Identification of the material parameters of soft tissues in the compressed leg

Elastic compression is recommended in prophylaxis and the treatment of venous disorder of the human leg. However, the mechanisms of compression are not completely understood and the response of internal tissues to the external pressure is partially unknown. To address this later issue, a 3D FE model of a human leg is developed. The geometry is derived from 3D CT scans. The FE model is made up of soft tissues and rigid bones. An inverse method is applied to identify the properties of soft tissues which are modelled as hyperelastic, near-incompressible, homogeneous and isotropic materials. The principle is to calibrate the constitutive properties using CT scans carried out with and without the presence of a compression sock. The deformed geometry computed by the calibrated FE model is in agreement with the geometry deduced from the CT scans. The model also provides the internal pressure distribution, which may lead to medical exploitation in the future.     

Dual regulation of macrophage migration inhibitory factor (MIF) expression in hypoxia by CREB and HIF-1

Macrophage migration inhibitory factor (MIF) is a well-described pro-inflammatory mediator that has also been implicated in the process of oncogenic transformation and tumor progression. However, despite the compelling evidence that MIF is overexpressed in, and contributes to, the pathology of inflammatory and malignant diseases the mechanisms that contribute to exaggerated expression of MIF have been poorly described. Here we show that hypoxia, and specifically HIF-1alpha, is a potent and rapid inducer of MIF expression. In addition, we demonstrate that hypoxia-induced MIF expression is dependent upon a HRE in the 5'UTR of the MIF gene but is further modulated by CREB expression. We propose a model where hypoxia-induced MIF expression is driven by HIF-1 but amplified by hypoxia-induced degradation of CREB. Given the importance of MIF in inflammatory and malignant diseases these data reveal a HIF-1-mediated pathway as a potential therapeutic target for suppression of MIF expression in hypoxic tissues.     

Evidence of key tinnitus-related brain regions documented by a unique combination of manganese-enhanced MRI and acoustic startle reflex testing

Animal models continue to improve our understanding of tinnitus pathogenesis and aid in development of new treatments. However, there are no diagnostic biomarkers for tinnitus-related pathophysiology for use in awake, freely moving animals. To address this disparity, two complementary methods were combined to examine reliable tinnitus models (rats repeatedly administered salicylate or exposed to a single noise event): inhibition of acoustic startle and manganese-enhanced MRI. Salicylate-induced tinnitus resulted in wide spread supernormal manganese uptake compared to noise-induced tinnitus. Neither model demonstrated significant differences in the auditory cortex. Only in the dorsal cortex of the inferior colliculus (DCIC) did both models exhibit supernormal uptake. Therefore, abnormal membrane depolarization in the DCIC appears to be important in tinnitus-mediated activity. Our results provide the foundation for future studies correlating the severity and longevity of tinnitus with hearing loss and neuronal activity in specific brain regions and tools for evaluating treatment efficacy across paradigms.     

Human Regulatory Subunit RIβ of cAMP-Dependent Protein Kinases: Expression, Holoenzyme Formation and Microinjection into Living Cells

The human regulatory subunit RIβ of cAMP-dependent protein kinases was expressed in Escherichia coli as a fusion protein with glutathione S -transferase. Purification was performed by affinity chromatography on glutathione-agarose beads after cleavage with thrombin. The human recombinant Riff protein migrated at 55 kDa on SDS-PAGE and displayed immunoreactivity with an anti-human RIβ antiserum. Furthermore, the purified recombinant RIβ protein was shown to exist as a dimer that was able to form holoenzyme with the catalytic subunit Cα. The rate of RIβ₂Cα₂ holoenzyme formation was faster in the presence than in the absence of MgATP. The kinase activity measured before and after adding cAMP to the holoenzyme showed that the presence of cAMP resulted in holoenzyme dissociation and release of active Cα-subunit, due to cAMP binding to RIβ. Compared to a RIα₂Cα₂ holoenzyme, the RIβ₂Cα₂ holoenzyme exhibited a more than twofold higher sensitivity to cAMP. The subcellular localization of Riff was analyzed in quiescent REF-52 fibroblasts and Wistar rat thyroid (WRT) cells after microinjection of fluorescently labeled proteins into the cytoplasm. A cytoplasmic distribution was observed when free RIβ was injected, whereas free Cα injected into the cytoplasm appeared in the nucleus. When holoenzymes with labeled Riff and unlabeled Cα, or unlabeled RIβ and labeled Cα, were injected, unstimulated cells showed fluorescence in the cytoplasm of both cell types. REF-52 cells stimulated with 8-bromo-cAMP (8-Br-cAMP) and WRT cells treated with thyrotropin (TSH) showed fluorescence mainly in the cytoplasm when RIβ was the labeled subunit of the in vivo dissociated bioenzyme. In contrast, nuclear fluorescence was evident from the release and translocation of labeled Cα from the holoenzyme complex after stimulation with 8-Br-cAMP or TSH.     

Trophoblast cell line resistance to NK lysis mainly involves an HLA class I-independent mechanism

The lack of classical HLA molecules on trophoblast prevents allorecognition by maternal T lymphocytes, but poses the problem of susceptibility to NK lysis. Expression of the nonclassical class I molecule, HLA-G, on cytotrophoblast may provide the protective effect. However, the class I-negative syncytiotrophoblast escapes NK lysis by maternal PBL. In addition, while HLA-G-expressing transfectants of LCL.721.221 cells are protected from lymphokine-activated killer lysis, extravillous cytotrophoblast cells and HLA-G-expressing choriocarcinoma cells (CC) are not. The aim of this work was therefore to clarify the role of HLA class I expression on trophoblast cell resistance to NK lysis and on their susceptibility to lymphokine-activated killer lysis. Our results showed that both JAR (HLA class I-negative) and JEG-3 (HLA-G- and HLA-Cw4-positive) cells were resistant to NK lysis by PBL and were equally lysed by IL-2-stimulated PBL isolated from a given donor. In agreement, down-regulating HLA class I expression on JEG-3 cells by acid treatment, masking these molecules or the putative HLA-G (or HLA-E) receptor CD94/NKG2 and the CD158a/p58.1 NKR with mAbs, and inducing self class I molecule expression on JAR cells did not affect NK or LAK lysis of CC. These results demonstrate that the resistance of CC to NK lysis mainly involves an HLA class I-independent mechanism(s). In addition, we show that the expression of a classical class I target molecule (HLA-B7) on JAR cells is insufficient to induce lysis by allospecific polyclonal CTL.     

Structure-activity relationship of a novel class of naphthyl amide KATP channel openers

We have discovered a novel series of N-[2-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-naphthalen-1-yl] amides that are potent openers of K(ATP) channels and investigated structure-activity relationships (SAR) around the 1,2-disubstituted naphthyl core. A-151892, a prototype compound of this series, was found to be a potent and efficacious potassium channel opener in vitro in transfected Kir6.2/SUR2B cells and pig bladder strips. Additionally, A-151892 was found to selectively inhibit unstable bladder contractions in vivo in an obstructed rat model of myogenic bladder function     

RhoA-mediated Ca2+ sensitization in erectile function

A Rho-kinase inhibitor increases corpus cavernosum (CC) pressure in an in vivo rat model (Chitaley, K., Wingard, C. J., Webb, R. C., Branam, H., Stopper, V. S., Lewis, R. W., and Mills, T. M. (2001) Nat. Med. 7, 119-122) suggesting that Rho-mediated Ca(2+) sensitization of CC smooth muscle maintains the flaccid (contracted) state. We directly demonstrate Ca(2+) sensitization of permeabilized rabbit and human CC and identify a highly expressed molecular component of this pathway. Ca(2+) sensitization of force induced by endothelin or GTPgammaS was significantly greater in CC than in rabbit ileum smooth muscle and was accompanied by a 17-fold higher RhoA content. Pull-down assays with the RhoA binding domain of mDia showed the high RhoA content of CC to be available for activation by GTPgammaS. Ca(2+) sensitization induced by endothelin, phenylephrine, or GTPgammaS was completely relaxed by the Rho kinase inhibitor Y-27632. Human and rabbit CC both express the phosphatase inhibitor CPI-17, the myosin phosphatase regulatory (MYPT-1) and catalytic (PP1delta) subunits, and two isoforms of Rho kinase. We suggest that high expression of RhoA contributes, through RhoA-mediated Ca(2+) sensitization, to the flaccid state of CC that can be reversed by a water-soluble, orally active Rho kinase inhibitor suitable for therapy of erectile dysfunction.     

Selectin-like kinetics and biomechanics promote rapid platelet adhesion in flow: the GPIb(alpha)-vWF tether bond

The ability of platelets to tether to and translocate on injured vascular endothelium relies on the interaction between the platelet glycoprotein receptor Ib alpha (GPIb(alpha)) and the A1 domain of von Willebrand factor (vWF-A1). To date, limited information exists on the kinetics that govern platelet interactions with vWF in hemodynamic flow. We now report that the GPIb(alpha)-vWF-A1 tether bond displays similar kinetic attributes as the selectins including: 1) the requirement for a critical level of hydrodynamic flow to initiate adhesion, 2) short-lived tethering events at sites of vascular injury in vivo, and 3) a fast intrinsic dissociation rate constant, k(0)(off) (3.45 +/- 0.37 s(-1)). Values for k(off), as determined by pause time analysis of transient capture/release events, were also found to vary exponentially (4.2 +/- 0.8 s(-1) to 7.3 +/- 0.4 s(-1)) as a function of the force applied to the bond (from 36 to 217 pN). The biological importance of rapid bond dissociation in platelet adhesion is demonstrated by kinetic characterization of the A1 domain mutation, I546V that is associated with type 2B von Willebrand disease (vWD), a bleeding disorder that is due to the spontaneous binding of plasma vWF to circulating platelets. This mutation resulted in a loss of the shear threshold phenomenon, a approximately sixfold reduction in k(off), but no significant alteration in the ability of the tether bond to resist shear-induced forces. Thus, flow dependent adhesion and rapid and force-dependent kinetic properties are the predominant features of the GPIb(alpha)-vWF-A1 tether bond that in part may explain the preferential binding of platelets to vWF at sites of vascular injury, the lack of spontaneous platelet aggregation in circulating blood, and a mechanism to limit thrombus formation.     

A 3-year plankton DNA metabarcoding survey reveals marine biodiversity patterns in Australian coastal waters

Aim

To use a long-term collection of bulk plankton samples to test the capacity of DNA metabarcoding to characterize the spatial and seasonal patterns found within a range of zooplankton communities, and investigate links with concurrent abiotic data collected as part of Australia's Integrated Marine Observing System (IMOS) programme.

Location

Samples were sourced seasonally for 3 years from nine Pan-Australian marine sites (n = 90).

Methods

Here, we apply a multi-assay metabarcoding approach to environmental DNA extracted from bulk plankton samples. Six assays (targeting 16SrRNA and COI genes) were used to target, amplify and sequence the zooplankton diversity found within each sample. The data generated from each assay were filtered and clustered into OTUs prior to analysis. Abiotic IMOS data collected contemporaneously enabled us to explore the physical and chemical drivers of community composition.

Results

From over 25 million sequences, we identified in excess of 500 distinct taxa and detected clear spatial differences. We found that site and sea surface temperature are the most consistent predictors of differences between zooplankton communities. We detected endangered and invasive species such as the bryozoan Membranipora membranacea and the mollusc Maoricolpus roseus, and seasonal occurrences of species such as humpback whales (Megaptera novaeangliae). We also estimated the number of samples required to detect any significant seasonal changes. For OTU richness, this was found to be assay dependent and for OTU assemblage, a minimum of nine samples per season would be required.

Main Conclusion

Our results demonstrate the ability of DNA to capture and map zooplankton community changes in response to seasonal and spatial stressors and provide vital evidence to environmental stakeholders. We confirm that a metabarcoding method offers a practical opportunity for an ecosystem-wide approach to long-term biomonitoring and understanding marine biomes where morphological analysis is not feasible.     

Structure-activity studies for a novel series of tricyclic dihydropyridopyrazolones and dihydropyridoisoxazolones as K(ATP) channel openers

In search of a novel chemotype of K(ATP) channel openers a series of tricyclic dihydropyridopyrazolones and dihydropyridoisoxazolones was synthesized. It was found that cyclopentanone in the left hand portion of the molecule was 4-fold more potent than cyclohexanone. Introduction of gem-dimethyl groups as well as incorporation of oxygen in the cyclohexanone ring in the left hand portion of the molecule increased the potency 10-fold. In the right hand portion of the molecule, the NH-group of the pyrazolone can be effectively substituted by oxygen increasing the activity 5-fold. Incorporation of a methyl group adjacent to the dihydropyridine (DHP) nitrogen not only significantly boosted activity, but also provided an additional benefit of increased metabolic stability. In vitro tests on the tissue from pig bladder strips provided further confirmation of K(ATP) activity of these compounds.