► We developed a technology for dual-color single-molecule sensitive analysis of microarrays. ► The platform is well suitable for amplification-free expression profiling of minute samples. ► Expression changes found in a rare subpopulation of multiple myeloma CD138 negative progenitor cells could be validated using qPCR.
We present a highly sensitive bioanalytical microarray assay that enables the analysis of small genomic sample material. By combining an optimized cDNA purification step with single molecule cDNA detection on the microarray, the platform has improved sensitivity compared to conventional systems, allowing amplification-free determination of expression profiles with as little as 600 ng total RNA. Total RNA from cells was reverse transcribed into fluorescently labeled cDNA and purified employing a precipitation method that minimizes loss of cDNA material. The microarray was scanned on a fluorescence chip-reader with single molecule sensitivity. Using the newly developed platform we were able to analyze the RNA expression profile of a subpopulation of rare multiple myeloma CD138 negative progenitor (MM CD138neg) cells. The high-sensitivity microarray approach led to the identification of a set of 20 genes differentially expressed in MM CD138neg cells. Our work demonstrates the applicability of a straight-forward single-molecule DNA array technology to current topics of molecular and cellular cancer research, which are otherwise difficult to address due to the limited amount of sample material.
Expression profiling; Microarray; Subpopulation analysis; Single molecule detection; Fluorescence microscopy
In a world where resources are scarce and urban areas consume the vast majority of these resources, it is vital to make cities greener and more sustainable. Advanced systems to improve and automate processes within a city will play a leading role in smart cities. From smart design of buildings, which capture rain water for later use, to intelligent control systems, which can monitor infrastructures autonomously, the possible improvements enabled by sensing technologies are immense. Ubiquitous sensing poses numerous challenges, which are of a technological or social nature. This paper presents an overview of the state of the art with regards to sensing in smart cities. Topics include sensing applications in smart cities, sensing platforms and technical challenges associated with these technologies. In an effort to provide a holistic view of how sensing technologies play a role in smart cities, a range of applications and technical challenges associated with these applications are discussed. As some of these applications and technologies belong to different disciplines, the material presented in this paper attempts to bridge these to provide a broad overview, which can be of help to researchers and developers in understanding how advanced sensing can play a role in smart cities.
advanced sensing; sensor networks; smart cities; internet of things
The scientific literature in the area of laser acupuncture is rather large; however, the actual mechanisms and effects have not yet been proven in detail. Since the early days of laser acupuncture, there are still many open questions concerning technical parameters of this innovative technique. In this paper, we report about the most important technical parameters (wavelength, output power, power density, energy density, dose range, and continuous or pulsed laser) for laser acupuncture and present quantitative results for optimal laser stimulation, which allow eliciting reproducible effects in the periphery and in the brain. There are several position statements on laser acupuncture and also several review articles in scientific literature concerning clinical effectiveness of laser acupuncture. For example, the Australian Medical Acupuncture College stated recently that “the optimal energy density for laser acupuncture and biostimulation, based on current clinical experience, is 4 J/cm2”. However, our results of previous research studies and of this paper clearly show that dose must be adjusted according to the individual responses.
Intermittent androgen suppression (IAS) therapy for prostate cancer patients attempts to maintain the hormone dependence of the tumor cells by cycles alternating between androgen suppression (AS) and treatment cessation till a certain prostate-specific antigen (PSA) threshold is reached. Side effects are expected to be reduced, compared to standard continuous androgen suppression (CAS) therapy. The present study examined the effect of IAS on bone metabolism by determinations of serum procollagen I N-terminal peptide (PINP), a biochemical marker of collagen synthesis. A total of 105 treatment cycles of 58 patients with prostate cancer stages ≥pT2 was studied assessing testosterone, PSA and PINP levels at monthly intervals. During phases of AS lasting for up to nine months PSA levels were reversibly reduced, indicating apoptotic regression of the prostatic tumors. Within the first cycle PINP increased at the end of the AS period and peaked in the treatment cessation phase. During the following two cycles a similar pattern was observed for PINP, except a break in collagen synthesis as indicated by low PINP levels in the first months off treatment. Therefore, measurements of the serum PINP concentration indicated increased bone matrix synthesis in response to >6 months of AS, which uninterruptedly continued into the first treatment cessation phase, with a break into each of the following two pauses. In summary, synthesis of bone matrix collagen increases while degradation decreases during off-treatment phases in patients undergoing IAS. Although a direct relationship between bone matrix turnover and risk of fractures is difficult to establish, IAS for treatment of biochemical progression of prostate tumors is expected to reduce osteoporosis in elderly men often at high risk for bone fractures representing a highly suitable patient population for this kind of therapy.
intermittent androgen suppression; prostate cancer; prostate-specific antigen; bone turnover; PINP
A common goal of outdoor augmented reality (AR) is the presentation of annotations that are registered to anchor points in the real world. We present an enhanced approach for registering and tracking such anchor points, which is suitable for current generation mobile phones and can also successfully deal with the wide variety of viewing conditions encountered in real life outdoor use. The approach is based on on-the-fly generation of panoramic images by sweeping the camera over the scene. The panoramas are then used for stable orientation tracking, while the user is performing only rotational movements. This basic approach is improved by several new techniques for the re-detection and tracking of anchor points. For the re-detection, specifically after temporal variations, we first compute a panoramic image with extended dynamic range, which can better represent varying illumination conditions. The panorama is then searched for known anchor points, while orientation tracking continues uninterrupted. We then use information from an internal orientation sensor to prime an active search scheme for the anchor points, which improves matching results. Finally, global consistency is enhanced by statistical estimation of a global rotation that minimizes the overall position error of anchor points when transforming them from the source panorama in which they were created, to the current view represented by a new panorama. Once the anchor points are redetected, we track the user's movement using a novel 3-degree-of-freedom orientation tracking approach that combines vision tracking with the absolute orientation from inertial and magnetic sensors. We tested our system using an AR campus guide as an example application and provide detailed results for our approach using an off-the-shelf smartphone. Results show that the re-detection rate is improved by a factor of 2 compared to previous work and reaches almost 90% for a wide variety of test cases while still keeping the ability to run at interactive frame rates.
► Enhanced approach to register and track annotations' anchor points. ► Panoramic image with extended dynamic range to increase image quality. ► Use of internal orientation sensor to prime an active search for the anchor points. ► Minimizing position error of anchor points by aligning panoramas. ► Tracking using a 3-degree-of-freedom orientation tracking based on sensor fusion.
Augmented reality; Annotation; Tracking; Mobile phone
Age-related gene expression patterns of Homo sapiens as well as of model organisms such as Mus musculus, Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster are a basis for understanding the genetic mechanisms of ageing. For an effective analysis and interpretation of expression profiles it is necessary to store and manage huge amounts of data in an organized way, so that these data can be accessed and processed easily.
GiSAO.db (Genes involved in senescence, apoptosis and oxidative stress database) is a web-based database system for storing and retrieving ageing-related experimental data. Expression data of genes and miRNAs, annotation data like gene identifiers and GO terms, orthologs data and data of follow-up experiments are stored in the database. A user-friendly web application provides access to the stored data. KEGG pathways were incorporated and links to external databases augment the information in GiSAO.db. Search functions facilitate retrieval of data which can also be exported for further processing.
We have developed a centralized database that is very well suited for the management of data for ageing research. The database can be accessed at https://gisao.genome.tugraz.at and all the stored data can be viewed with a guest account.
AIM: To determine free and conjugated serum bile acid (BA) levels in inflammatory bowel disease (IBD) subgroups with defined clinical manifestations.
METHODS: Comprehensive serum BA profiling was performed in 358 IBD patients and 310 healthy controls by liquid chromatography coupled to electrospray ionization tandem mass spectrometry.
RESULTS: Serum levels of hyodeoxycholic acid, the CYP3A4-mediated detoxification product of the secondary BA lithocholic acid (LCA), was increased significantly in Crohn’s disease (CD) and ulcerative colitis (UC), while most other serum BA species were decreased significantly. Total BA, total BA conjugate, and total BA glycoconjugate levels were decreased only in CD, whereas total unconjugated BA levels were decreased only in UC. In UC patients with hepatobiliary manifestations, the conjugated primary BAs glycocholic acid, taurocholic acid, and glycochenodeoxycholic acid were as significantly increased as the secondary BAs LCA, ursodeoxycholic acid, and tauroursodeoxycholic acid compared to UC patients without hepatobiliary manifestations. Finally, we found that in ileocecal resected CD patients, the unconjugated primary BAs, cholic acid and chenodeoxycholic acid, were increased significantly compared to controls and patients without surgical interventions.
CONCLUSION: Serum BA profiling in IBD patients that indicates impaired intestinal barrier function and increased detoxification is suitable for advanced diagnostic characterization and differentiation of IBD subgroups with defined clinical manifestations.
Bile acids; Liquid chromatography; Tandem mass spectrometry; Inflammatory bowel disease; Crohn’s disease; Ulcerative colitis
Platinum(IV) compounds like oxoplatin (cis, cis, trans-diammine-dichlorido-dihydroxido-platinum(IV)) show increased stability and therefore can be applied orally. In a panel of 38 human cancer cell lines this drug induced S-phase arrest and cell death with IC50 values 2.5-fold higher than cisplatin. Oxoplatin may be converted to cisplatin by intracellular reducing agents, however, exposure to 0.1 M HCl mimicking gastric acid yielded cis-diammine-tetrachlorido-platinum(IV) exhibiting twofold increased activity. Similar results were obtained for another platinum(IV) compound, JM 149 (ammine-dichlorido-(cyclohexylamine)-dihydroxido-platinum(IV)), but not for its parent drug JM 216/satraplatin. Genome-wide expression profiling of H526 small cell lung cancer cells treated with these platinum species revealed clear differences in the expression pattern of affected genes between oxoplatin and cisplatin. In conclusion, oxoplatin constitutes a potent oral agent that is either reduced or converted to distinct active compounds, for example, by gastric acid or acidic areas prevailing in solid tumors, in dependence of the respective pharmaceutical formulation.
12R-lipoxygenase (12R-LOX) and the epidermal LOX-3 (eLOX-3) constitute a novel LOX pathway involved in terminal differentiation in skin. This view is supported by recent studies showing that inactivating mutations in 12R-LOX and eLOX-3 are linked to the development of autosomal recessive congenital ichthyosis. We show that 12R-LOX deficiency in mice results in a severe impairment of skin barrier function. Loss of barrier function occurs without alterations in proliferation and stratified organization of the keratinocytes, but is associated with ultrastructural anomalies in the upper granular layer, suggesting perturbance of the assembly/extrusion of lamellar bodies. Cornified envelopes from skin of 12R-LOX–deficient mice show increased fragility. Lipid analysis demonstrates a disordered composition of ceramides, in particular a decrease of ester-bound ceramide species. Moreover, processing of profilaggrin to monomeric filaggrin is impaired.
This study indicates that the 12R-LOX–eLOX-3 pathway plays a key role in the process of epidermal barrier acquisition by affecting lipid metabolism, as well as protein processing.
During August 2006, a protracted outbreak of Salmonella (S.) Enteritidis infections in a large Hamburg nursing home was investigated.
A site visit of the home was conducted and food suppliers' premises tested for Salmonella. Among nursing home residents a cohort study was carried out focusing on foods consumed in the three days before the first part of the outbreak. Instead of relying on residents' memory, data from the home's patient food ordering system was used as exposure data. S. Enteritidis isolates from patients and suspected food vehicles were phage typed and compared.
Within a population of 822 nursing home residents, 94 case patients among residents (1 fatality) and 17 among staff members were counted 6 through 29 August. The outbreak peaked 7 through 9 August, two days after a spell of very warm summer weather. S. Enteritidis was consistently recovered from patients' stools throughout the outbreak. Among the food items served during 5 through 7 August, the cohort study pointed to afternoon cake on all three days as potential risk factors for disease. Investigation of the bakery supplying the cake yielded S. Enteritidis from cakes sampled 31 August. Comparison of the isolates by phage typing demonstrated both isolates from patients and the cake to be the exceedingly rare phage type 21c.
Cake (various types served on various days) contaminated with S. Enteritidis were the likely vehicle of the outbreak in the nursing home. While the cakes were probably contaminated with low pathogen dose throughout the outbreak period, high ambient summer temperatures and failure to keep the cake refrigerated led to high pathogen dose in cake on some days and in some of the housing units. This would explain the initial peak of cases, but also the drawn out nature of the outbreak with cases until the end of August. Suggestions are made to nursing homes, aiding in outbreak prevention. Early outbreak detection is crucial, such that counter measures can be swift and drawn-out outbreaks of nosocomial food-borne infections avoided.
In the search for novel bioactive compounds from sponge-derived microorganisms, we have recently identified two structurally and biosynthetically unprecedented fungal metabolites, the novel-type alkaloids sorbicillactone A and sorbicillactone B. Sorbicillactone A is active against leukemia cells without showing notable cytotoxicity. Therefore, we have developed an efficient process for its biotechnological production and isolation on a large scale supplying sufficient material for the ongoing preclinical investigations and structure-activity relationship (SAR) studies.
sorbicillactone A; anti-leukemic activity; large-scale production; BIOTECmarin
It is commonly accepted that embryonic segmentation of vertebrates is regulated by a segmentation clock, which is induced by the cycling genes Hes1 and Hes7. Their products form dimers that bind to the regulatory regions and thereby repress the transcription of their own encoding genes. An increase of the half-life of Hes7 protein causes irregular somite formation. This was shown in recent experiments by Hirata et al. In the same work, numerical simulations from a delay differential equations model, originally invented by Lewis, gave additional support. For a longer half-life of the Hes7 protein, these simulations exhibited strongly damped oscillations with, after few periods, severely attenuated the amplitudes. In these simulations, the Hill coefficient, a crucial model parameter, was set to 2 indicating that Hes7 has only one binding site in its promoter. On the other hand, Bessho et al. established three regulatory elements in the promoter region.
We show that – with the same half life – the delay system is highly sensitive to changes in the Hill coefficient. A small increase changes the qualitative behaviour of the solutions drastically. There is sustained oscillation and hence the model can no longer explain the disruption of the segmentation clock. On the other hand, the Hill coefficient is correlated with the number of active binding sites, and with the way in which dimers bind to them. In this paper, we adopt response functions in order to estimate Hill coefficients for a variable number of active binding sites. It turns out that three active transcription factor binding sites increase the Hill coefficient by at least 20% as compared to one single active site.
Our findings lead to the following crucial dichotomy: either Hirata's model is correct for the Hes7 oscillator, in which case at most two binding sites are active in its promoter region; or at least three binding sites are active, in which case Hirata's delay system does not explain the experimental results. Recent experiments by Chen et al. seem to support the former hypothesis, but the discussion is still open.
The quantification of single nucleotide polymorphism (SNP) allele frequencies in pooled DNA samples using real time PCR is a promising approach for large-scale diagnostics and genotyping. The limits of detection (LOD) and limits of quantification (LOQ) for mutant SNP alleles are of particular importance for determination of the working range, which, in the case of allele-specific real time PCR, can be limited by the variance of calibration data from serially diluted mutant allele samples as well as by the variance of the 100% wild-type allele samples (blank values). In this study, 3σ and 10σ criteria were applied for the calculation of LOD and LOQ values. Alternatively, LOQ was derived from a 20% threshold for the relative standard deviation (%RSD) of measurements by fitting a curve for the relationship between %RSD and copy numbers of the mutant alleles. We found that detection and quantification of mutant alleles were exclusively limited by the variance of calibration data since the estimated LODcalibration (696 in 30 000 000 copies, 0.0023%), LOQ20%RSD (1470, 0.0049%) and LOQcalibration (2319, 0.0077) values were significantly higher than the LODblank (130, 0.0004%) and LOQblank (265, 0.0009%) values derived from measurements of wild-type allele samples. No significant matrix effects of the genomic background DNA on the estimation of LOD and LOQ were found. Furthermore, the impact of large genome sizes and the general application of the procedure for the estimation of LOD and LOQ in quantitative real time PCR diagnostics are discussed.
Using the standing droplet technique in the renal proximal convolution and simultaneous microperfusion of the peritubular capillaries, the zero net flux transtubular concentration difference of taurocholate (ΔCTC−) at 45 s was determined as a measure of active bile acid reabsorption in vivo. Starting with 0.1 mmol/liter taurocholate in both perfusates the control ΔCTC− of 0.042 mmol/liter fell to 0.006 mmol/liter (P < 0.001) when the Na+ concentration in the perfusates was reduced to zero. Removal of bicarbonate from the perfusates to alter pH had no influence on ΔCTC−. When glycocholate was added to the perfusates ΔCTC− was decreased, while probenecid increased ΔCTC−.
These observations were extended by studies performed with brush border membrane vesicles derived from renal cortex. The initial (20 s) uptake of 0.01 mmol/liter taurocholate in the presence of a Nao+ > Nai+ gradient was stimulated twofold compared with its uptake in the absence of a Na+ gradient. Uptake of taurocholate was osmotically and temperature sensitive. Membranes preloaded with unlabeled glycocholate showed accelerated entry of labeled taurocholate (trans-stimulation) only in the presence of Na+. Replacement of Na+ in the media with K+, Li+, and choline+ decreased initial taurocholate uptake by 49, 53, and 62%, respectively. Stimulation of taurocholate transport by cation gradient diffusion potentials was unlikely inasmuch as the addition of valinomycin under K+ gradient conditions had no effect. A transmembrane pH gradient (pHo < pHi) did not influence initial uptake of taurocholate. Finally, in the presence of Na+ taurocholate transport showed cis-inhibition with unlabeled bile acids and saturation kinetics with respect to increasing taurocholate concentrations. The micropuncture and vesicle data indicate that the net transport of taurocholate in the proximal tubule is the result of an electroneutral Na+-taurocholate cotransport across the brush border membrane.
To identify new genetic regulators of cellular aging and senescence, we performed genome-wide comparative RNA profiling with selected human cellular model systems, reflecting replicative senescence, stress-induced premature senescence, and distinct other forms of cellular aging. Gene expression profiles were measured, analyzed, and entered into a newly generated database referred to as the GiSAO database. Bioinformatic analysis revealed a set of new candidate genes, conserved across the majority of the cellular aging models, which were so far not associated with cellular aging, and highlighted several new pathways that potentially play a role in cellular aging. Several candidate genes obtained through this analysis have been confirmed by functional experiments, thereby validating the experimental approach. The effect of genetic deletion on chronological lifespan in yeast was assessed for 93 genes where (i) functional homologues were found in the yeast genome and (ii) the deletion strain was viable. We identified several genes whose deletion led to significant changes of chronological lifespan in yeast, featuring both lifespan shortening and lifespan extension. In conclusion, an unbiased screen across species uncovered several so far unrecognized molecular pathways for cellular aging that are conserved in evolution.
aging; evolution; replicative lifespan; replicative senescence; senescence; yeast
Using perfusion techniques in single proximal tubule segments of rat kidney, the relationship between net sodium movement and active transport of ions, as measured by the short-circuit method, has been studied. In addition, the role of the colloid-osmotic pressure gradient in proximal transtubular fluid and sodium movement has been considered. Furthermore, the limiting concentration gradient against which sodium movement can occur and the relationship between intratubular sodium concentration and fluid transfer have been investigated. Comparison of the short-circuit current with the reabsorptive movement of sodium ions indicates that this process is largely, perhaps exclusively, active in nature. No measurable contribution of the normally existing colloid-osmotic pressure gradient to transtubular water movement was detected. On the other hand, fluid movement across the proximal tubular epithelium is dependent upon the transtubular sodium gradient and is abolished when a mean concentration difference of 50 mEq/liter is exceeded.
Mammalian torpor saves enormous amounts of energy, but a widely assumed cost of torpor is immobility and therefore vulnerability to predators. Contrary to this assumption, some small marsupial mammals in the wild move while torpid at low body temperatures to basking sites, thereby minimizing energy expenditure during arousal. Hence, we quantified how mammalian locomotor performance is affected by body temperature. The three small marsupial species tested, known to use torpor and basking in the wild, could move while torpid at body temperatures as low as 14.8–17.9°C. Speed was a sigmoid function of body temperature, but body temperature effects on running speed were greater than those in an ectothermic lizard used for comparison. We provide the first quantitative data of movement at low body temperature in mammals, which have survival implications for wild heterothermic mammals, as directional movement at low body temperature permits both basking and predator avoidance.
body temperature; lizard; marsupial; running speed; torpor
Structure and function are closely related in the healthy human brain. In patients with chronic heroin exposure, brain imaging studies have identified long-lasting changes in gray matter (GM) volume. More recently, we showed that acute application of heroin in dependent patients results in hypoperfusion of fronto-temporal areas compared with the placebo condition. However, the relationship between structural and cerebral blood flow (CBF) changes in heroin addiction has not yet been investigated. Moreover, it is not known whether there is any interaction between the chronic structural changes and the short and long-term effects on perfusion caused by heroin. Using a double-blind, within-subject design, heroin or placebo (saline) was administered to 14 heroin-dependent patients from a stable heroin-assisted treatment program, in order to observe acute short-term effects. Arterial spin labeling (ASL) was used to calculate perfusion quantification maps in both treatment conditions, while Voxel-Based Morphometry (VBM) was conducted to calculate regional GM density. VBM and ASL data were used to calculate homologous correlation fields by Biological Parametric Mapping (BPM) and a whole-brain Pearson r correlation. We correlated each perfusion condition (heroin and placebo) separately with a VBM sample that was identical for the two treatment conditions. It was assumed that heroin-associated perfusion is manifested in short-term effects, while placebo-associated perfusion is more related to long-term effects. In order to restrict our analyses to fronto-temporal regions, we used an explicit mask for our analyses. Correlation analyses revealed a significant positive correlation in frontal areas between GM and both perfusion conditions (heroin and placebo). Heroin-associated perfusion was also negatively correlated with GM in the inferior temporal gyrus on both hemispheres. These findings indicate that, in heroin-dependent patients, low GM volume is positively associated with low perfusion within frontal regions.
heroin addiction; biological parametric mapping; arterial spin labeling; voxel-based morphometry
According to the cognitive behavioral model of severe health anxiety (hypochondriasis) four central maintaining mechanisms are how the individual perceives the risk of disease and how negative its consequences would be, attention to bodily sensations, and intolerance of uncertainty. The aim of the present study was to investigate the mediating role of these putative mechanisms in Internet-delivered CBT for severe health anxiety. We analyzed data from an RCT where participants were randomized to Internet-delivered CBT (n=40) or to a control condition (n=41). Mediators and outcome, i.e. health anxiety, were assessed weekly throughout the treatment, enabling fulfillment of the criterion of temporal precedence of changes occurring in the mediator in relation to the outcome to be met. The results showed that reduced perceived risk of disease, less attention to bodily symptoms, and reduced intolerance of uncertainty significantly mediated improvement in health anxiety. The study supports the validity of the cognitive behavioral model of health anxiety. The findings have theoretical and clinical implications as they indicate processes that may be causally related to the improvements observed after CBT for health anxiety.
0.5% to 10% of clean surgeries result in surgical-site infections, and attempts to reduce this rate have had limited success. Germicidal UV lamps, with a broad wavelength spectrum from 200 to 400 nm are an effective bactericidal option against drug-resistant and drug-sensitive bacteria, but represent a health hazard to patient and staff. By contrast, because of its limited penetration, ∼200 nm far-UVC light is predicted to be effective in killing bacteria, but without the human health hazards to skin and eyes associated with conventional germicidal UV exposure.
The aim of this work was to test the biophysically-based hypothesis that ∼200 nm UV light is significantly cytotoxic to bacteria, but minimally cytotoxic or mutagenic to human cells either isolated or within tissues.
A Kr-Br excimer lamp was used, which produces 207-nm UV light, with a filter to remove higher-wavelength components. Comparisons were made with results from a conventional broad spectrum 254-nm UV germicidal lamp. First, cell inactivation vs. UV fluence data were generated for methicillin-resistant S. aureus (MRSA) bacteria and also for normal human fibroblasts. Second, yields of the main UV-associated pre-mutagenic DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts) were measured, for both UV radiations incident on 3-D human skin tissue.
We found that 207-nm UV light kills MRSA efficiently but, unlike conventional germicidal UV lamps, produces little cell killing in human cells. In a 3-D human skin model, 207-nm UV light produced almost no pre-mutagenic UV-associated DNA lesions, in contrast to significant yields induced by a conventional germicidal UV lamp.
As predicted based on biophysical considerations, 207-nm light kills bacteria efficiently but does not appear to be significantly cytotoxic or mutagenic to human cells. Used appropriately, 207-nm light may have the potential for safely and inexpensively reducing surgical-site infection rates, including those of drug-resistant origin.
The chaperone function of the ER-residing heat shock protein gp96 plays an important role in protein physiology and has additionally important immunological functions due to its peptide-binding capacity. Low amounts of gp96 stimulate immunity; high quantities induce tolerance by mechanisms not fully understood. A lack of gp96 protein in intestinal macrophages (IMACs) from Crohn`s disease (CD) patients correlates with loss of tolerance against the host gut flora, leading to chronic inflammation. Since gp96 shows dose-dependent direction of immunological reactions, we studied primary IMACs and developed cell models to understand the regulation of gp96 expression. Induction of gp96-expression was higher in in vitro differentiated dendritic cells (i.v.DCs) than in in vitro differentiated macrophages (i.v.MACs), whereas monocytes (MOs) expressed only low gp96 levels. The highest levels of expression were found in IMACs. Lipopolysaccharide (LPS), muramyl dipeptide (MDP), tumour necrosis factor (TNF), and Interleukin (IL)-4 induced gp96-expression, while IL12, IL-17, IL-23 and interferon (IFN)-γ were not effective indicating that Th1 and Th17 cells are probably not involved in the induction of gp96. Furthermore, gp96 was able to induce its own expression. The ER-stress inducer tunicamycin increased gp96-expression in a concentration- and time-dependent manner. Both ulcerative colitis (UC) and CD patients showed significantly elevated gp96 mRNA levels in intestinal biopsies which correlated positively with the degree of inflammation of the tissue. Since gp96 is highly expressed on the one hand upon stress induction as during inflammation and on the other hand possibly mediating tolerance, these results will help to understand the whether gp96 plays a role in the pathophysiology of inflammatory bowel disease (IBD).
We aimed to investigate the role of the nuclear carrier and binding proteins, transportin-1 (TRN1) and transportin-2 (TRN2), TATA-binding protein-associated factor 15 (TAF15) and Ewing’s Sarcoma protein (EWS) in inclusion body formation in cases of Frontotemporal Lobar Degeneration (FTLD) associated with Fused in Sarcoma protein (FTLD-FUS).
Eight cases of FTLD-FUS (5 cases of atypical FTLD-U (aFTLD-U), 2 of Neuronal Intermediate Filament Inclusion Body Disease (NIFID) and 1 of Basophilic Inclusion Body Disease (BIBD)) were immunostained for FUS, TRN1, TRN2, TAF15 and EWS. 10 cases of FTLD associated with TDP-43 inclusions served as reference cases.
The inclusion bodies in FTLD-FUS contained TRN1 and TAF15 and, to a lesser extent, EWS, but not TRN2. The patterns of immunostaining for TRN1 and TAF15 were very similar to that of FUS. None of these proteins was associated with tau or TDP-43 aggregations in FTLD.
Data suggest that FUS, TRN1 and TAF15 may participate in a functional pathway in an interdependent way, and imply that the function of TDP-43 may not necessarily be in parallel with, or complementary to, that of FUS, despite each protein sharing many similar structural elements.
Frontotemporal Lobar degeneration; Fused in Sarcoma; TDP-43; transportins; TATA-binding protein-associated factor 15; Ewing’s sarcoma protein
Stem cell therapy holds great promise for treating neurodegenerative disease, but major barriers to effective therapeutic strategies remain. A complete understanding of the derived phenotype is required for predicting cell response once introduced into the host tissue. We sought to identify major axonal guidance cues present in neurons derived from the transient overexpression of neurogenin-1 (Neurog1) in mouse embryonic stem cells (ESCs). Neurog1 upregulated the netrin-1 axon guidance receptors DCC (deleted in colorectal cancer) and neogenin (NEO1). Quantitative polymerase chain reaction results showed a 2-fold increase in NEO1 mRNA and a 36-fold increase in DCC mRNA in Neurog1-induced compared with control ESCs. Immunohistochemistry indicated that DCC was primarily expressed on cells positive for the neuronal marker TUJ1. DCC was preferentially localized to the cell soma and growth-cones of induced neurons. In contrast, NEO1 expression showed less specificity, labeling both TUJ1-positive and TUJ1-negative cells as well as uninduced control cells. Axonal outgrowth was directed preferentially toward aggregates of HEK293 cells secreting a recombinant active fragment of netrin-1. These data indicate that DCC and NEO1 are downstream products of Neurog1 and may guide the integration of Neurog1-induced ESCs with target cells secreting netrin-1. Differential expression profiles for netrin receptors could indicate different roles for this guidance cue on neuronal and non-neuronal cells.
GLIC is a bacterial member of the large family of pentameric ligand-gated ion channels. To study ion conduction through GLIC and other membrane channels, we combine the one-dimensional potential of mean force for ion passage with a Smoluchowski diffusion model, making it possible to calculate single-channel conductance in the regime of low ion concentrations from all-atom molecular dynamics (MD) simulations. We then perform MD simulations to examine sodium ion conduction through the GLIC transmembrane pore in two systems with different bulk ion concentrations. The ion potentials of mean force, calculated from umbrella sampling simulations with Hamiltonian replica exchange, reveal a major barrier at the hydrophobic constriction of the pore. The relevance of this barrier for ion transport is confirmed by a committor function that rises sharply in the barrier region. From the free evolution of Na+ ions starting at the barrier top, we estimate the effective diffusion coefficient in the barrier region, and subsequently calculate the conductance of the pore. The resulting diffusivity compares well with the position-dependent ion diffusion coefficient obtained from restrained simulations. The ion conductance obtained from the diffusion model agrees with the value determined via a reactive-flux rate calculation. Our results show that the conformation in the GLIC crystal structure, with an estimated conductance of ~1 picosiemens at 140 mM ion concentration, is consistent with a physiologically open state of the channel.
AIM: To compare clinical success and complications of uncovered self-expanding metal stents (SEMS) vs covered SEMS (cSEMS) in obstruction of the small bowel.
METHODS: Technical success, complications and outcome of endoscopic SEMS or cSEMS placement in tumor related obstruction of the duodenum or jejunum were retrospectively assessed. The primary end points were rates of stent migration and overgrowth. Secondary end points were the effect of concomitant biliary drainage on migration rate and overall survival. The data was analyzed according to the Strengthening the Reporting of Observational Studies in Epidemiology guidelines.
RESULTS: Thirty-two SEMS were implanted in 20 patients. In all patients, endoscopic stent implantation was successful. Stent migration was observed in 9 of 16 cSEMS (56%) in comparison to 0/16 SEMS (0%) implantations (P = 0.002). Stent overgrowth did not significantly differ between the two stent types (SEMS: 3/16, 19%; cSEMS: 2/16, 13%). One cSEMS dislodged and had to be recovered from the jejunum by way of laparotomy. Time until migration between SEMS and cSEMS in patients with and without concomitant biliary stents did not significantly differ (HR = 1.530, 95%CI 0.731-6.306; P = 0.556). The mean follow-up was 57 ± 71 d (range: 1-275 d).
CONCLUSION: SEMS and cSEMS placement is safe in small bowel tumor obstruction. However, cSEMS is accompanied with a high rate of migration in comparison to uncovered SEMS.
Endoscopy; Digestive system; Intestinal neoplasms; Self-expandable metal stents; Tumor obstruction; Self-expandable metal stents complications