Normalized cDNA libraries from a porcine model of orthopedic implant-associated infection

Staphylococcal infections that result from an alteration in a patient's immune response at the surgical site are a major problem in procedures that incorporate biomaterials in trauma surgery and joint replacement. Diagnosis of infection based on pathogen detection is difficult and exacerbated by increasing numbers of partially or totally resistant strains of nosocomial pathogens, particularly Staphylococcus aureus. Expression profiling of a host's cellular immune response could facilitate the identification of the pathways involved in pathogen recognition and eradication and could lead to more rational design of drugs and therapies. To this end, we constructed and characterized ten individually tagged and directionally cloned cDNA libraries from peripheral blood cells (PBC), spleen (Sp), thymus (Th), lymph node (LN), and bone marrow (BM) from immunologically naive and challenged pigs as part of an implant-associated orthopedic model of deep infection. Three of these libraries were normalized at C _{0 t} values 5, 10, 20, and 30. The libraries comprise more than 20 million primary transformants with an average insert length >1.4 kb. Cluster analysis of 7620 ESTs revealed 1029 clusters containing an average of 3.6 sequences and 3846 singletons. Gene discovery is estimated to be ∼64%. Searches of public databases resulted in 49.3% annotated porcine sequences, of which 22.2% had significant homologies to ESTs from a variety of species, and 28.5% were without a significant match in any public database. We also identified 9.1% ESTs as involved in host cell and organism defense and 11.5% related to cell signaling and communication. These sequences, together with the 28.5% appearing as novel, are of specific interest to the infectious disease process.     

The Giardia ventrolateral flange is a lamellar membrane protrusion that supports attachment

Attachment to the intestinal epithelium is critical to the lifestyle of the ubiquitous parasite Giardia lamblia. The ventrolateral flange is a sheet-like membrane protrusion at the interface between parasites and attached surfaces. This structure has been implicated in attachment, but its role has been poorly defined. Here, we identified a novel actin associated protein with putative WH2-like actin binding domains we named Flangin. Flangin complexes with Giardia actin (GlActin) and is enriched in the ventrolateral flange making it a valuable marker for studying the flanges’ role in Giardia biology. Live imaging revealed that the flange grows to around 1 μm in width after cytokinesis, then remains uniform in size during interphase, grows in mitosis, and is resorbed during cytokinesis. A flangin truncation mutant stabilizes the flange and blocks cytokinesis, indicating that flange disassembly is necessary for rapid myosin-independent cytokinesis in Giardia. Rho family GTPases are important regulators of membrane protrusions and GlRac, the sole Rho family GTPase in Giardia, was localized to the flange. Knockdown of Flangin, GlActin, and GlRac result in flange formation defects. This indicates a conserved role for GlRac and GlActin in forming membrane protrusions, despite the absence of canonical actin binding proteins that link Rho GTPase signaling to lamellipodia formation. Flangin-depleted parasites had reduced surface contact and when challenged with fluid shear force in flow chambers they had a reduced ability to remain attached, confirming a role for the flange in attachment. This secondary attachment mechanism complements the microtubule based adhesive ventral disc, a feature that may be particularly important during mitosis when the parental ventral disc disassembles in preparation for cytokinesis. This work supports the emerging view that Giardia’s unconventional actin cytoskeleton has an important role in supporting parasite attachment.     

Differential regulation of somatodendritic and nerve terminal dopamine release by serotonergic innervation of substantia nigra

Nigrostriatal dopaminergic neurons release dopamine from dendrites in substantia nigra and axon terminals in striatum. The cellular mechanisms for somatodendritic and axonal dopamine release are similar, but somatodendritic and nerve terminal dopamine release may not always occur in parallel. The current studies used in vivo microdialysis to simultaneously measure changes in dendritic and nerve terminal dopamine efflux in substantia nigra and ipsilateral striatum respectively, following intranigral application of various drugs by reverse dialysis through the nigral probe. The serotonin releasers (+/-)-fenfluramine (100 micro m) and (+)-fenfluramine (100 micro m) significantly increased dendritic dopamine efflux without affecting extracellular dopamine in striatum. The non-selective serotonin receptor agonist 1-(m-chlorophenyl)-piperazine (100 micro m) elicited a similar pattern of dopamine release in substantia nigra and striatum. NMDA (33 micro m) produced an increase in nigral dopamine of a similar magnitude to mCPP or either fenfluramine drug. However, NMDA also induced a concurrent increase in striatal dopamine. The D2 agonist quinpirole (100 micro m) had a parallel inhibitory effect on dopamine release from dendritic and terminal sites as well. Taken together, these data suggest that serotonergic afferents to substantia nigra may evoke dendritic dopamine release through a mechanism that is uncoupled from the impulse-dependent control of nerve terminal dopamine release.     

A Pilot Randomized Controlled Trial of a Clinic and Home‐Based Behavioral Intervention to Decrease Obesity in Preschoolers

We evaluated the efficacy of a 6‐month clinic and home‐based behavioral intervention (Learning about Activity and Understanding Nutrition for Child Health; LAUNCH) to reduce obesity in preschool children ≥95th BMI percentile compared to enhanced standard of care (Pediatrician Counseling; PC). LAUNCH was a family‐based behavioral intervention that taught parents to use child behavior management strategies to increase healthy eating and activity for their children and themselves. PC presented the same diet and activity recommendations, but was delivered in a one‐time PC session. Eighteen children aged 2–5 years (mean 4.71 ± 1.01) with an average BMI percentile of 98 (±1.60) and an overweight parent were randomized to LAUNCH or PC. Assessments were conducted at baseline, 6 months (end of LAUNCH treatment) and 12 months (6 months following LAUNCH treatment). LAUNCH showed a significantly greater decrease on the primary outcomes of child at month 6 (post‐treatment) BMI z (?0.59 ± 0.17), BMI percentile (?2.4 ± 1.0), and weight gain (?2.7 kg ± 1.2) than PC and this difference was maintained at follow‐up (month 12). LAUNCH parents also had a significantly greater weight loss (?5.5 kg ± 0.9) at month 6 and 12 (?8.0 kg ± 3.5) than PC parents. Based on the data from this small sample, an intensive intervention that includes child behavior management strategies to improve healthy eating and activity appears more promising in reducing preschool obesity than a low intensity intervention that is typical of treatment that could be delivered in primary care.     

A fluorescence-based high-throughput assay for antimicrotubule drugs

With the advent of combinatorial chemistry and the extensive libraries of potential drugs produced from it, there is a growing need for rapid sensitive, high-throughput screening for drug potency. Microtubules are important targets for anticancer agents, and new antimicrotubule compounds are of continued interest in drug development. The in vitro potency of antimicrotubule drugs may be evaluated by measuring the extent of tubulin assembly. The extent of polymerization is proportional to the turbidity of the solution, which usually has been measured as apparent absorption. The turbidity method has inherent problems that hinder its adaptation to a high-throughput format, such as a requirement for high protein concentrations and a high coefficient of variation. We present here a high-throughput assay for antimicrotubule activity in which fluorescence is used to monitor microtubule assembly. Both assembly-inhibiting and assembly-promoting compounds can be evaluated. The assay is rapid and easy to perform, and the data are reliable, with good accuracy and reproducibility.