Proteomics of rat biological fluids--the tenth anniversary update

In addition to a remarkable sexual dimorphism of serum and urine proteomes, the rat is exceptional for the wide difference between the serum patterns during an acute phase reaction vs baseline conditions. This feature allows monitoring with high sensitivity onset and progression of any pathological state that involves an inflammatory component as well as assessing the outcome of any therapeutic intervention. Reference maps have been defined for the proteomes of serum, urine, cerebrospinal fluid and bronchoalveolar lavage fluid. For both serum and urine most of the proteomic investigations have dealt with toxicological testing, for BALF with allergic or irritative reactions, whereas with CSF the main aim was the characterization of rat models of neurological disorders. When surveying more than ten years of literature on rat biological fluid proteomics, it is puzzling to see how seldom a consistent analytical plan has been set up for the comparative investigation on two or more types of sample, whether to fully characterize a disease model or to evaluate pharmacological/toxicological effects of a drug. It is also regrettable that in several cases only a negligible part of the results is discussed at length whereas most data are not even made known to the scientific community.     

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 μm in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 μm below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.     

Blood collection for biochemical analysis in adult zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies. There are relatively few publications related to adult zebrafish physiology of organs and systems, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. Recently, Gupta et al. described how to dissect adult zebrafish organs, Kinkle et al. described how to perform intraperitoneal injections, and Pugach et al. described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish. The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.     

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient''s blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient''s blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 °C, the other sample represented the patient''s baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 °C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. ^2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.     

CO2‐caused change in plant species composition rivals the shift in vegetation between mid‐grass and tallgrass prairies

Atmospheric CO₂ enrichment usually changes the relative contributions of plant species to biomass production of grasslands, but the types of species favored and mechanisms by which change is mediated differ among ecosystems. We measured changes in the contributions of C₃ perennial forbs and C₄ grasses to aboveground biomass production of tallgrass prairie assemblages grown along a field CO₂ gradient (250–500 μmol mol^?1) in central Texas USA. Vegetation was grown on three soil types and irrigated each season with water equivalent to the growing season mean of precipitation for the area. We predicted that CO₂ enrichment would increase the forb contribution to community production, and favor tall‐grasses over mid‐grasses by increasing soil water content and reducing the frequency with which soil water fell below a limitation threshold. CO₂ enrichment favored forbs over grasses on only one of three soil types, a Mollisol. The grass fraction of production increased dramatically across the CO₂ gradient on all soils. Contribution of the tall‐grass Sorghastrum nutans to production increased at elevated CO₂ on the two most coarse‐textured of the soils studied, a clay Mollisol and sandy Alfisol. The CO₂‐caused increase in Sorghastrum was accompanied by an offsetting decline in production of the mid‐grass Bouteloua curtipendula. Increased CO₂ favored the tall‐grass over mid‐grass by increasing soil water content and apparently intensifying competition for light or other resources (Mollisol) or reducing the frequency with which soil water dipped below threshold levels (Alfisol). An increase in CO₂ of 250 μmol mol^?1 above the pre‐industrial level thus led to a shift in the relative production of established species that is similar in magnitude to differences observed between mid‐grass and tallgrass prairies along a precipitation gradient in the central USA. By reducing water limitation to plants, atmospheric CO₂ enrichment may alter the composition and even structure of grassland vegetation.     

An in vivo selection method to optimize trans-splicing ribozymes

Group I intron ribozymes can repair mutated mRNAs by replacing the 3′-terminal portion of the mRNA with their own 3′-exon. This trans-splicing reaction has the potential to treat genetic disorders and to selectively kill cancer cells or virus-infected cells. However, these ribozymes have not yet been used in therapy, partially due to a low in vivo trans-splicing efficiency. Previous strategies to improve the trans-splicing efficiencies focused on designing and testing individual ribozyme constructs. Here we describe a method that selects the most efficient ribozymes from millions of ribozyme variants. This method uses an in vivo rescue assay where the mRNA of an inactivated antibiotic resistance gene is repaired by trans-splicing group I intron ribozymes. Bacterial cells that express efficient trans-splicing ribozymes are able to grow on medium containing the antibiotic chloramphenicol. We randomized a 5′-terminal sequence of the Tetrahymena thermophila group I intron and screened a library with 9 × 10⁶ ribozyme variants for the best trans-splicing activity. The resulting ribozymes showed increased trans-splicing efficiency and help the design of efficient trans-splicing ribozymes for different sequence contexts. This in vivo selection method can now be used to optimize any sequence in trans-splicing ribozymes.     

Neurohormonal and cytokine fluctuations following transcatheter closure for an atrial septal defect

Introduction

Inflammation and neurohormonal activation are considered to be involved in the development of earlier and/or later complications in congenital heart disease patients, even after a successful repair of the lesion. It is not yet clarified what is the role of the therapeutic interventions in the occurrence of such a response and how it could be associated with possible postoperative complications.

Aim

We sought to assess the inflammatory and neurohormonal response to transcatheter closure of secundum type atrial septal defects (ASD) over a six-month follow-up period. We also evaluated the association between the respective markers and catheterization data as well as echocardiographic measurements.

Methods

Plasma concentrations of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), N-terminal-proatrial natriuretic peptide (NT-proANP) and N-terminal-probrain natriuretic peptide (NT-proBNP) were assessed and echocardiographic measurements were performed in twenty-eight patients with atrial septal defect prior to, and at the first, second and sixth months post transcatheter closure. Thirty-three age-matched healthy volunteers were also enrolled.

Results

IL-6 plasma levels, although higher preoperatively, [physical logarithm (ln) IL-6: 3.37 ± 0.66 vs 2.92 ± 0.44 pg/ml, p = 0.015], reached control levels postoperatively, at the end of the third month, whereas TNF-α and IL-10 were not influenced by the procedure. NT-proANP levels were elevated preoperatively compared to the control group (ln NT-proANP 3.78 ± 0.572 vs 3.48 ± 0.30, p = 0.031), with a further significant increase during the 1st month (ln NT-proANP 3.78 ± 0.572 vs 4.2 ± 0.42, p = 0.006), following the pattern of the left atrial volume enlargement, and remained high even 6 months after the procedure .On the other hand, the initially normal concentrations of NT-proBNP, after a transient significant increase during the first month postoperatively (ln NT-proBNP 3.56 ± 0.94 vs 4.58 ± 0.91, p < 0.0001) returned to the controls’ levels at the end of the third month. Preoperative concentrations of NT-proANP positively correlated with NT-proBNP concentrations and pulmonary to systemic flow ratio (Qp/Qs).

Conclusions

Transcatheter closure could improve, on a mid- term basis, the inflammatory process but natriuretic peptides’ secretion continues in parallel with left atrial volume increase. Further follow up is required to determine the long-term progress of the inflammatory and neurohormonal response to the procedure.