Baring it all: undressing Cambrian ‘Orsten’ phosphatocopine crustaceans using synchrotron radiation X‐ray tomographic microscopy

Synchrotron radiation X‐ray tomographic microscopy (SRXTM) was used to virtually dissect and peel the shields off of the microscopic, bivalved phosphatocopine crustaceans in the Cambrian ‘Orsten’ type of preservation of Sweden. Doing so opened up for an array of concealed internal structures to be observed in a fully enclosed specimen of Hesslandona ventrospinata and a semi‐enclosed specimen of Hesslandona angustata. For comparison, also a head‐larva stage specimen of H. angustata, with shields in ‘butterfly position’, was analysed. The X‐ray tomographic data sets revealed excellently preserved structures, such as labrum, sternum, antennae, mandibular and post‐mandibular limbs with their minute setae, all of which were more or less disguised by the enclosing shields. This, moreover, allowed assignment to growth stages of the specimens, which is impossible based solely on external morphology and size. Micro‐spherules observed inside the shields of the semi‐enclosed H. angustata specimen may represent remains of food particles, and the feeding biology of phosphatocopines is discussed in detail. Our analyses suggest that phosphatocopines were particle feeders. The SRXTM technique offers the ability to three‐dimensionally reconstruct the morphology in high resolution, construct virtual serial sections and study concealed structures. The resulting data allow for new structures to be revealed for previously known taxa and for new taxa to be identified, with the added benefit of not destroying the specimens in the process. Hence, we do not longer have to rely on serendipitous finds of broken and/or open phosphatocopine specimens to elucidate their diagnostic ventral morphology.     

Influence of Calcium and Magnesium on Manganese Absorption

Mutual effects between Mn, Ca, and Mg were studied during steady-state absorption experiments with excised barley roots. Calcium appeared to enhance the rate of Mn absorption; whereas, Mg had a highly depressive effect. The combination of both Ca and Mg was even more inhibitory to Mn absorption than Mg alone. Manganese had no effect on the usual negligible Ca absorption by this tissue, but effectively inhibited the absorption of Mg. Although divalent cation absorption from the Ca-Mg-Mn system was essentially nil, K absorption was greatly stimulated in the presence of these cations.These mutual effects and others reported in the literature are explained by the hypothesis that selectivity in ion absorption results from cation-induced conformational changes in the structure of the carrier molecule.     

Diaphragm adaptations in patients with COPD

Inspiratory muscle weakness in patients with COPD is of major clinical relevance. For instance, maximum inspiratory pressure generation is an independent determinant of survival in severe COPD. Traditionally, inspiratory muscle weakness has been ascribed to hyperinflation-induced diaphragm shortening. However, more recently, invasive evaluation of diaphragm contractile function, structure, and biochemistry demonstrated that cellular and molecular alterations occur, of which several can be considered pathologic of nature. Whereas the fiber type shift towards oxidative type I fibers in COPD diaphragm is regarded beneficial, rendering the overloaded diaphragm more resistant to fatigue, the reduction of diaphragm fiber force generation in vitro likely contributes to diaphragm weakness. The reduced diaphragm force generation at single fiber level is associated with loss of myosin content in these fibers. Moreover, the diaphragm in COPD is exposed to oxidative stress and sarcomeric injury. This review postulates that the oxidative stress and sarcomeric injury activate proteolytic machinery, leading to contractile protein wasting and, consequently, loss of force generating capacity of diaphragm fibers in patients with COPD. Interestingly, several of these presumed pathologic alterations are already present early in the course of the disease (GOLD I/II), although these patients appear not limited in their daily life activities. Treatment of diaphragm dysfunction in COPD is complex since its etiology is unclear, but recent findings indicate the ubiquitin-proteasome pathway as a prime target to attenuate diaphragm wasting in COPD.     

Continuum description of the Poisson׳s ratio of ligament and tendon under finite deformation

Ligaments and tendons undergo volume loss when stretched along the primary fiber axis, which is evident by the large, strain-dependent Poisson?s ratios measured during quasi-static tensile tests. Continuum constitutive models that have been used to describe ligament material behavior generally assume incompressibility, which does not reflect the volumetric material behavior seen experimentally. We developed a strain energy equation that describes large, strain dependent Poisson?s ratios and nonlinear, transversely isotropic behavior using a novel method to numerically enforce the desired volumetric behavior. The Cauchy stress and spatial elasticity tensors for this strain energy equation were derived and implemented in the FEBio finite element software (www.febio.org). As part of this objective, we derived the Cauchy stress and spatial elasticity tensors for a compressible transversely isotropic material, which to our knowledge have not appeared previously in the literature. Elastic simulations demonstrated that the model predicted the nonlinear, upwardly concave uniaxial stress–strain behavior while also predicting a strain-dependent Poisson?s ratio. Biphasic simulations of stress relaxation predicted a large outward fluid flux and substantial relaxation of the peak stress. Thus, the results of this study demonstrate that the viscoelastic behavior of ligaments and tendons can be predicted by modeling fluid movement when combined with a large Poisson?s ratio. Further, the constitutive framework provides the means for accurate simulations of ligament volumetric material behavior without the need to resort to micromechanical or homogenization methods, thus facilitating its use in large scale, whole joint models.     

Quantitative electroelution of oligonucleotides and large DNA fragments from gels and purification by electrodialysis

We have designed a new device [Biotrap (Elutrap in the U.S.A. and Canada), available from Schleicher & Schuell] for electroelution, -concentration , and -dialysis of DNA and other charged macromolecules above 5 kDa. In an electric field, the DNA migrates in an open channel out of the gel slice through a microporous membrane, BT2, into the trap section, where it is retained by a very dense, non-adsorbant, and inert membrane BT1. Specifically designed for use in an electric field, the matrix of this new membrane is much denser than that of dialysis membranes. In contrast to dialysis membranes, BT1 will not adsorb large DNA fragments nor allow passage of small DNA fragments when subjected to an electric field. In the absence of an electric field, BT1 and BT2 effectively seal the trap, maintaining the final elution volume of the purified sample. The trap can contain from 200-600 microliter and is collected from above with a pipet. In the experiments described here, 85-95% of oligonucleotides (14-mer) and large (150 kb) DNA fragments were recovered, independent of fragment length. The purity of the eluted DNA was demonstrated by restriction enzyme digestion, nick-translation, primer extension, end-labeling with polynucleotide kinase, and ligation. Electrodialysis was successfully used for the complete removal of common contaminants inhibiting the polynucleotide kinase reaction and for the removal of CsCl from DNA samples.     

Middle ear defects associated with the double knock out mutation of murine goosecoid and Msx1 genes.

A number of developmental regulatory genes, including homeobox genes, are dynamically expressed in the mammalian cephalic ectomesenchyme during craniofacial morphogenesis. Owing to the vast amount of gene knock out experiments, functions of such genes are now being revealed in the mammalian skeletal patterning process. The murine goosecoid (Gsc) and Msx1 genes are expressed during craniofacial development and each mutant mouse displays intriguing facial abnormalities including those of middle ear ossicles, suggesting that both genes play roles in spatial programming of craniofacial regions. In order to examine whether these genes could function in concert to direct particular craniofacial morphogenesis, double knock out mice were analyzed. The phenotype of the double mutant mice was restricted to the first arch derivatives and was apparently additive of the single gene mutant mice, implying region specific genetic interactions of these homeobox genes expressed in overlapping regions of middle ear forming ectomesenchyme. Our results also suggested that the patterning of distal portions of the malleus depends on the tympanic membrane, for which normal expressions of both the genes are prerequisite.     

Eya1 is required for the morphogenesis of mammalian thymus,parathyroid and thyroid

Eyes absent (Eya) genes regulate organogenesis in both vertebrates and invertebrates. Mutations in human EYA1 cause congenital Branchio-Oto-Renal (BOR) syndrome, while targeted inactivation of murine Eya1 impairs early developmental processes in multiple organs, including ear, kidney and skeletal system. We have now examined the role of Eya1 during the morphogenesis of organs derived from the pharyngeal region, including thymus, parathyroid and thyroid. The thymus and parathyroid are derived from 3rd pharyngeal pouches and their development is initiated via inductive interactions between neural crest-derived arch mesenchyme, pouch endoderm, and possibly the surface ectoderm of 3rd pharyngeal clefts. Eya1 is expressed in all three cell types during thymus and parathyroid development from E9.5 and the organ primordia for both of these structures failed to form in Eya1(-/-) embryos. These results indicate that Eya1 is required for the initiation of thymus and parathyroid gland formation. Eya1 is also expressed in the 4th pharyngeal region and ultimobranchial bodies. Eya1(-/-) mice show thyroid hypoplasia, with severe reduction in the number of parafollicular cells and the size of the thyroid lobes and lack of fusion between the ultimobranchial bodies and the thyroid lobe. These data indicate that Eya1 also regulates mature thyroid gland formation. Furthermore, we show that Six1 expression is markedly reduced in the arch mesenchyme, pouch endoderm and surface ectoderm in the pharyngeal region of Eya1(-/-) embryos, indicating that Six1 expression in those structures is Eya1 dependent. In addition, we show that in Eya1(-/-) embryos, the expression of Gcm2 in the 3rd pouch endoderm is undetectable at E10.5, however, the expression of Hox and Pax genes in the pouch endoderm is preserved at E9.5-10.5. Finally, we found that the surface ectoderm of the 3rd and 4th pharyngeal region show increased cell death at E10.5 in Eya1(-/-) embryos. Our results indicate that Eya1 controls critical early inductive events involved in the morphogenesis of thymus, parathyroid and thyroid.     

Description of an incompatibility mutant of Escherichia coli

A mutant Hfr strain of Escherichia coli which has an impaired incompatibility function but is normal for other F factor functions has been isolated. This Inc(-) Hfr permits the maintenance and transfer of both the integrated F factor and an F' factor. F' factors have been isolated from the integrated F factor of the Inc(-) Hfr strain. When these episomes were tested in matings with Hfr or F' strains, they did not differ in any observed way from wild-type F' factors.