Passive Rotation Angle Motion Validation for an Ankle-Foot Orthosis Multi-Jointed Surrogate Lower Limb Design.

Ankle-foot orthoses (AFO) are devices that assist lower limb motion. Mechanical testing an AFO would ideally load the device while worn on the leg, since AFO function is dependent on intimate leg contact. However, this is not appropriate for cyclic or load-to-failure applications. A surrogate lower limb (SLL) was designed for this AFO testing application, to provide anthropometric 3D movement when subjected to standard test loads. This novel four-joint SLL was inspired by the Rizzoli foot model, which segments the lower limb into five sections. SLL joint prototypes were validated by measuring rotation angles and comparing with typical anatomical ranges of motion. The 3D printed models were within acceptable variability of human joint movement and, therefore, were appropriate for use in the final SSL.

Sows affect their piglets' faecal microbiota until fattening but not their Salmonella enterica shedding status.

Recent studies have shown that Salmonella shedding status affects sows' microbiota during gestation and that these modifications are reflected in the faecal microbiota of their piglets at weaning. The aims of this study were: (a) to evaluate the persistence, up to the fattening period, of the previously measured link between the microbiota of piglets and their mothers' Salmonella shedding status; and (b) measure the impact of the measured microbiota variations on their Salmonella excretion at this stage. To achieve this, 76 piglets born from 19 sows for which the faecal microbiota was previously documented, were selected in a multisite production system. The faecal matter of these swine was sampled after 4 weeks, at the fattening stage. The Salmonella shedding status and faecal microbiota of these animals were described using bacteriological and 16S rRNA gene amplicon sequencing respectively. The piglet digestive microbiota association with the Salmonella shedding status of their sows did not persist after weaning and did not affect the risk of Salmonella excretion during fattening, while the birth mother still affected the microbiota of the swine at fattening. This supports the interest in sows as a target for potentially transferrable microbiota modifications.

Salmonella shedding status of the sow affects the microbiota of their piglets at weaning.

AIM: To observe the transfer of the digestive microbiota from sow to piglet, describe the impact of the sow's Salmonella shedding on this transfer and identify transferred populations that could be associated with the future Salmonella status of the piglets. METHODS AND RESULTS: Salmonella shedding status of 19 sows was determined at the beginning and end of gestation. Four piglets were randomly selected from each sow. Using MiSeq, the microbiotas of the sows at the end of gestation and of their piglets 1 day before weaning were described. Results showed that the Salmonella shedding of the sows, the birth mother, the lairage room, the parity and the contamination of the lairage environment were associated to the microbiota of the piglets (permanova P < 0·05). Several genera were associated with piglets born from negative or positive sows. CONCLUSION: There is a link between the microbiota of the sows at the end of gestation and the microbiota of their piglets at weaning, and the Salmonella shedding of the sow is associated with the microbiota of the piglets. SIGNIFICANCE AND IMPACT OF THE STUDY: Salmonella status of the sows affects the microbiota of their piglets and could affect the long-term Salmonella colonization resistance of these animals and their health.

The strange case of the earliest silver extraction by European colonists in the New World.

La Isabela, the first European town in the New World, was established in 1494 by the second expedition of Christopher Columbus but was abandoned by 1498. The main motive for settlement was to find and exploit deposits of precious metals. Archaeological evidence of silver extraction at La Isabela seemed to indicate that the expedition had located and tested deposits of silver-bearing lead ore in the Caribbean. Lead isotope analysis refutes this hypothesis but provides new evidence of the desperation of the inhabitants of La Isabela just before its abandonment.

Heat shock-induced alterations in phosphorylation of the largest subunit of RNA polymerase II as revealed by monoclonal antibodies CC-3 and MPM-2.

The phosphorylation of the carboxy-terminal domain of the largest subunit of RNA polymerase II plays an important role in the regulation of transcriptional activity and is also implicated in pre-mRNA processing. Different stresses, such as a heat shock, induce a marked alteration in the phosphorylation of this domain. The expression of stress genes by RNA polymerase II, to the detriment of other genes, could be attributable to such modifications of the phosphorylation sites. Using two phosphodependent antibodies recognizing distinct hyperphosphorylated forms of RNA polymerase II largest subunit, we studied the phosphorylation state of the subunit in different species after heat shocks of varying intensities. One of these antibodies, CC-3, preferentially recognizes the carboxy-terminal domain of the largest subunit under normal conditions, but its reactivity is diminished during stress. In contrast, the other antibody used, MPM-2, demonstrated a strong reactivity after a heat shock in most species studied. Therefore, CC-3 and MPM-2 antibodies discriminate between phosphoisomers that may be functionally different. Our results further indicate that the pattern of phosphorylation of RNA polymerase II in most species varies in response to environmental stress.

Antiangiogenic effects of the oral administration of liquid cartilage extract in humans.

BACKGROUND: The antiangiogenic properties of shark cartilage extracts have been demonstrated in animal models but there are no data in human subjects. MATERIALS AND METHODS: A placebo or one of two doses of a liquid shark cartilage extract was orally administered daily, from Day 1 to Day 23 of the study protocol, to 29 healthy male volunteers randomized into three groups. On Day 12, a polyvinyl alcohol sponge threaded in a perforated silicone tubing was inserted subcutaneously on the anterior side of the arm and removed on Day 23. Evaluation of endothelial cell density, with factor VIII immunostaining, an indirect measurement of angiogenesis, was performed on histological sections of the implant using a semiquantitative numerical scale ranging from 1 (low density) to 5 (high density). The hydroxyproline content of the sponges was measured by HPLC. RESULTS: The mean endothelial cell density was significantly lower in groups that had received the liquid cartilage extract: grades 2.24 +/- 0.10, 2.47 +/- 0.10, and 3.15 +/- 0.11 for 7 and 21 ml liquid cartilage extract and placebo, respectively (P < 0.01 for both comparisons). No grade 1 was observed in the placebo group, whereas 9 treated subjects received a grade 1. Hydroxyproline content of the sponges did not differ between groups and there was no significant correlation between hydroxyproline content and endothelial cell density in the sponges. CONCLUSIONS: These results demonstrate that the liquid cartilage extract contains an antiangiogenic component bioavailable in humans by oral administration. This is the first report of an inhibition of wound angiogenesis in healthy men.

Antiangiogenic properties of a novel shark cartilage extract: potential role in the treatment of psoriasis.

BACKGROUND: A number of inflammatory and immune diseases are associated with vascular changes. Psoriasis, as an example, is a common inflammatory skin disease with dilation of capillaries as an early histological change. In more developed psoriatic lesions there is proliferation of blood vessels and neovascularization. The use of agents that target these vascular changes represents a novel therapeutic strategy in the treatment of inflammatory diseases. Since cartilage is an avascular tissue, it has been hypothesized that there may be factors found in cartilage that inhibit blood vessel formation. OBJECTIVE: The objectives of this study were 1) to determine whether extracts of cartilage could inhibit angiogenesis, and 2) since altered angiogenesis is associated with certain diseases, including psoriasis, to examine whether inhibition of angiogenesis could potentially contribute to the treatment of psoriasis. METHODS: Extracts of shark cartilage were prepared by homogenization and ultrafiltration to derive the active agent termed AE -941. This agent was tested for antiangiogenesis activity using the embryonic vascularization test, which is a modification of the ex vivo chick embryo culture (CAM). Since one of the first steps in angiogenesis is degradation by metalloproteinases of the basement membrane of capillaries, AE -941 was tested for collagenase activity using a fluorogenic peptide substrate. Anti-inflammatory properties were tested using a cutaneous irritation model in humans. RESULTS: A dose dependent inhibition in embryonic neovascularization as well as in collagenase activity by AE -941 was demonstrated. When test compounds were applied on the forearms of test subjects, AE -941 was shown to have anti-inflammatory properties. Anecdotal data suggested that topical AE -941 had a beneficial effect in psoriasis. CONCLUSION: Our results show that AE -941 has anti-angiogenic and anti-inflammatory properties. Antiangiogenesis agents such as AE -941 provide an entirely new class of agents to treat cutaneous and systemic diseases associated with altered vascularity.

Stimulation of gastric acid secretion by cAMP in a novel alpha-toxin-permeabilized gland model.

It is generally believed that histamine-stimulated gastric acid secretion involves a transient elevation of intracellular Ca2+ and the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) cascade through phosphorylation, whose actions ultimately effect the fusion of H(+)-K(+)-adenosinetriphosphatase (ATPase)-containing vesicles to the apical plasma membrane of parietal cells. To dissect the signaling events underlying gastric acid secretion, we have developed a permeabilized gastric gland model using Staphylococcus alpha-toxin. The advantage of this model is its ability to retain cytosolic components that are required for the secretory machinery. Here we show that acid secretion in alpha-toxin-permeabilized glands is a cAMP-dependent process, reaching a maximal stimulation at 100 microM cAMP. The cAMP-elicited acid secretion, as monitored by the accumulation of the weak base aminopyrine (AP), required functional mitochondria or exogenously supplied ATP. Maximal stimulation elicited by cAMP for AP uptake by permeabilized glands was 51-85% of intact glands. Moreover, secretory activity was potentiated by 0.1 mM ATP. The recruitment of H(+)-K(+)-ATPase-rich tubulovesicles into the apical plasma membrane was measured using biochemical and morphological assays, thus validating the cell activation processes in response to cAMP. From this permeabilized model, [gamma-32P]ATP was used to directly phosphorylate target proteins. A number of proteins whose phosphorylation-dephosphorylation is specifically modulated by cAMP were found. These studies establish the first permeabilized gland model in which the resting-to-secreting transition can be triggered and show that cAMP-mediated phosphorylation is correlated with secretory activity.

A heterogeneous set of FMR1 proteins is widely distributed in mouse tissues and is modulated in cell culture.

The fragile X syndrome is an X-linked inherited disease and is the result of transcriptional inactivation of the FMR1 gene and the absence of its encoded FMR protein (FMRP). Using a specific monoclonal antibody directed against human FMRP, we have studied the steady-state levels of its murine homolog in several tissues and organs of adult and young mice. In immunoblot analyses, the antibody recognizes a heterogeneous subset of proteins with apparent molecular weights ranging from 80 to 70 kDa. These proteins are detected in all the 27 tissues tested; however, the relative proportion of each polypeptide recognized varies between tissues, and a significantly higher expression is observed in young animals. Northern blot analysis of RNA extracted from selected tissues from adult mouse shows that these tissues express the major 4.8 kb mRNA, although at different levels, and contain several additional shorter transcripts, particularly in muscular tissues. We also report that expression of the FMR1 gene is modulated in proliferating and quiescent primary mouse kidney cell cultures with an inverse relationship between levels of FMR1 mRNA and of its encoded proteins. This suggests that FMRPs are highly stable in quiescent cells and that FMR1 expression is likely post-transcriptionally controlled. Our results document the widespread expression of the FMR1 gene, and suggest that it is controlled by different mechanisms implicated in cell growth and differentiation.

Direct measurement of extracellular proton flux from isolated gastric glands.

We used the microphysiometer, a sensitive extracellular pH sensor, to resolve luminal (or apical) H+ secretion and basolateral release of OH- as well as liberation of acidic metabolites in rabbit gastric glands. Stimulation of glands via the adenosine 3',5'-cyclic monophosphate pathway produced a biphasic change in the extracellular acidification rate (EAR): after an initial transient decrease below the unstimulated baseline (-40.9 +/- 3.4%), the EAR increased to a steady-state maximal plateau (+98.1 +/- 5.3%) within 30 min (n = 37). We interpret the biphasic EAR profile as an initial excess of basolaterally released OH- followed by delayed luminal efflux of simultaneously produced H+. The elevated EAR at steady state reflected liberation of metabolic acid attributed to H(+)-K(+)-ATPase enzymatic activity. The presence of H2-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid prevented OH- release and reduced steady-state EAR. Basolateral OH- release and steady-state EAR were also inhibited by the H(+)-K(+)-ATPase inactivators omeprazole and SCH-28080. Inhibition of Na+/H+ exchange did not reduce steady-state EAR and did not affect apical H+ production, as judged by the accumulation of the weak base aminopyrine. Sodium thiocyanate (1 mM), which short circuits intraluminal H+ accumulation, blocked OH- release, demonstrating its dependence on H(+)-OH- separation at the apical membrane. A computerized model was developed to illustrate how the observed biphasic EAR profile would result from a delayed luminal efflux of H+ due to transitory intraluminal compartmentalization.

Acid secretion in alpha-toxin-permeabilized gastric glands.

Rabbit gastric glands were treated with alpha-toxin to test for permeabilization of basolateral membrane and retention of functional activity of parietal cells. Treatment with up to 400 U alpha-toxin/mL resulted in a dose-dependent increase in permeabilization, as judged by nuclear uptake of trypan blue (960 daltons), while causing relatively little loss of cytoplasmic macromolecules in the size range of lactate dehydrogenase (134,000 daltons). In the presence of cAMP and ATP, alpha-toxin-permeabilized resting gastric glands were stimulated to accumulate aminopyrine by approximately 10-fold over glands incubated without added nucleotides. Aminopyrine accumulation in stimulated permeabilized glands was inhibited by specific H+,K(+)-ATPase inhibitors, omeprazole and SCH-28080, and by the selective inhibitor of protein kinase A, H-89 (IC50 = 7.17 +/- 2.05 microM; n = 4). Aminopyrine accumulation in the alpha-toxin-treated glands was dependent on both exogenous ATP and cAMP; however, when no exogenous ATP was present, cAMP-activated aminopyrine accumulation reached approximately 50% of maximum, and at levels of ATP > 0.05 mM, maximal aminopyrine accumulation occurred without exogenous cAMP. In the presence of ATP alone, aminopyrine accumulation in permeabilized glands achieved 61.1 +/- 3.2% (n = 10; range, 50-70%) of the values measured on paired samples of intact glands stimulated with histamine plus isobutylmethylxanthine. These results demonstrate the functional responsiveness of alpha-toxin-permeabilized resting gastric glands. The participation of a protein kinase A dependent pathway during activation of permeabilized parietal cell is proposed.

Ezrin-calpain I interactions in gastric parietal cells.

Gastric ezrin, a membrane-cytoskeletal linker with sequence homology to talin and erythrocyte band 4.1, has been associated with the remodeling of parietal cell apical membrane that occurs with adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase stimulation. Here we examine the interrelationship between parietal cell ezrin and Ca(2+)-dependent protease activity. Addition of Ca2+ to sonicated gastric gland preparations rendered a relatively selective proteolysis of the 80-kDa ezrin, accompanied by the appearance of a 55-kDa breakdown product. Ca(2+)-dependent proteolysis of ezrin was blocked by E64, a cysteine protease inhibitor, or calpastatin, indicating calpain as the responsible protease. Degradation of ezrin in intact gastric glands was achieved by varying extracellular [Ca2+] and [ionomycin]. Ezrin degradation in situ was rapid and relatively selective, although Ca(2+)-dependent degradation of some spectrin-like bands was also observed. The effect of activated calpain I on parietal cell function was assessed by probing the secretory response to histamine stimulation using [14C]aminopyrine uptake, along with parallel measurements of calpain activity, over a wide range of ionomycin. Activation of calpain, as evidenced by loss of parietal cell ezrin, was correlated with decreased AP uptake by stimulated gastric glands, supporting a role for ezrin in the oxyntic secretory process. The calpain-ezrin interaction established here, and the similarities of calpain with talin and erythrocyte band 4.1, suggest a common feature to this family of ezrin/band 4.1/talin proteins that have been implicated in membrane-cytoskeletal association.

Monoclonal antibody CC-3 recognizes phosphoproteins in interphase and mitotic cells.

Among a library of monoclonal antibodies (mAbs) recognizing developmental markers in the chick embryo, mAb CC-3 was selected because of its differential immunostaining of mitotic cells. The intracellular distribution of the CC-3 antigen (CC-3a) throughout the cell cycle was visualized by immunolocalization. In interphase cells CC-3a resided in the nucleus and was arranged in distinct extranucleolar clusters. At prophase, the nuclear reactivity of CC-3a considerably increased and subsequently extended to the cytoplasm at metaphase. From metaphase through anaphase, most of the reactivity was associated with the mitotic apparatus. During cytokinesis CC-3a was detected in the mid-body and also in discrete speckles dispersed throughout the cytoplasm. The initial interphase pattern was then restored in the two daughter nuclei. Immunoblot analysis demonstrated that a 255-kDa phosphoprotein was present only in the interphase nucleus and that a complete new set of phosphoproteins accounted for the mitotic cell reactivity. The binding of CC-3 was dependent on the phosphorylation of its antigens. CC-3a is an evolutionary conserved molecule; it is present in such phylogenetically distant species as Drosophila and humans. Furthermore, the unique behavior of CC-3 on sections of normal, embryonic, and regenerative tissue and in cell culture immunostaining make it a reliable tool to identify mitotic foci.

A forty-three-year follow-up of a vitallium scaphoid arthroplasty.

Over the years various treatment options for scaphoid nonunions have been proposed. We present as a historical note the long-term follow-up of one such treatment, the insertion of a vitallium scaphoid.

Localization of molecules with restricted patterns of expression in morphogenesis: an immunohistochemical approach.

In a search for molecules with restricted patterns of expression during development, monoclonal antibodies were raised against different transitory structures of the chick embryo. Mice were immunized with cell suspensions from lightly homogenized embryonic tissues explanted from morphogenetically active regions. A convenient immunohistochemical assay was used to screen the hybridoma supernatants on a large scale. It relied on the use of poly(ethylene glycol) as embedding medium. Its water miscibility allowed, in a one-step incubation with antibody-containing supernatants, the dewaxing and rehydration of the tissue sections as well as antibody binding. We report here the usefulness of this approach in selecting monoclonals with unique patterns of immunoreactivity. In this study, cephalic neural crest cells in early or late phase of migration, together with their surrounding tissues, were used as immunogens. The monoclonal antibodies obtained have been classified into regional, cell-lineage, cell-cycle or extracellular material-associated markers. The information provided by the direct visualization of the immunoreactivity of the various monoclonal antibodies on tissue sections, as early as the first round of screening, allows rapid determination of the subsequent strategy to be followed for further characterization of the individual markers.

The globular region of histone H5 is equally accessible to antibodies in relaxed and condensed chromatin.

The accessibility of histone H5 in chromatin was examined with monoclonal antibodies recognizing several epitopes of the globular region (GH5) of the histone (Rózalski, M., Lafleur, L., and Ruiz-Carrillo, A. (1985) J. Biol. Chem. 260, 14379-14385). The stoichiometry of the chromatin-antibody complexes indicated that while 0-86% of the H5 molecules were able to react, depending on the particular epitope, the extent of antibody binding to relaxed chromatin (in 5 mM KCl) and condensed chromatin (in 100 mM KCl or 0.35 mM MgCl2) was virtually identical. This indicates that the topography of H5 does not change during the conformational transition of chromatin. The data suggest that H5 is not completely internalized in the 30-nm fiber or that the fiber is flexible enough to allow full exposure of the GH5 epitopes. Several control experiments, including monoclonal antibody binding, sedimentation analysis, DNase II digestion, and glutaraldehyde cross-linking, showed that epitope accessibility is not due to H5 exchange or to perturbation of the chromatin fiber. The accessibility of GH5 suggests ways in which inactive chromatin may be unfolded in vivo.