genital epithelial barrier function.

The Cervicovaginal Microbiota-Host Interaction Modulates Chlamydia trachomatis Infection.

The mechanism(s) by which -dominated cervicovaginal microbiota provide a barrier to infection remain(s) unknown. Here we evaluate the impact of different spp. identified via culture-independent metataxonomic analysis of -infected women on infection in a three-dimensional (3D) cervical epithelium model.

The vaginal microbiota, host defence and reproductive physiology.

The interaction between the human host and the vaginal microbiota is highly dynamic. Major changes in the vaginal physiology and microbiota over a woman's lifetime are largely shaped by transitional periods such as puberty, menopause and pregnancy, while daily fluctuations in microbial composition observed through culture-independent studies are more likely to be the results of daily life activities and behaviours. The vaginal microbiota of reproductive-aged women is largely made up of at least five different community state types.

A Temperature-Jump Optical Trap for Single-Molecule Manipulation.

To our knowledge, we have developed a novel temperature-jump optical tweezers setup that changes the temperature locally and rapidly. It uses a heating laser with a wavelength that is highly absorbed by water so it can cover a broad range of temperatures. This instrument can record several force-distance curves for one individual molecule at various temperatures with good thermal and mechanical stability.

Host response to respiratory bacterial pathogens as identified by integrated analysis of human gene expression data.

Respiratory bacterial pathogens are one of the leading causes of infectious death in the world and a major health concern complicated by the rise of multi-antibiotic resistant strains. Therapeutics that modulate host genes essential for pathogen infectivity could potentially avoid multi-drug resistance and provide a wider scope of treatment options. Here, we perform an integrative analysis of published human gene expression data generated under challenges from the gram-negative and Gram-positive bacteria pathogens, Pseudomonas aeruginosa and Streptococcus pneumoniae, respectively.

Tension induces a base-paired overstretched DNA conformation.

Mixed-sequence DNA molecules undergo mechanical overstretching by approximately 70% at 60-70 pN. Since its initial discovery 15 y ago, a debate has arisen as to whether the molecule adopts a new form [Cluzel P, et al. (1996) Science 271:792-794; Smith SB, Cui Y, Bustamante C (1996) Science 271:795-799], or simply denatures under tension [van Mameren J, et al.

Identification of common biological pathways and drug targets across multiple respiratory viruses based on human host gene expression analysis.

Background: Pandemic and seasonal respiratory viruses are a major global health concern. Given the genetic diversity of respiratory viruses and the emergence of drug resistant strains, the targeted disruption of human host-virus interactions is a potential therapeutic strategy for treating multi-viral infections. The availability of large-scale genomic datasets focused on host-pathogen interactions can be used to discover novel drug targets as well as potential opportunities for drug repositioning.

Single-molecule derivation of salt dependent base-pair free energies in DNA.

Accurate knowledge of the thermodynamic properties of nucleic acids is crucial to predicting their structure and stability. To date most measurements of base-pair free energies in DNA are obtained in thermal denaturation experiments, which depend on several assumptions. Here we report measurements of the DNA base-pair free energies based on a simplified system, the mechanical unzipping of single DNA molecules.

Proofreading dynamics of a processive DNA polymerase.

Replicative DNA polymerases present an intrinsic proofreading activity during which the DNA primer chain is transferred between the polymerization and exonuclease sites of the protein. The dynamics of this primer transfer reaction during active polymerization remain poorly understood. Here we describe a single-molecule mechanical method to investigate the conformational dynamics of the intramolecular DNA primer transfer during the processive replicative activity of the Phi 29 DNA polymerase and two of its mutants.

Chemical and toxicologic assessment of organic contaminants in surface water using passive samplers.

Passive sampling methodologies were used to conduct a chemical and toxicologic assessment of organic contaminants in the surface waters of three geographically distinct agricultural watersheds. A selection of current-use agrochemicals and persistent organic pollutants, including polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and organochlorine pesticides, were targeted using the polar organic chemical integrative sampler (POCIS) and the semipermeable membrane device passive samplers. In addition to the chemical analysis, the Microtox assay for acute toxicity and the yeast estrogen screen (YES) were conducted as potential assessment tools in combination with the passive samplers.

Recent advances in optical tweezers.

It has been over 20 years since the pioneering work of Arthur Ashkin, and in the intervening years, the field of optical tweezers has grown tremendously. Optical tweezers are now being used in the investigation of an increasing number of biochemical and biophysical processes, from the basic mechanical properties of biological polymers to the multitude of molecular machines that drive the internal dynamics of the cell. Innovation, however, continues in all areas of instrumentation and technique, with much of this work focusing on the refinement of established methods and on the integration of this tool with other forms of single-molecule manipulation or detection.

Force unfolding kinetics of RNA using optical tweezers. I. Effects of experimental variables on measured results.

Experimental variables of optical tweezers instrumentation that affect RNA folding/unfolding kinetics were investigated. A model RNA hairpin, P5ab, was attached to two micron-sized beads through hybrid RNA/DNA handles; one bead was trapped by dual-beam lasers and the other was held by a micropipette. Several experimental variables were changed while measuring the unfolding/refolding kinetics, including handle lengths, trap stiffness, and modes of force applied to the molecule.

DNA translocation and loop formation mechanism of chromatin remodeling by SWI/SNF and RSC.

ATP-dependent chromatin-remodeling complexes (remodelers) modulate gene transcription by regulating the accessibility of highly packaged genomic DNA. However, the molecular mechanisms involved at the nucleosomal level in this process remain controversial. Here, we monitor the real-time activity of single ySWI/SNF or RSC complexes on single, stretched nucleosomal templates under tensions above 1 pN forces.

Molecular handles for the mechanical manipulation of single-membrane proteins in living cells.

We have developed a procedure to selectively biotinylate a specific membrane protein, enabling its attachment to external force probes and thus allowing its mechanical manipulation within its native environment. Using potassium channels as model membrane proteins in oocytes, we have found that Maleimide-PEG3400-biotin is the crosslinker with highest conjugation selectivity and accessibility to external probes. Neutravidin-coated beads provide for directed attachment while avoiding nonspecific interactions with the cell.

Acceptable noise level, phoneme recognition in noise, and measures of auditory efferent activity.

Acceptable noise level (ANL) is unrelated to sentence recognition in noise but may be related to phoneme recognition in noise (PRN). Individual differences in efferent activity in medial olivocochlear bundle (MOCB) and acoustic reflex (AR) pathways may account for intersubject variability in ANL and PRN. Monotic and dichotic ANL, monotic PRN, contralateral suppression of transient evoked otoacoustic emissions, and ipsilateral and contralateral acoustic reflex thresholds were measured in 31 adults with normal hearing.

Probing the mechanical folding kinetics of TAR RNA by hopping, force-jump, and force-ramp methods.

Mechanical unfolding and refolding of single RNA molecules have previously been observed in optical traps as sudden changes in molecular extension. Two methods have been traditionally used: "force-ramp", with the applied force continuously changing, and "hopping". In hopping experiments the force is held constant and the molecule jumps spontaneously between two different states.

Temperature control methods in a laser tweezers system.

Two methods of temperature control of a dual-beam optical-tweezers system are compared. In the first method, we used a 975 nm infrared laser to raise the temperature 5.6 degrees C/100 mW in a nonheating (830 nm) optical trap.

The bacterial condensin MukBEF compacts DNA into a repetitive, stable structure.

Condensins are conserved proteins containing SMC (structural maintenance of chromosomes) moieties that organize and compact chromosomes in an unknown mechanism essential for faithful chromosome partitioning. We show that MukBEF, the condensin in Escherichia coli, cooperatively compacts a single DNA molecule into a filament with an ordered, repetitive structure in an adenosine triphosphate (ATP) binding-dependent manner. When stretched to a tension of approximately 17 piconewtons, the filament extended in a series of repetitive transitions in a broad distribution centered on 45 nanometers.

Structural transitions and elasticity from torque measurements on DNA.

Knowledge of the elastic properties of DNA is required to understand the structural dynamics of cellular processes such as replication and transcription. Measurements of force and extension on single molecules of DNA have allowed direct determination of the molecule's mechanical properties, provided rigorous tests of theories of polymer elasticity, revealed unforeseen structural transitions induced by mechanical stresses, and established an experimental and conceptual framework for mechanical assays of enzymes that act on DNA. However, a complete description of DNA mechanics must also consider the effects of torque, a quantity that has hitherto not been directly measured in micromanipulation experiments.

Chirality sensing by Escherichia coli topoisomerase IV and the mechanism of type II topoisomerases.

Escherichia coli topoisomerase (Topo) IV is an essential type II Topo that removes DNA entanglements created during DNA replication. Topo IV relaxes (+) supercoils much faster than (-) supercoils, promoting replication while sparing the essential (-) supercoils. Here, we investigate the mechanism underlying this chiral preference.