In situ nuclear magnetic resonance microimaging of live biofilms in a microchannel.

Biofilms are comprised of microbial cells and an extracellular polymeric substance (EPS) matrix that supports interactions between community members and with the local environment. The highly hydrated EPS matrix makes the application of many biofilm visualization techniques difficult. Hence, to better visualize how biofilms interact with their environment, there is a need for imaging techniques to monitor hydrated state biofilm dynamics. We employed an in situ dynamic approach to construct label-free images of biofilms. In situ imaging was conducted using a vacuum compatible microfluidic reactor, SALVI (System for Analysis at the Liquid Vacuum Interface), for biofilm growth; real-time confocal laser scanning microscopy analysis; and nuclear magnetic resonance (NMR) microimaging and spectroscopy. We integrated SALVI microchannel fluids and live biofilms to demonstrate in situ measurement capabilities, including velocity mapping, diffusion coefficient mapping, relaxometry, localized spectroscopy, relaxation times, porosity, and two- and three-dimensional imaging within the microchannel at high spatial resolution. We monitored organic acids adjacent to biofilms, suggesting that kinetic rate and substrate-product yield ratio studies are possible using the SALVI microfluidic reactor for growth characterizations. The integration of NMR microimaging studies into the SALVI platform demonstrates that a multimodal microfluidic platform can serve as an avenue to explore complex biological phenomena, such as biofilm attachment to surfaces, with detailed quantitative physical and chemical mapping. The further incorporation of other SALVI-compatible technologies, such as liquid time-of-flight secondary ion mass spectrometry imaging, with NMR microimaging will produce a powerful correlative approach to monitor in situ biofilm chemistry and dynamics at different spatial scales.

Biologic meshes in perineal reconstruction following extra-levator abdominoperineal excision (elAPE).

Recent improvements in the outcome for low rectal cancer have focused on the reconstruction of the perineal defect following greater acceptance of the need for a wider perineal excision encompassing the levator ani complex. In this article we look at the use of biologic materials to close the perineal defect and compare this with the use of other techniques.

Mechanistic insight into biopolymer induced iron oxide mineralization through quantification of molecular bonding.

Microbial production of iron (oxyhydr)oxides on polysaccharide rich biopolymers occurs on such a vast scale that it impacts the global iron cycle and has been responsible for major biogeochemical events. Yet the physiochemical controls these biopolymers exert on iron (oxyhydr)oxide formation are poorly understood. Here we used dynamic force spectroscopy to directly probe binding between complex, model and natural microbial polysaccharides and common iron (oxyhydr)oxides. Applying nucleation theory to our results demonstrates that if there is a strong attractive interaction between biopolymers and iron (oxyhydr)oxides, the biopolymers decrease the nucleation barriers, thus promoting mineral nucleation. These results are also supported by nucleation studies and density functional theory. Spectroscopic and thermogravimetric data provide insight into the subsequent growth dynamics and show that the degree and strength of water association with the polymers can explain the influence on iron (oxyhydr)oxide transformation rates. Combined, our results provide a mechanistic basis for understanding how polymer-mineral-water interactions alter iron (oxyhydr)oxides nucleation and growth dynamics and pave the way for an improved understanding of the consequences of polymer induced mineralization in natural systems.

Ecological effects of a major oil spill on panamanian coastal marine communities.

In 1986 more than 8 million liters of crude oil spilled into a complex region of mangroves, seagrasses, and coral reefs just east of the Caribbean entrance to the Panama Canal. This was the largest recorded spill into coastal habitats in the tropical Americas. Many population of plants and animals in both oiled and unoiled sites had been studied previously, thereby providing an unprecedented measure of ecological variation before the spill. Documenation of the spread of oil and its biological begun immediately. Intertidal mangroves, algae, and associated invertebrates were covered by oil and died soon after. More surprisingly, there was also extensive mortality of shallow subtidal reef corals and infauna of seagrass beds. After 1.5 years only some organisms in areas exposed to the open sea have recovered.

Immunological Methods to Study Monoclonal Antibody Activity in Chronic Lymphocytic Leukaemia.

Over recent decades it has become increasingly apparent that malignant cells, including chronic lymphocytic leukemia (CLL) cells, do not exist in isolation. Rather they coalesce with numerous "normal" cells of the body and, in the case of CLL, inhabit key immunological niches within secondary lymphoid organs (SLO), where a plethora of stromal and immune cells mediate their growth and survival. With the advent and approval of targeted immune therapies such as monoclonal antibodies (mAb), which elicit their efficacy by engaging immune-mediated effector mechanisms, it is important to develop accurate methods to measure their activities. Here, we describe a series of reliable assays capable of measuring important antibody-mediated effector functions: antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) that measure these immune activities.

Effects of dissociated glucocorticoids on OPG and RANKL in osteoblastic cells.

Glucocorticoids are effective anti-inflammatory and immunosuppressive agents, but their use is often associated with debilitating side effects such as glucocorticoid-induced osteoporosis. Newly developed glucocorticoid analogues such as the so-called dissociated glucocorticoids are potent immunosuppressants and have the potential for fewer side effects. The effects of these new analogues on osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) in osteoblastic cells have not been studied. OPG and RANKL are osteoblast-derived proteins pivotal to the regulation of bone mass. RANKL stimulates bone resorption by increasing osteoclast differentiation, activation and survival. OPG is the decoy receptor for RANKL and thus inhibits bone resorption. Here, we show that dexamethasone, prednisolone, deflazacort and the dissociated glucocorticoids, RU24858, RU40066, RU24782, AL438-F1 and ZK216348 significantly inhibit OPG production in two human osteoblastic cell lines (MG63 and hFOB). The potency for OPG inhibition was ligand and cell-type specific. In both cell types, dexamethasone and prednisolone were the most potent ligands inhibiting OPG production with IC(50)s of approximately 0.1 nM and 10 nM respectively. In MG63 cells, deflazacort and the RU compounds were the next most potent ligands followed by AL438-F1 and ZK216348. In hFOB cells, however, the RU compounds were the least potent ligands with an IC(50) 74 times higher than in MG63 cells. In contrast, the level of maximum inhibition or effectiveness of OPG inhibition did not vary between cell types but did vary according to the ligand. Dexamethasone, prednisolone, deflazacort and the RU compounds all inhibited OPG production by a maximum of approximately 70-80%, whereas AL438-F1 and ZK 216348 inhibited OPG production by a maximum of only 40-50% at 1 microM. All of the dissociated glucocorticoids and deflazacort were poor stimulators of RANKL gene expression stimulating by only approximately 1-3-fold compared to 7-fold by prednisolone. These data demonstrate that deflazacort and the dissociated glucocorticoids are weak stimulators of the RANKL:OPG ratio compared to prednisolone. Therefore, these compounds have the potential to cause less bone loss than that seen with prednisolone, though this was not investigated here.

Making better use of technological advances to meet stakeholder needs.

Controlling transboundary diseases requires an inclusive and collaborative international approach. Decisions should be taken (and seen to be taken) on advice from multidisciplinary teams of scientists and representatives from all groups significantly affected by the disease (the 'stakeholders'). Changes in trade and travel mean that, unless a new model is developed for disease prevention, there is a real possibility that transboundary animal diseases will become increasingly difficult to control. The traditional government approach of dealing almost exclusively with the commercial sector of the livestock industry is no longer sufficient, and new ways must be found to include all sectors, including 'grey' husbandry (fragmented, disparate groups whose involvement with animals may range from the legal and responsible to the unsanctioned and/or illegal). The increasing convergence of human and animal health issues makes it imperative to make the best possible use of new tools. The particular challenges confronting veterinary science are: preventing the introduction of disease, rapidly identifying disease and controlling epidemics. This paper focuses on the United Kingdom to investigate the inadequacies of current approaches, identify needs, offer recommendations and propose a new approach to disease control, which emphasises global considerations. The objectives are: better participation across the entire sector, better communication, better science and better decision-making, all of which should lead to better security from disease.

A bio-assay for effectors of osteoclast differentiation in serum from patients with bone disease.

UNLABELLED: Osteoclast differentiation and activity, and hence bone loss, depend on two opposing cytokines. Receptor activator of NF-(kappa)B ligand (RANKL) produced by osteoblasts and T-cells stimulates, while osteoprotegerin inhibits. Both of these cytokines are found in serum. Our aim was to develop a functional assay for any factors present in human serum that can affect osteoclast differentiation and to assess whether any such factors vary in diseases in which bone loss occurs. METHODS: Using a culture model of osteoclast differentiation in the presence of macrophage colony stimulating factor and soluble RANKL, we have measured the effects of different human sera on osteoclast differentiation. The production of a marker enzyme for the osteoclast, tartrate-resistant acid phosphatase (TRAP), was used to follow osteoclast differentiation. RESULTS: In general, human serum stimulates osteoclast differentiation as indicated by TRAP activity, but in patients with low bone density this stimulation was attenuated. Sera from 40 female subjects with low bone mineral density showed significantly lower TRAP cell differentiation activity than sera from the healthy female controls. CONCLUSION: We describe a functional bio-assay for factors in human serum which can affect osteoclast differentiation. This assay may have application in monitoring the effects of therapy in bone disease.

The dynamics of red cell chimaerism in histoincompatible parabiosed mice.

A new technique for the quantitation of isoenzymes was applied to assess the proportions of red cells in the circulation of parabiosed mice. Two parent-F1 hybrid combinations showing phosphoglucose isomerase polymorphism were examined at successive stages of parabiosis and after separation. Once red cell populations became mixed, 3 days after union, the ratio of red cell phenotypes was never significantly different from one partner to the other, although parental red cells became predominant after about 20 days. However, F1 hybrid red cells could always be detected. After separation of parabiosed mice there was a return to the original composition although this took longer than would be expected on the basis of reported red cell life span. Packed cell volume measurements indicated that a parental polycythaemia and F1 hybrid anaemia developed in one strain combination but not in the other. Evidence was adduced to support the hypothesis of a difference in red cell flux being responsible for the generation of this polycythaemia-anaemia.

Tissue repopulation during cure of osteopetrotic (mi/mi) mice using normal and defective (We/Wv) bone marrow.

Resorption of petrotic bone in osteopetrotic (mi/mi) mice was brought about by transplantation of bone marrow to X-irradiated recipients. In an attempt to learn more about the donor cell line involved in this process, both normal and defective marrow were used. The consequent repopulation of the lympho-myeloid complex was monitored by isoenzymes of glucose phosphate isomerase. The progress of normal marrow grafts was contrasted with that of a defective marrow (We/Wv). Despite the observation with We/Wv marrow showed reduced ability to form colonies in the spleen of an irradiated recipient, this marrow was as effective as normal marrow in inducing resorption of petrotic bone. The primordial stem cell for the osteoclast (haematopoietic stem cell?) is thus not a CFUS. Chimaeras with resolution of osteopetrosis by We/Wv bone marrow may exhibit erythropoiesis from residual stem cells of the host but leucocytes and platelets from the donor.

Effects of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate on mouse osteoclasts.

A group of 5-day-old mice were injected intraperitoneally with (3-amino-1-hydroxypropylidine)-1,1-bisphosphonate (APD). Morphologic changes were observed in vitally stained osteoclasts on parietal bones 3 days later, and these were judged to be degenerative. At this time significantly increased numbers of nuclei per osteoclast and total numbers of osteoclast nuclei were observed. However, at 4 days after the injection of APD, the total numbers of osteoclasts were significantly reduced relative to controls. When parietal bones were maintained in culture, APD reduced osteoclast numbers and inhibited cell-mediated 45Ca2+ release. Exposure of bones to parathyroid hormone increased the number of osteoclasts counted 1 day later. This effect was not blocked by APD. Calcitonin prevented the reduction in osteoclast numbers due to APD in vitro. We conclude that APD has a direct effect on resorbing mouse osteoclasts.

Osteoclasts are not the major source of interleukin-6 in mouse parietal bones.

Interleukin-6 (IL-6) is produced by bone cells and has been shown to stimulate the proliferation of osteoclast progenitors. Which cells in bone produce IL-6 is controversial. This article tests the hypothesis that tartrate-resistant acid phosphatase-positive osteoclasts (TRAP + OC) in neonatal mouse parietal bones are the major source of IL-6. Bones were preincubated with indomethacin to decrease the number of TRAP + OC and the amount of IL-6 produced. Incubation with parathyroid hormone or prostaglandin E2 increased the number of TRAP + OC and the amount of IL-6 produced. Calcitonin and 17 beta-estradiol inhibited this increase in TRAP + OC but had no effect on IL-6 production. 1,25-dihydroxy-vitamin D3 also stimulated an increase in TRAP + OC number but did not cause increased IL-6 production. Both the endocranial and ectocranial membranes of these bones produced large amounts of IL-6. TRAP activity in extracts of endocranial membranes was 14-fold that of the ectocranial membrane and, histochemically, some TRAP + cells could be detected here. However, the ectocranial membranes produced more IL-6 than the endocranial membranes. We conclude that TRAP + OC are not a major source of IL-6 in this system.

Osteoprotegerin is produced when prostaglandin synthesis is inhibited causing osteoclasts to detach from the surface of mouse parietal bone and attach to the endocranial membrane.

Osteoclast differentiation and activation is controlled, at least in part, by the counterbalancing influences of osteoprotegerin ligand (OPGL) and osteoprotegerin (OPG). Nonsteroidal anti-inflammatory drugs have been shown to inhibit bone loss in vivo and bone resorption in vitro, and this is associated with a loss of osteoclasts from the bone surface. We test the hypothesis that OPG mediates the inhibition of osteoclast activity that occurs with indomethacin in the mouse calvaria. Recombinant human OPG, like indomethacin, was found to cause osteoclasts to detach from the bone surface and attach to the adjacent endocranial membrane (periosteum). Recombinant human OPG also inhibited the stimulatory effect of prostaglandin E2 (PGE2), parathyroid hormone (PTH), and 1,25-dihydroxyvitamin D3 (1,25D3) on osteoclast adhesion to bone after an incubation with indomethacin. A function-blocking antibody to OPG and soluble human OPGL both inhibited the effect of indomethacin, leaving active osteoclasts on the bone. OPG activity was detected in the culture medium from indomethacin-treated bones and PTH, PGE2, 1,25D3, and dexamethasone all inhibited the production of OPG activity. We conclude that, in the absence of specific stimulators of bone resorption, OPG is produced by the mouse calvaria in vitro, which inhibits bone resorption by causing osteoclasts to detach from the bone surface.

Calmodulin-binding protein detection using a non-radiolabeled calmodulin fusion protein.

Calmodulin-binding proteins are involved in numerous cellular signaling pathways. The biotinylated-calmodulin overlay is a nonradioactive method widely used to detect calmodulin-binding proteins in tissue and cell samples. This method has several limitations; therefore, we developed a nonradioactive calmodulin-binding protein detection overlay using an S-tag-labeled calmodulin fusion protein. An expression system was used to generate a calmodulin fusion protein with an S-tag label, a 15 amino acid sequence that binds to a 105 amino acid S-protein. The S-protein is conjugated to horseradish peroxidase for final detection with a chemiluminescent substrate. The S-tag calmodulin was compared to purified calmodulin and biotinylated calmodulin in a calmodulin-dependent phosphodiesterase assay. The results of the calmodulin-dependent phosphodiesterase assay indicate that S-tag calmodulin induces higher phosphodiesterase activity than biotinylated calmodulin and lower activity than purified calmodulin. A comparison of the biotinylated and S-tag calmodulin overlay assays indicate that S-tag calmodulin is more sensitive than biotinylated calmodulin in the detection of calcineurin, a known calmodulin-binding protein. The overlay assay results also indicate that the S-tag calmodulin and biotinylated calmodulin detect similar calmodulin-binding proteins in colon epithelial cells. In conclusion, the S-tag calmodulin overlay assay is a consistent, sensitive, and rapid nonradioactive method to detect calmodulin-binding proteins.

GIL: a red cell antigen of very high frequency.

A new high-frequency red cell antigen has been identified and named GIL. GIL differs from all high-frequency antigens included in the International Society of Blood Transfusion classification. There is very little family information and GIL has not been shown to be an inherited character. Five women with anti-GIL have been found. All had been pregnant at least twice. Red blood cells of two of the babies gave positive direct antiglobulin tests, but there were no clinical signs of hemolytic disease. Anti-GIL may have been responsible for a hemolytic transfusion reaction and results of monocyte monolayer assays of two of the anti-GIL suggested a potential to cause destruction of transfused GIL+ RBCs.

Self-feeding performance in nursing home residents.

In a study of cognitively impaired nursing home residents, excess disability was found in the specific mealtime task of drinking liquids and among those eating a puréed diet. Nursing home staff tended to rely on spoonfeeding, a process in which the resident is a passive recipient of care rather than an active participant in it, as an intervention among residents who were partially able to feed themselves. Feeding techniques other than spoonfeeding--including verbal and nonverbal prompts, and physical guiding--can support residents' participation in feeding even when independence is no longer possible.

Institutionalized elderly. Relaxation, locus of control, self-esteem.

Progressive relaxation significantly moved elders toward a perception of internal locus of control. Progressive relaxation and activity programs significantly increased elders' self-esteem. Progressive relaxation was significantly more effective than the activity group in increasing self-esteem. Changes in locus of control and self-esteem were not correlated.

Time perception and the Stroop task.

Three experiments were conducted to assess the effects of the Stroop task (color-word incongruities) on observers' estimates of 30-sec. inspection periods. The experiments differed in psychophysical procedure; the three classic methods of production, reproduction, and verbal estimation were employed. Observers underestimated the passage of time, compared to doing nothing, when they were engaged on the Stroop task. However, judgments of duration on the Stroop task were shorter than those made in the control condition of naming color dots only when the method of production was employed. These findings are similar to results with mental arithmetic tasks and contribute to the understanding of the relationship between cognitive processing and time perception.

Ultrasonography guided rectus sheath catheters versus epidural analgesia for open colorectal cancer surgery in a single centre.

INTRODUCTION: Epidural anaesthesia (EA) has been the accepted standard for postoperative analgesia in open abdominal surgery. However, it is not without significant risk. This study aimed to audit the effect of EA and ultrasonography placed rectus sheath catheters (RSCs) on analgesia as well as the incidence of postoperative complications following open colorectal cancer surgery. METHODS: A three-year retrospective case note review was undertaken of all patients undergoing open colorectal cancer surgery at the Royal Devon and Exeter Hospital NHS Foundation Trust who received either EA or RSC for postoperative analgesia under the care of the senior authors. A single surgeon and single anaesthetist were practitioners. RESULTS: The case notes of 120 patients were reviewed retrospectively: 85 patients had EA and 24 RSC while 11 patients were excluded from the study. The EA group experienced a significantly higher incidence of hypotension (systolic blood pressure <130 mmHg) than the RSC group on the first postoperative day (p=0.0001). There was no significant difference in pain score or opiate sparing properties between the groups (p=0.92). There was no significant difference in postoperative respiratory tract infection, anastomotic leak or wound complications between the groups (p=0.2, p=1.0 and p=0.5 respectively). The RSC group had a higher incidence of ileus than the EA group (4/24 vs 2/85, p=0.026). However, the numbers were too small to draw a reliable conclusion. CONCLUSIONS: The use of ultrasonography guided RSCs has demonstrated effective postoperative analgesia equivalent to EA with the potential benefits of a reduced incidence of hypotension. A prospective randomised trial is now underway to compare RSC and EA in open abdominal and pelvic surgery.