Dilutional control of prothrombin activation at physiologically relevant shear rates

The generation of proteolyzed prothrombin species by preassembled prothrombinase in phospholipid-coated glass capillaries was studied at physiologic shear rates (100–1000 s⁻¹). The concentration of active thrombin species (α-thrombin and meizothrombin) reaches a steady state, which varies inversely with shear rate. When corrected for shear rate, steady-state levels of active thrombin species exhibit no variation and a Michaelis-Menten analysis reveals that chemistry of this reaction is invariant between open and closed systems; collectively, these data imply that variations with shear rate arise from dilutional effects. Significantly, the major products observed include nonreactive species arising from the loss of prothrombin's phospholipid binding domain (des F1 species). A numerical model developed to investigate the spatial and temporal distribution of active thrombin species within the capillary reasonably approximates the observed output of total thrombin species at different shears; it also predicts concentrations of active thrombin species in the wall region sufficient to account for observed levels of des FI species. The predominant feedback formation of nonreactive species and high levels of the primarily anticoagulant intermediate meizothrombin (∼40% of total active thrombin species) may provide a mechanism to prevent thrombus propagation downstream of a site of thrombosis or hemorrhage.     

Modern-day challenges in therapeutic protein production

The market for therapeutic proteins is on the rise, plagued by several challenges related to production amounts and costs. Solutions to these problems are widely thought to come from academia, governments and production companies. This conference aimed to bring experts in the industry together under one roof, in order to demystify several novel technologies in therapeutic protein development. Key topics included analytical tools for protein stability and ligand interactions, measurement of protein aggregates as small as 30 nm and reducing production costs, just to name a few. The need to eliminate protein aggregates early during bioprocessing was emphasized. Finally, several companies presented novel technologies related to therapeutic protein development.     

Staphylococcus aureus CidA and LrgA proteins exhibit holin-like properties

The Staphylococcus aureus cid and lrg operons are known to be involved in biofilm formation by controlling cell lysis and the release of genomic DNA, which ultimately becomes a structural component of the biofilm matrix. Although the molecular mechanisms controlling cell death and lysis are unknown, it has been hypothesized that the cidA and lrgA genes encode holin- and antiholin-like proteins and function to regulate these processes similarly to bacteriophage-induced death and lysis. In this study, we focused on the biochemical and molecular characterization of CidA and LrgA with the goal of testing the holin model. First, membrane fractionation and fluorescent protein fusion studies revealed that CidA and LrgA are membrane-associated proteins. Furthermore, similarly to holins, CidA and LrgA were found to oligomerize into high-molecular-mass complexes whose formation was dependent on disulfide bonds formed between cysteine residues. To determine the function of disulfide bond-dependent oligomerization of CidA, an S. aureus mutant in which the wild-type copy of the cidA gene was replaced with the cysteine mutant allele was generated. As determined by β-galactosidase release assays, this mutant exhibited increased cell lysis during stationary phase, suggesting that oligomerization has a negative impact on this process. When analyzed for biofilm development and maturation, this mutant displayed increased biofilm adhesion in a static assay and a greater amount of dead-cell accumulation during biofilm maturation. These studies support the model that CidA and LrgA proteins are bacterial holin-/antiholin-like proteins that function to control cell death and lysis during biofilm development.     

IgE responsiveness to Dermatophagoides farinae in West Highland white terrier dogs is associated with region on CFA35

Immunoglobulin E (IgE)-mediated hypersensitivity against environmental allergens, commonly including Dermatophagoides farinae, is associated with atopic diseases in both humans and dogs. We have recently identified a family of clinically healthy West Highland white terriers (WHWTs) with high-serum D. farinae-IgE levels. In this study, we investigated the genetic mechanism controlling IgE responsiveness in dogs by performing a genome-wide association study (GWAS) using the Affymetrix V2 Dog SNP array in 31 high-IgE and 24 low-IgE responder WHWTs. A gene-dropping simulation method, using SIB-PAIR software, showed significant allelic association between serum D. farinae-specific IgE levels and a 2.3-Mb area on CFA35 (best empirical P = 1 × 10(-5)). A nearby candidate gene, CD83, encodes a protein which has important immunological functions in antigen presentation and regulation of humoral immune responses. We sequenced this gene in 2 high-IgE responders and 2 low-IgE responders and identified an intronic polymorphic repeat sequence with a predicted functional effect, but the association was insufficient to explain the GWAS association signal in this population (P = 1 × 10(-3)). Further studies are necessary to investigate the significance of these findings for IgE responsiveness and atopic disease in the dog.     

Selection on the human bitter taste gene, TAS2R16, in Eurasian populations

Bitter taste is one of the most important senses alerting humans to noxious foods. In gatherer communities, sensitivity to bitterness is presumably advantageous because of various noxious plants. TAS2R16 is the gene coding the taste receptor molecules for some of the most common toxins in plants. A previous study of this gene indicated selection has increased the frequency of a derived allele in this gene that arose before the human expansion out of Africa. We have applied a different methodology for detecting selection, the Long Range Haplotype (LRH) analysis, to TAS2R16 in a larger sampling of populations from around the world. The haplotype with the derived alleles at both the functional polymorphism and a polymorphism in the regulatory region of TAS2R16 showed evidence for recent positive selection in most of the Eurasian populations, though the highest selection signal occurs in Mbuti Pygmies, an African hunter-gatherer group. In Eurasia, only populations of Mesopotamia and the southeast coast of China have no signals of selection. The evidence of recent selection found in most Eurasian populations differs from the geographic pattern seen in the earlier study of selection. One can speculate that the difference may result from a gathering lifestyle extending into the most recent 10,000 yrs and the need to recognize newly encountered bitter natural toxins as populations expanded into new environments and the biota changes with the ending of the most recent ice age. Alternatively, the promoter region variant may be a marker for altered function beyond what the derived amino acid allele conferred.     

Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy

In this protocol, we describe a 3D imaging technique known as 'volume electron microscopy' or 'focused ion beam scanning electron microscopy (FIB/SEM)' applied to biological tissues. A scanning electron microscope equipped with a focused gallium ion beam, used to sequentially mill away the sample surface, and a backscattered electron (BSE) detector, used to image the milled surfaces, generates a large series of images that can be combined into a 3D rendered image of stained and embedded biological tissue. Structural information over volumes of tens of thousands of cubic micrometers is possible, revealing complex microanatomy with subcellular resolution. Methods are presented for tissue processing, for the enhancement of contrast with osmium tetroxide/potassium ferricyanide, for BSE imaging, for the preparation and platinum deposition over a selected site in the embedded tissue block, and for sequential data collection with ion beam milling; all this takes approximately 90 h. The imaging conditions, procedures for alternate milling and data acquisition and techniques for processing and partitioning the 3D data set are also described; these processes take approxiamtely 30 h. The protocol is illustrated by application to developing chick cornea, in which cells organize collagen fibril bundles into complex, multilamellar structures essential for transparency in the mature connective tissue matrix. The techniques described could have wide application in a range of fields, including pathology, developmental biology, microstructural anatomy and regenerative medicine.