Microporous cell‐laden hydrogels for engineered tissue constructs

In this article, we describe an approach to generate microporous cell‐laden hydrogels for fabricating biomimetic tissue engineered constructs. Micropores at different length scales were fabricated in cell‐laden hydrogels by micromolding fluidic channels and leaching sucrose crystals. Microengineered channels were created within cell‐laden hydrogel precursors containing agarose solution mixed with sucrose crystals. The rapid cooling of the agarose solution was used to gel the solution and form micropores in place of the sucrose crystals. The sucrose leaching process generated homogeneously distributed micropores within the gels, while enabling the direct immobilization of cells within the gels. We also characterized the physical, mechanical, and biological properties (i.e., microporosity, diffusivity, and cell viability) of cell‐laden agarose gels as a function of engineered porosity. The microporosity was controlled from 0% to 40% and the diffusivity of molecules in the porous agarose gels increased as compared to controls. Furthermore, the viability of human hepatic carcinoma cells that were cultured in microporous agarose gels corresponded to the diffusion profile generated away from the microchannels. Based on their enhanced diffusive properties, microporous cell‐laden hydrogels containing a microengineered fluidic channel can be a useful tool for generating tissue structures for regenerative medicine and drug discovery applications. Biotechnol. Bioeng. 2010; 106: 138–148. © 2010 Wiley Periodicals, Inc.     

Pollination strategies in Cretan Arum lilies

Flowers of the genus Arum are known to attract dung‐breeding flies and beetles through olfactory deceit. In addition to this strategy, the genus has evolved several other pollination mechanisms. The present study aimed to characterize the pollination strategies of the Cretan Arum species by investigating the flowering phenology, thermogeny, inflorescence odours, and the pollinating fauna. The results obtained show that Arum cyrenaicum and Arum concinnatum emit a strong dung smell and exhibit the distinctive features associated with this pollination syndrome. Both species are highly thermogenic, have a similar odour profile and attract small‐bodied Diptera. Although sharing the same habitat, these two plant species are never found growing sympatrically as a result of the early blooming period of A. cyrenaicum. By contrast, Arum creticum and Arum idaeum have evolved a more traditional and mutually beneficial pollination mechanism. The stinking smell has been replaced by a more flower‐like odour that attracts bees (Lasioglossum sp.) and, occasionally, bugs (Dionconotus cruentatus). Although attracting the same pollinator, the main compound present in the odour of A. creticum is different from that of A. idaeum. Principal component analysis (PCA), based on physiologically active components of the flower odours determined by testing on the antenna of the Lasioglossum bee, revealed two different clusters, indicating that pollinators can potentially discriminate between the odours of the two species. A further PCA on the main floral odour volatiles as identified by gas chroatography‐mass spectroscopy from all the Arum species under investigation displayed odour‐based similarities and differences among the species. The PCA‐gas chomotography‐electroantennographic detection active peaks analysis showed that the two species, A. creticum and A. idaeum, form two groups and are clearly separated from A. cyrenaicum and A. concinnatum, which, conversely, cluster together. The evolutionary forces and selective pressures leading to diversification of pollination mechanisms in the Cretan Arum spp. are discussed. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 991–1001.     

Thermoregulatory variation among populations of bats along a latitudinal gradient

Most studies of hibernation physiology sample individuals from populations within a single geographic area, yet some species have large ranges meaning populations likely experience area-specific levels of energetic challenges. As well, few studies have assessed within-season variation. Since physiological adjustments often are influenced by environmental factors, and the types of environments vary with geography, we expected variance in hibernation patterns among geographically separated populations. Our specific goal was to measure intraspecific variation in torpid metabolic rate (TMR) and body temperature (T _b) as a function of ambient temperature (T _a) for a non-migratory and migratory species to determine whether there is a continuum in physiological responses based on latitude. We chose big brown (Eptesicus fuscus) and eastern red bats (Lasiurus borealis) as model species and sampled individuals from populations throughout each species’ winter range. In both species, individuals from southern populations maintained higher TMR at cooler T _as and lower TMR at warmer T _as than those from northern populations. Big brown bats from southern populations regulated T _b during torpor at higher levels and there was no significant difference in T _b between populations of eastern red bats. Although metabolic responses were similar across the gradient between species, the effect was more dramatic in big brown bats. Our data demonstrate a continuum in thermoregulatory response, ranging from classic hibernation in northern populations to a pattern more akin to daily torpor in southern populations. Our research highlights the potential usefulness of bats as model organisms to address questions about within-species physiological variation in wild populations.     

Organellar vs cellular control of mitochondrial dynamics

Mitochondrial dynamics, the fusion and fission of individual mitochondrial units, is critical to the exchange of the metabolic, genetic and proteomic contents of individual mitochondria. In this regard, fusion and fission events have been shown to modulate mitochondrial bioenergetics, as well as several cellular processes including fuel sensing, ATP production, autophagy, apoptosis, and the cell cycle. Regulation of the dynamic events of fusion and fission occur at two redundant and interactive levels. Locally, the microenvironment of the individual mitochondrion can alter its ability to fuse, divide or move through the cell. Globally, nuclear-encoded processes and cellular ionic and second messenger systems can alter or activate mitochondrial proteins, regulate mitochondrial dynamics and concomitantly change the condition of the mitochondrial population. In this review we investigate the different global and local signals that control mitochondrial biology. This discussion is carried out to clarify the different signals that impact the status of the mitochondrial population.     

High prevalence of autoantibodies to RNA helicase A in Mexican patients with systemic lupus erythematosus

Introduction

Autoantibodies to RNA helicase A (RHA) were reported as a new serological marker of systemic lupus erythematosus (SLE) associated with early stage of the disease. Anti-RHA and other autoantibodies in Mexican SLE patients and their correlation with clinical and immunological features were examined.

Methods

Autoantibodies in sera from 62 Mexican SLE patients were tested by immunoprecipitation of ³⁵S-labeled K562 cell extract and enzyme-linked immunosorbent assay (anti-U1RNP/Sm, ribosomal P, β2GPI, and dsDNA). Anti-RHA was screened based on the immunoprecipitation of the 140-kDa protein, the identity of which was verified by Western blot using rabbit anti-RHA serum. Clinical and immunological characteristics of anti-RHA-positive patients were analyzed.

Results

Anti-RHA was detected in 23% (14/62) of patients, a prevalence higher than that of anti-Sm (13%, 8/62). Prevalence and levels of various autoantibodies were not clearly different between anti-RHA (+) vs. (-) cases, although there was a trend of higher levels of anti-RHA antibodies in patients without anti-U1RNP/Sm (P = 0.07). Both anti-RHA and -Sm were common in cases within one year of diagnosis; however, the prevalence and levels of anti-RHA in patients years after diagnosis did not reduce dramatically, unlike a previous report in American patients. This suggests that the high prevalence of anti-RHA in Mexican patients may be due to relatively stable production of anti-RHA.

Conclusions

Anti-RHA was detected at high prevalence in Mexican SLE patients. Detection of anti-RHA in races in which anti-Sm is not common should be clinically useful. Racial difference in the clinical significance of anti-RHA should be clarified in future studies.     

Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2- 3Galbeta1-3GalNAc sequences

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)- GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc- Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.      

Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (ρO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; ρO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.     

Adult Respiratory Distress Syndrome

Many causes for the adult respiratory distress syndrome (ARDS) have been reported, all with common pathologic, pathophysiologic and biochemical end results. The final common pathway may involve changes in lung content of a critical enzyme, superoxide dismutase, or alterations in surfactant metabolism, or both. The early assumption that the disorder is partially due to oxygen toxicity from inspired oxygen concentrations greater than 60 percent is consistent with findings of recent biochemical studies. Although the lung normally maintains its alveoli dry, during ARDS increased permeability of small pulmonary vessels results in primary pulmonary edema, in contrast to edema from increased vascular pressure. These data have been obtained mainly in animals; whether they apply to humans with ARDS is not certain. Tissue oxygenation is improved by increasing end-expiratory pressure in an animal model of ARDS, more effectively during spontaneous breathing than during mechanical ventilation. During spontaneous breathing, adverse ventilatory effects were caused by stimulation of pulmonary reflexes.