We find that conjugation and chemical composition can alter fundamental aspects of aptamer residence in circulation and distribution to tissues. Though the primary effect of PEGylation was on aptamer clearance, the prolonged systemic exposure afforded by presence of the 20 kDa moiety appeared to facilitate distribution of aptamer to tissues, particularly those of highly perfused organs.

We constructed a library of >10(12) unique, covalently coupled mRNA-protein molecules by randomizing three exposed loops of an immunoglobulin-like protein, the tenth fibronectin type III domain (10Fn3). The antibody mimics that bound TNF-alpha were isolated from the library using mRNA display. Ten rounds of selection produced 10Fn3 variants that bound TNF-alpha with dissociation constants (K(d)) between 1 and 24 nM. After affinity maturation, the lowest K(d) measured was 20 pM. Selected antibody mimics were shown to capture TNF-alpha when immobilized in a protein microarray. 10Fn3-based scaffold libraries and mRNA-display allow the isolation of high-affinity, specific antigen binding proteins; potential applications of such binding proteins include diagnostic protein microarrays and protein therapeutics.

Major ABO incompatibility may lead to delayed reticulocyte engraftment, resulting in prolonged transfusion dependency and increased risks of transmission of infection and iron overload. Therefore, therapeutic strategies should be taken into consideration to allow erythroid reconstitution in these patients.

Hydrogels that provide mechanical support and sustainedly release therapeutics have been used to treat tendon injuries. However, most hydrogels are insufficiently tough, release drugs in bursts, and require cell infiltration or suturing to integrate with surrounding tissue. Here, we report that a hydrogel serving as a high-capacity drug depot and combining a dissipative tough matrix on one side and a chitosan adhesive surface on the other side supports tendon gliding and strong adhesion (larger than 1,000 J/m2) to tendon on opposite surfaces of the hydrogel, as we show with porcine and human tendon preparations during cyclic-friction loadings. The hydrogel is biocompatible, strongly adheres to patellar, supraspinatus and Achilles tendons of live rats, boosted healing and reduced scar formation in a rat model of Achilles-tendon rupture, and sustainedly released the corticosteroid triamcinolone acetonide in a rat model of patellar tendon injury, reducing inflammation, modulating chemokine secretion, recruiting tendon stem and progenitor cells, and promoting macrophage polarization to the M2 phenotype. Hydrogels with ‘Janus’ surfaces and sustained-drug-release functionality could be designed for a range of biomedical applications.

The hypothesis that forced-air warming preserves core temperature better than circulating-water mattresses was tested in: (a) 16 adults undergoing major maxillofacial surgery, including radical node resection and flap reconstruction; (b) 53 adults undergoing hip arthroplasty, having approximately 25% of their body surface area available for warming; (c) 20 infants undergoing minor maxillofacial surgery; and (d) 10 young children undergoing pelvic or femoral osteotomies. Patients having each type of surgery were randomly assigned to forced-air warming (approximately 40 degrees C) or conductive warming using a full-length circulating-water mattress at 40 degrees C. Forced-air warming was applied to the legs of the adults undergoing maxillofacial surgery and to one arm, the shoulders, and the neck in the adults undergoing hip arthroplasty; a U-shaped, tubular forced-air cover was positioned around the pediatric patients. Core temperatures increased in all patients given forced-air warming and decreased or remained constant in those without active warming. Furthermore, we needed to decrease the temperature of the warmer from high to medium (approximately 37 degrees C) in most patients assigned to forced-air warming to prevent hyperthermia. After 15 h of anesthesia, rectal temperatures in the adults undergoing maxillofacial surgery were 3.4 degrees C higher in the forced-air group (P < 0.01). After 4 h of anesthesia, esophageal temperatures had increased 0.8 +/- 0.5 degrees C in the patients warmed with forced-air and decreased 0.8 +/- 0.3 degrees C in those warmed by circulating-water mattresses (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Reportedly, during spinal anesthesia, the shivering threshold is reduced approximately 1 degree C but the vasoconstriction threshold remains normal. Such divergence between the shivering and vasoconstriction thresholds is an unusual pattern of thermoregulatory impairment and suggests that the mechanisms of impairment during regional anesthesia may be especially complex. Accordingly, we sought to define the pattern of thermoregulatory impairment during spinal anesthesia by measuring response thresholds. Seven healthy women volunteered to participate on two study days. On one day, we evaluated thermoregulatory responses to hypothermia and hyperthermia during spinal anesthesia; on the other day, responses were evaluated without anesthesia. Upper body skin temperature was kept constant throughout the study. The volunteers were warmed via the lower body and cooled by central venous infusion of cold fluid. The core temperatures triggering a sweating rate of 40 g.m-2 x h-1, a finger flow of 0.1 mL/min, and a marked and sustained increase in oxygen consumption were considered the thermoregulatory thresholds for sweating, vasoconstriction, and shivering, respectively. Spinal anesthesia significantly decreased the thresholds for vasoconstriction and shivering, and the decrease in each was approximately 0.5 degree C. The range of temperatures not triggering thermoregulatory responses (those between sweating and vasoconstriction) was 0.9 +/- 0.6 degree C during spinal anesthesia. The synchronous decrease in the shivering and vasoconstriction thresholds during spinal anesthesia is consistent with thermoregulatory impairment resulting from altered afferent thermal input.

We describe the synthesis of a novel type of mRNA template and its use in the preparation of mRNA-protein fusions. A light-induced psoralen crosslinking reaction was used to attach a puromycin-containing oligonucleotide to the 3'-end of an mRNA template. The photo-crosslinked template was found to undergo efficient mRNA-protein fusion formation in rabbit reticulocyte lysate. Fusion formation was subsequently tested with templates carrying puromycin linkers of different length and chemical composition. Short linkers with multiple triethyleneglycol phosphate building blocks allowed the most efficient fusion formation under a wide range of salt conditions. The present method simplifies the preparation of mRNA-protein fusions and thus significantly accelerates the in vitro protein evolution procedure which involves repetitive cycles of fusion production and selection.

Two molecular sensors that specifically recognize ADP in a background of over 100-fold molar excess of ATP are described. These sensors are nucleic-acid based and comprise a general method for monitoring protein kinase activity. The ADP-aptamer scintillation proximity assay is configured in a single-step, homogeneous format while the allosteric ribozyme (RiboReporter) sensor generates a fluorescent signal upon ADP-dependent ribozyme self-cleavage. Both systems perform well when configured for high-throughput screening and have been used to rediscover a known protein kinase inhibitor in a high-throughput screening format.