Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.     

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others^1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition⁴, and it can be difficult to manage clinically⁵. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria^6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota⁸. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal^9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV^11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform¹³. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample¹⁴. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.Download video file.^{(68M, mov)}     

MicroRNA expression profiling identifies activated B cell status in chronic lymphocytic leukemia cells

Chronic lymphocytic leukemia (CLL) is thought to be a disease of resting lymphocytes. However, recent data suggest that CLL cells may more closely resemble activated B cells. Using microRNA (miRNA) expression profiling of highly-enriched CLL cells from 38 patients and 9 untransformed B cells from normal donors before acute CpG activation and 5 matched B cells after acute CpG activation, we demonstrate an activated B cell status for CLL. Gene set enrichment analysis (GSEA) identified statistically-significant similarities in miRNA expression between activated B cells and CLL cells including upregulation of miR-34a, miR-155, and miR-342-3p and downregulation of miR-103, miR-181a and miR-181b. Additionally, decreased levels of two CLL signature miRNAs miR-29c and miR-223 are associated with ZAP70(+) and IgV(H) unmutated status and with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the direction of change of individual miRNAs may predict clinical course in CLL.     

Rational truncation of an RNA aptamer to prostate-specific membrane antigen using computational structural modeling

RNA aptamers represent an emerging class of pharmaceuticals with great potential for targeted cancer diagnostics and therapy. Several RNA aptamers that bind cancer cell-surface antigens with high affinity and specificity have been described. However, their clinical potential has yet to be realized. A significant obstacle to the clinical adoption of RNA aptamers is the high cost of manufacturing long RNA sequences through chemical synthesis. Therapeutic aptamers are often truncated postselection by using a trial-and-error process, which is time consuming and inefficient. Here, we used a "rational truncation" approach guided by RNA structural prediction and protein/RNA docking algorithms that enabled us to substantially truncateA9, an RNA aptamer to prostate-specific membrane antigen (PSMA),with great potential for targeted therapeutics. This truncated PSMA aptamer (A9L; 41mer) retains binding activity, functionality, and is amenable to large-scale chemical synthesis for future clinical applications. In addition, the modeled RNA tertiary structure and protein/RNA docking predictions revealed key nucleotides within the aptamer critical for binding to PSMA and inhibiting its enzymatic activity. Finally, this work highlights the utility of existing RNA structural prediction and protein docking techniques that may be generally applicable to developing RNA aptamers optimized for therapeutic use.     

Hierarchy and value: An introduction

Martha J. Kaplan. 1995. Neither Cargo Nor Cult: Ritual Politics and Colonial Imagination in Fiji. London and Durham: Duke University Press, xviii + 236 pp.

Martha Mundy. 1995. Domestic Government: Kinship, Community and Polity in North Yemen. London: I.B. Tauris Publishers. 317 pp.

Thomas Csordas (ed.). 1994. Embodiment and Experience: The Existential Ground of Culture and Self. Cambridge: Cambridge University Press. xi + 294 pp.

Philip L. Kohl &; Clare Fawcett (eds). 1995. Nationalism, Politics, and the Practice of Archaeology. Cambridge: Cambridge University Press. xi + 329 pp.

Joanna B. Eicher (ed.). 1995. Dress and Ethnicity: Change Across Space and Time. Oxford: Berg Publishers. xiv + 316 pp.

Anatoly M. Khazanov. 1995. After the USSR: Ethnicity, Nationalism, and Politics in the Commonwealth of Independent States. Madison: The University of Wisconsin Press. 334 pp.

Pertti J. Anttonen &; Reimund Kvideland (eds). 1994. Nordic Frontiers: Recent Issues in the Study of Modern Traditional Culture in the Nordic Countries. Turku: Nordic Institute of Folklore. 238 pp.

Hugo G. Nutini. 1995. The Wages of Conquest: The Mexican Aristocracy in the Context of Western Aristocracies. Ann Arbor: University of Michigan Press. 444 pp.     

Variability in the developmental life history of the genus Gorilla

Life history is influenced by factors both intrinsic (e.g., body and relative brain size) and extrinsic (e.g., diet, environmental instability) to organisms. In this study, we examine the prediction that energetic risk influences the life history of gorillas. Recent comparisons suggest that the more frugivorous western lowland gorilla shows increased infant dependence, and thus a slower life history, than the primarily folivorous mountain gorilla to buffer against the risk of starvation during periods of food unpredictability. We further tested this hypothesis by incorporating additional life history data from wild western lowland gorillas and captive western lowland gorillas with the assumption that the latter live under ecological conditions of energetic risk that more closely resemble those of mountain gorillas and thus should show faster life histories than wild members of the species. Overall, we found captive western lowland and wild mountain gorillas to have faster developmental life histories than wild western lowland gorillas, weaning their infants approximately a year earlier and thus reducing interbirth intervals by a year. These results provide support that energetic risk plays an important role in determining gorilla life history. Unlike previous assertions, gorillas do not have substantially faster life histories, at least at the genus level, than other great apes. This calls for a re‐evaluation of theories concerning comparative ape life history and evolution and highlights the need for data from additional populations that vary in energetic risk. Am J Phys Anthropol 152:165–172, 2013. © 2013 Wiley Periodicals, Inc.     

The Platform Protein Is Essential for Type IV Pilus Biogenesis

A systematic genetic analysis was performed to identify the inner membrane proteins essential for type IV pilus (T4P) expression in Pseudomonas aeruginosa. By inactivating the retraction aspect of pilus function, genes essential for T4P assembly were discriminated. In contrast to previous studies in the T4P system of Neisseria spp., we found that components of the inner membrane subcomplex consisting of PilMNOP were not essential for surface pilus expression, whereas the highly conserved inner membrane protein PilC was essential. Here, we present data that PilC may coordinate the activity of cytoplasmic polymerization (PilB) and depolymerization (PilT) ATPases via their interactions with its two cytoplasmic domains. Using in vitro co-affinity purification, we show that PilB interacts with the N-terminal cytoplasmic domain of PilC. We hypothesized that PilT similarly interacts with the PilC C-terminal cytoplasmic domain. Overexpression of that domain in the wild-type protein reduced twitching motility by ∼50% compared with the vector control. Site-directed mutagenesis of conserved T4P-specific residues in the PilC C-terminal domain yielded mutant proteins that supported wild-type pilus assembly but had a reduced capacity to support twitching motility, suggesting impairment of putative PilC-PilT interactions. Taken together, our results show that PilC is an essential inner membrane component of the T4P system, controlling both pilus assembly and disassembly.     

Evaluating the Return in Ecosystem Services from Investment in Public Land Acquisitions

We evaluate the return on investment (ROI) from public land conservation in the state of Minnesota, USA. We use a spatially-explicit modeling tool, the Integrated Valuation of Ecosystem Services and Tradeoffs (InVEST), to estimate how changes in land use and land cover (LULC), including public land acquisitions for conservation, influence the joint provision and value of multiple ecosystem services. We calculate the ROI of a public conservation acquisition as the ratio of the present value of ecosystem services generated by the conservation to the cost of the conservation. For the land scenarios analyzed, carbon sequestration services generated the greatest benefits followed by water quality improvements and recreation opportunities. We found ROI values ranged from 0.21 to 5.28 depending on assumptions about future land use change, service values, and discount rate. Our study suggests conservation is a good investment as long as investments are targeted to areas with low land costs and high service values.