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BackgroundBase of tongue cancer incidence and patient survival is increasing why treatment sequelae becomes exceedingly important. Osteoradionecrosis (ORN) is a late adverse effect of radiotherapy and brachytherapy (BT) could be a risk factor. Brachytherapy is used in three out of six health care regions in Sweden.AimsInvestigate if patients treated in regions using BT show an increased risk for ORN and whether brachytherapy has any impact on overall survival.Material and MethodsWe used data from the Swedish Head and Neck Cancer Register between 2008-2014. Due to the nonrandomized nature of the study and possible selection bias we compared the risk for ORN in brachy vs non-brachy regions.ResultsFifty out of 505 patients (9.9%) developed ORN; eight of these were treated in nonbrachy regions (16%), while 42 (84%) were treated in brachy regions. Neither age, sex, TNM-classification/stage, p16, smoking, neck dissection, or chemotherapy differed between ORN and no-ORN patients. The risk for ORN was significantly higher for patients treated in brachy regions compared to non-brachy regions (HR = 2,63, p = .012), whereas overall survival did not differ (HR = 0.95, p = .782).Conclusions and SignificanceBrachytherapy ought to be used cautiously for selected patients or within prospective randomized studies.

Microhabitat conditions determine the magnitude and speed of microbial processes but have been challenging to investigate. In this study we used microfluidic devices to determine the effect of the spatial distortion of a pore space on fungal and bacterial growth, interactions, and substrate degradation. The devices contained channels differing in bending angles and order. Sharper angles reduced fungal and bacterial biomass, especially when angles were repeated in the same direction. Substrate degradation was only decreased by sharper angles when fungi and bacteria were grown together. Investigation at the cellular scale suggests that this was caused by fungal habitat modification, since hyphae branched in sharp and repeated turns, blocking the dispersal of bacteria and the substrate. Our results demonstrate how the geometry of microstructures can influence microbial activity. This can be transferable to soil pore spaces, where spatial occlusion and microbial feedback on microstructures is thought to explain organic matter stabilization., Arellano Caicedo et al. use microfluidic devices to determine how spatial distortion of pore spaces in soil organic matter affects bacterial and fungal growth and substrate degradation. Their newly conceptualized chip design allows the quantification of bacterial and fungal biomass, and ecosystem function organic matter cycling with help of fluorescent markers in different microstructures.

A method to determine the absolute activity of 137Cs in irradiated nuclear fuel is presented. Using a well-known point-like calibration source in combination with measurements of the gamma-ray intensity from the nuclear fuel and Monte Carlo calculations based on the nominal measurement geometry, the activity content can be determined without prior knowledge of the intrinsic detection efficiency of the gamma-ray detector. The presented method is tested using measurements of the 137Cs intensity from spent nuclear fuel of the pressurized water type at the central interim storage in Sweden. Using an assumption of homogeneous distribution of 137Cs throughout the fuel, we demonstrate a linear relationship between measured activity and the activity calculated by a state-of-the-art simulation code. For future studies, we suggest some factors that potentially can decrease the uncertainty in the correlation between measured and calculated activity.

The aim of this randomised controlled trial was to compare the costs and benefits of computer-based 2-dimensional and 3-dimensional predictions in orthognathic surgery. Subjects aged 18-30 years with severe class III malocclusion had their treatment planned with both 2- and 3-dimensional techniques. They were randomised in a 1:1 ratio for one or other planning technique. Costs (financial, time, and dose of radiation) were compared with benefits (accuracy and health-related quality of life (HRQoL)). In total, 57 subjects (27 women and 30 men, mean (range) age 21 (18-28) years) completed the study. Comparisons showed no significant difference in total time spent, but a large advantage for the 2-dimensional technique in financial costs (p 0.001); it also required a significantly lower dose of radiation (p 0.001). The cost-effectiveness analysis showed a reduction in time of 0.53 minutes/HRQoL-point gained, and an increased economic cost of US$15/HRQoL point gained for the 3-dimensional technique. It also showed that the two techniques consumed an equal amount of time, but that the 2-dimensional technique had lower financial costs, and the 3-dimensional technique a larger dose of radiation.

OBJECTIVE The present study assessed somatosensory changes related to trigeminal nerve damage using extensive evaluation tools and assessed the effect of such damage on the patients' psychosocial status and quality of life compared with healthy participants. METHODS In 37 patients with intraorally or extraorally presenting trigeminal nerve damage diagnosed as painful or nonpainful posttraumatic trigeminal neuropathy, psychophysical tests like quantitative sensory testing (QST) and qualitative sensory testing and the electrophysiological "nociceptive-specific" blink reflex were performed. The patients and 20 healthy participants completed a set of questionnaires assessing their psychosocial status and quality of life. RESULTS A loss or gain of somatosensory function was seen in at least 1 QST parameter in >88.9% of the patients. Patients in whom extraoral QST was performed showed an overall loss of somatosensory function, whereas intraoral QST showed a general gain of somatosensory function. Qualitative sensory testing identified a side-to-side difference in the tactile and pinprick stimulation in >77% of the patients. An abnormal "nociceptive-specific" blink reflex response was seen in 42.1% to 71.4% of patients dependent on the trigeminal branch stimulated, though comparisons with healthy reference values showed ambiguous results. Compared with the healthy participants, patients showed higher scores for pain catastrophizing, symptoms of depression and anxiety, limited jaw function, more somatic symptoms, and significantly impaired oral health-related quality of life (all P

The main objective of the present study was to assess the accuracy of two- and three-dimensional prediction techniques in orthognathic surgery. It was also a test of the very planning sequence. The scientific question was how well does the software support the surgeon in his way to find the perfect correction of the facial appearance while normalizing the occlusion? Thirty patients with a class III occlusion were included in this prospective study. Surgical planning with both techniques were undertaken for all patients. Surgery was performed according to the two-dimensional technique. The cephalometric measurements from two-dimensional and three dimensional predictions were compared with the postoperative results at the 12 months follow-up respectively. Together with an analysis of tracing error, placements of 2020 markers, 1860 measurements and 1280 comparisons was performed. The analysis showed an equally high accuracy for the studied techniques. The highest accuracy was found in the anterior maxilla. There was a tendency for an overestimation for the three-dimensional technique and an underestimation for the two-dimensional technique. CONCLUSIONS: The present study indicates an equal high accuracy in predicting facial outcome for both studied techniques. However, in those patients with asymmetric malocclusion and/or facial appearance the three-dimensional technique has an obvious advantage.

Introduction Thorough treatment planning is essential for a good clinical outcome in orthognathic treatment. The planning is often digital. Both 2-dimensional (2D) and 3-dimensional (3D) software options are available. The aim of this randomized 2-arm parallel double-blinded active-controlled clinical trial was to compare the outcomes of computer-based 2D and 3D planning techniques according to patient-reported health related quality of life. The hypothesis was that a 3D technique would give a better treatment outcome compared with a 2D technique. Methods Orthognathic treatment for 62 subjects, aged 18 to 28 years, with severe Class III malocclusion was planned with both 2D and 3D techniques. After treatment planning but before surgery, the patients were randomly allocated via blind collection of 1 enveloped card for each subject in a 1:1 ratio to the test (3D) or the control (2D) group. Thus, the intervention was according to which planning technique was used. The primary outcome was patient-reported outcome measures. The secondary outcome was relationship between patient-reported outcome measures and cephalometric accuracy. Questionnaires on the patient's health-related quality of life (HRQoL) were distributed preoperatively and 12 months after surgical treatment. The questionnaires were coded, meaning blinding throughout the analysis. Differences between groups were tested with the Fisher permutation test. The HRQoL was also compared with measurements of cephalometric accuracy for the 2 groups. Results Three subjects were lost to clinical follow-up, leaving 57 included. Of these, 55 subjects completed the questionnaires, 28 in the 2D and 27 in the 3D groups. No statistically significant difference regarding HRQoL was found between the studied planning techniques: the Oral Health Impact Profile total showed −3.69 (95% confidence interval, −19.68 to 12.30). Consistent results on HRQoL and cephalometric accuracy showed a difference between pretreatment and posttreatment that increased in both groups but to a higher level in the 3D group. A difference between pretreatment and posttreatment HRQoL was shown for both groups, indicating increased quality of life after treatment. This supports recent findings comparing 3D and 2D planning techniques. No serious harm was observed during the study. Conclusions Improvements of HRQoL were shown after treatment independent of which planning technique, 2D or 3D, was used. No statistically significant difference was found between the planning techniques. Registration This trial was not registered. Protocol The protocol was not published before trial commencement. Funding This project was supported by personal grants to Martin Bengtsson from the Scandinavian Association of Oral and Maxillofacial Surgeons (25000 SEK), the Southern Region of the Swedish Dental Association (50000 SEK), and the Swedish Association of Oral and Maxillofacial Surgeons (25000 SEK). The sponsors had no influence on the study design, analysis of the data, or the writing of the article.

Raw data from calorimetric measurements of five nuclear fuel assemblies of the PWR 17 × 17 type are provided. Measurements of the temperature both inside a calorimeter, in which the fuel assembly was placed, as well as outside, were performed as a function of time while water circulating inside the calorimeter heats up from radiation emitted in the radioactive decay of material in the fuel assembly. The data contain also measurements of dose rate in the water outside the calorimeter. Data from 38 measurements using an electrically heated model of a fuel assembly are also provided to be used for, e.g., calibration. The data can be used for validation of computer codes for modelling of nuclear systems, e.g. nuclear reactors, storage and transport of nuclear fuel or systems for geological disposal.

Using a high-purity Germanium gamma-ray energy spectroscopic detector system, time-stamped list-mode data sets were acquired during axial scanning of 19 boiling water reactor (BWR) and 28 pressurized water reactor (PWR) type of nuclear fuel assemblies. The data sets were collected during two measurements campaigns in September 2016 and March 2019 at the Central Interim Storage Facility for Spent Nuclear (Clab) in Sweden. A certified calibration source of 137Cs was positioned along the central line of sight between the measured fuel assembly and the detector. Data sets from measurements with only the calibration source and other background sources, i.e. without a nuclear fuel assembly present, are also included. The list-mode structure of the measured data allows for an axially-resolved as well as energy-spectral resolved intensity of nuclide-specific gamma lines emitted from the spent nuclear fuel. Data presented here can be used e.g. for validation of gamma-ray transport simulation tools or for development of methods to estimate parameters of the spent nuclear fuel based on data from gamma-ray spectroscopy.

The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.

Objective. Flexible neural probes are hypothesized to reduce the chronic foreign body response (FBR) mainly by reducing the strain-stress caused by an interplay between the tethered probe and the brain's micromotion. However, a large discrepancy of Young's modulus still exists (3-6 orders of magnitude) between the flexible probes and the brain tissue. This raises the question of whether we need to bridge this gap; would increasing the probe flexibility proportionally reduce the FBR? Approach. Using novel off-stoichiometry thiol-enes-epoxy (OSTE+) polymer probes developed in our previous work, we quantitatively evaluated the FBR to four types of probes with different softness: silicon (∼150 GPa), polyimide (1.5 GPa), OSTE+Hard (300 MPa), and OSTE+Soft (6 MPa). Main results. We observed a significant reduction in the fluorescence intensity of biomarkers for activated microglia/macrophages and blood-brain barrier (BBB) leakiness around the three soft polymer probes compared to the silicon probe, both at 4 weeks and 8 weeks post-implantation. However, we did not observe any consistent differences in the biomarkers among the polymer probes. Significance. The results suggest that the mechanical compliance of neural probes can mediate the degree of FBR, but its impact diminishes after a hypothetical threshold level. This infers that resolving the mechanical mismatch alone has a limited effect on improving the lifetime of neural implants. (Less)

The aim of the present study was to compare the accuracy of two-dimensional (2D) and three-dimensional (3D) prediction methods. The hypothesis was that a 3D technique would give a more accurate outcome of the postoperative result.Patients with severe class III occlusion were included and planed with both a 2D and 3D prediction technique. They were there after randomly subdivided into a control (2D) and test (3D) group and treated according to the technique randomized for. Cephalometric measurements from 2D and 3D predictions were compared with 12-month follow-up respectively. Together with an analysis of tracing error, placements of 3580 cephalometric markers, 2460 measurements, 680 intra-individual analyses and 1200 preop/postop comparisons were performed in 57 individuals.Statistically significant differences for accuracy between the two groups were seen for 11/NSL-112/NSL2 and for A-A2 (p 0.05). Both groups showed a high level of accuracy for SNA and SNB. The test group also showed a relatively high level of accuracy for 11/NSL and for the A-point. No prediction method achieves a perfect accuracy. As expected from this, measuring accuracy within each group showed statistically significant difference for all markers and cephalometric measurements (p 0.001). Mandibular markers showed greater differences than maxillary markers.The present study indicates an equal high accuracy in predicting facial outcome for both studied techniques. However, in patients with asymmetry the three-dimensional technique has an obvious advantage.

In early-stage atherosclerosis, the luminal surface of the arterial wall becomes rough because of detachment of endothelial cells and degeneration of the internal elastic layer. Therefore, it would be useful if minute luminal surface roughness of the carotid arterial wall, which occurs in the early stage of atherosclerosis, could be measured noninvasively with ultrasound. The injured luminal surface is believed to have roughness of a few hundred micrometers. However, in conventional ultrasonography, the axial resolution of a B-mode image depends on the ultrasonic wavelength (150 μm at ultrasonic center frequency of 10 MHz) because a B-mode image is constructed using the amplitude of the RF echo signal. Therefore, such surface roughness cannot be measured accurately from a conventional B-mode image. Recently, we successfully measured such minute surface profile transcutaneously using the phase shift of an ultrasonic echo from the carotid arterial wall. In our previous validation experiment, a silicone phantom with minute surface roughness of 10–20 μm was measured. However, the feasibility of our proposed method has never been validated using biological tissues. In the present study, luminal surface roughness of a porcine artery was measured and the result was evaluated by comparing it with the result measured using a stylus profilometer. The root mean squared difference between the surface roughness measured by ultrasound and the stylus profilometer was 10.5 μm. This result proves that our proposed method can be used to measure minute surface roughness of biological tissue.

A project to research the application of non-destructive assay (NDA) techniques for spent fuel assemblies is underway at the Central Interim Storage Facility for Spent Nuclear Fuel (for which the S ...

Glucagon secretion is inhibited by glucagon-like peptide-1 (GLP-1) and stimulated by adrenaline. These opposing effects on glucagon secretion are mimicked by low (1-10 nM) and high (10 muM) concentrations of forskolin, respectively. The expression of GLP-1 receptors in alpha cells is

The neuronal soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is essential for synaptic vesicle exocytosis, but its study has been limited by the neonatal lethality of murine SNARE knockouts. Here, we describe a viable mouse line carrying a mutation in the b-isoform of neuronal SNARE synaptosomal-associated protein of 25 kDa (SNAP-25). The causative I67T missense mutation results in increased binding affinities within the SNARE complex, impaired exocytotic vesicle recycling and granule exocytosis in pancreatic β-cells, and a reduction in the amplitude of evoked cortical excitatory postsynaptic potentials. The mice also display ataxia and impaired sensorimotor gating, a phenotype which has been associated with psychiatric disorders in humans. These studies therefore provide insights into the role of the SNARE complex in both diabetes and psychiatric disease.

Background: Gene expression has a strong stochastic element resulting in highly variable mRNA levels between individual cells, even in a seemingly homogeneous cell population. Access to fundamental information about cellular mechanisms, such as correlated gene expression, motivates measurements of multiple genes in individual cells. Quantitative reverse transcription PCR (RT-qPCR) is the most accessible method which provides sufficiently accurate measurements of mRNA in single cells. Results: Low concentration of guanidine thiocyanate was used to fully lyse single pancreatic β-cells followed by RT-qPCR without the need for purification. The accuracy of the measurements was determined by a quantitative noise-model of the reverse transcription and PCR. The noise is insignificant for initial copy numbers >100 while at lower copy numbers the noise intrinsic of the PCR increases sharply, eventually obscuring quantitative measurements. Importantly, the model allows us to determine the RT efficiency without using artificial RNA as a standard. The experimental setup was applied on single endocrine cells, where the technical and biological noise levels were determined. Conclusion: Noise in single-cell RT-qPCR is insignificant compared to biological cell-to-cell variation in mRNA levels for medium and high abundance transcripts. To minimize the technical noise in single-cell RT-qPCR, the mRNA should be analyzed with a single RT reaction, and a single qPCR reaction per gene.

This paper presents a new strategy to combine the power of antibody based capturing of target species in complex samples with the benefits of microfluidic reverse phase sample preparation on an integrated sample enrichment target (RP-ISET) and the analysis speed of MALDI MS. The immunoaffinity step is performed on an in-house developed 3D-structured high surface area porous silicon (PSi) matrix, which allows efficient antibody immobilization by surface adsorption without any coupling agents in 30-60 min. The hydrophilic nature of the porous silicon surface at the molecular level displays a low adsorption of background peptides when exposed to complex digests or plasma samples, improving the conditions for the antigen specific extraction and subsequent readout. At the same time, the hydrophobic behavior, due to the nanostructured surface, of the PSi material facilitates liquid confinement during the assay. Using a footprint conforming to the standard for 384 well microplates, direct adaption of the protocol into standard sample handling robots is possible. The performance of the proposed immunoaffinity PSi-ISET immunoMALDI (iMALDI) assay was evaluated by specific detection of angiotensin I at a 10 femtomol level in diluted plasma samples (10 μL, 1 nM).

OBJECTIVE Paracrine signaling via γ-aminobutyric acid (GABA) and GABAA receptors (GABAARs) has been documented in rodent islets. Here we have studied the importance of GABAergic signaling in human pancreatic islets. RESEARCH DESIGN AND METHODS Expression of GABAARs in islet cells was investigated by quantitative PCR, immunohistochemistry, and patch-clamp experiments. Hormone release was measured from intact islets. GABA release was monitored by whole-cell patch-clamp measurements after adenoviral expression of α1β1 GABAAR subunits. The subcellular localization of GABA was explored by electron microscopy. The effects of GABA on electrical activity were determined by perforated patch whole-cell recordings. RESULTS PCR analysis detected relatively high levels of the mRNAs encoding GABAAR α2, β3, γ2, and π subunits in human islets. Patch-clamp experiments revealed expression of GABAAR Cl− channels in 52% of β-cells (current density 9 pA/pF), 91% of δ-cells (current density 148 pA/pF), and 6% of α-cells (current density 2 pA/pF). Expression of GABAAR subunits in islet cells was confirmed by immunohistochemistry. β-Cells secreted GABA both by glucose-dependent exocytosis of insulin-containing granules and by a glucose-independent mechanism. The GABAAR antagonist SR95531 inhibited insulin secretion elicited by 6 mmol/l glucose. Application of GABA depolarized β-cells and stimulated action potential firing in β-cells exposed to glucose. CONCLUSIONS Signaling via GABA and GABAAR constitutes an autocrine positive feedback loop in human β-cells. The presence of GABAAR in non–β-cells suggests that GABA may also be involved in the regulation of somatostatin and glucagon secretion.

Even in an apparently homogeneous population of cells there are considerable differences between individual cells. A response to a stimulus of a cell population or tissue may be consistent and gradual while the single-cell response might be binary and apparently irregular. The origin of this variability may be preprogrammed or stochastic and a study of this phenomenon will require quantitative measurements of individual cells. Here, we describe a method to collect dispersed single cells either by glass capillaries or flow cytometry, followed by quantitative mRNA profiling using reverse transcription and real-time PCR. We present a single cell lysis protocol and optimized priming conditions for reverse transcription. The large cell-to-cell variability in single-cell gene expression measurements excludes it from standard data analysis. Correlation studies can be used to find common regulatory elements that are indistinguishable at the population level. Single-cell gene expression profiling has the potential to become common practice in many laboratories and a powerful research tool for deeper understanding of molecular mechanisms.

Background: Human embryonic stem cells (hESCs) require expression of transcription factor genes POU5F1 (POU class 5 homeobox 1), NANOG (Nanog homeobox), and SOX2 [SRY (sex determining region Y)-box 2] to maintain their capacity for self-renewal and pluripotency. Because of the heterogeneous nature of cell populations, it is desirable to study the gene regulation in single cells. Large and potentially important fluctuations in a few cells cannot be detected at the population scale with microarrays or sequencing technologies. We used single-cell gene expression profiling to study cell heterogeneity in hESCs. Methods: We collected 47 single hESCs from cell line SA121 manually by glass capillaries and 57 single hESCs from cell line HUES3 by flow cytometry. Single hESCs were lysed and reverse-transcribed. Reverse-transcription quantitative real-time PCR was then used to measure the expression POU5F1, NANOG, SOX2, and the inhibitor of DNA binding genes ID1, ID2, and ID3. A quantitative noise model was used to remove measurement noise when pairwise correlations were estimated. Results: The numbers of transcripts per cell varied >100-fold between cells and showed lognormal features. POU5F1 expression positively correlated with ID1 and ID3 expression (P < 0.05) but not with NANOG or SOX2 expression. When we accounted for measurement noise, SOX2 expression was also correlated with ID1, ID2, and NANOG expression (P < 0.05). Conclusions: We demonstrate an accurate method for transcription profiling of individual hESCs. Cell-to-cell variability is large and is at least partly nonrandom because we observed correlations between core transcription factors. High fluctuations in gene expression may explain why individual cells in a seemingly undifferentiated cell population have different susceptibilities for inductive cues.

Pancreatic islets have a central role in blood glucose homeostasis. In addition to insulin-producing beta-cells and glucagon-secreting alpha-cells, the islets contain somatostatin-releasing delta-cells(1). Somatostatin is a powerful inhibitor of insulin and glucagon secretion(2). It is normally secreted in response to glucose(3) and there is evidence suggesting its release becomes perturbed in diabetes(4). Little is known about the control of somatostatin release. Closure of ATP-regulated K+-channels (K-ATP-channels)(5) and a depolarization-evoked increase in cytoplasmic free Ca2+ concentration ([Ca2+](i))(6-8) have been proposed to be essential. Here, we report that somatostatin release evoked by high glucose (>= 10 mM) is unaffected by the K-ATP-channel activator diazoxide and proceeds normally in K-ATP-channel-deficient islets. Glucose-induced somatostatin secretion is instead primarily dependent on Ca2+-induced Ca2+-release (CICR). This constitutes a novel mechanism for K-ATP-channel-independent metabolic control of pancreatic hormone secretion.

Background The flexibility of implantable neural probes has increased during the last 10 years, starting with stiff materials such as silicone to more flexible materials like polyimide. We have developed a novel polymer based on Off-Stoichiometry Thiol-Enes + Epoxy (OSTE+, consisting of a thiol, two allyls, an epoxy resin and two initiators), which is up to 100 times more flexible than polyimide. Since a flexible neural probe should be more biocompatible than a stiff probe, an OSTE+ probe should be more biocompatible than one composed of a more rigid material. We have investigated the toxicity of OSTE+ as well as of OSTE+ that had been incubated in water for a week (OSTE+H2O) using MTT assays with mouse L929 fibroblasts. We found that OSTE+ showed cytotoxicity, but OSTE+H2O did not. Extracts were analyzed using LC-MS and GC-MS in order to identify leaked chemicals. Results Most constituents were found in extracts of OSTE+, whereas only initiators were found in OSTE+H2O extracts. The detected levels of each chemical found in the LC-MS and the GC-MS analysis were below the toxicity level when compared to MTT assays of all the individual chemicals, except for one of the initiators that had an IC50 value close to the detected levels. Conclusion Our notion is that the toxicity of OSTE+ was caused by one of the initiators, by impurities in the constituents or by synergistic effects of low doses of leaked chemicals. However, our conclusion is that if OSTE+ is incubated for one week in water, OSTE+ is not cytotoxic and suitable for further in vivo studies.

In this study,a silicon micro immobilised enzyme reactor (mu IMER) has been applied for hydrolysis of maltoheptaose as a model maltodextrin and starch using immobilised otamylase (from Aspergillus oryzae) and glycoamylase (from Aspergillus niger). The influence of several parameters was investigated such as immobilisation chemistry, buffer constituents, pH, temperature, flow rate and substrate concentration. The conversion efficiency profile of the substrate was measured and the long-term stability of the reactor was tested. For separation and detection of the formed hydrolysis products, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was used. The results show that the mu IMERs can also be used for hydrolysis of starch and also additionally be connected directly on-line with, e.g., liquid chromatography, making it possible to perform on-line characterisation and analysis of starch hydrolysis products. (Less)

The scientific, medical, and diagnostic communities have been presented the most powerful tool for quantitative nucleic acids analysis: real-time PCR [Bustin, S.A., 2004. A-Z of Quantitative PCR. IUL Press, San Diego, CA]. This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers in the mid 80:ies [Saiki, R.K., et al., 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science 230, 1350], for which Kary Mullis was awarded the 1993 year's Nobel prize in Chemistry. By PCR essentially any nucleic acid sequence present in a complex sample can be amplified in a cyclic process to generate a large number of identical copies that can readily be analyzed. This made it possible, for example, to manipulate DNA for cloning purposes, genetic engineering, and sequencing. But as an analytical technique the original PCR method had some serious limitations. By first amplifying the DNA sequence and then analyzing the product, quantification was exceedingly difficult since the PCR gave rise to essentially the same amount of product independently of the initial amount of DNA template molecules that were present. This limitation was resolved in 1992 by the development of real-time PCR by Higuchi et al. [Higuchi, R., Dollinger, G., Walsh, P.S., Griffith, R., 1992. Simultaneous amplification and detection of specific DNA-sequences. Bio-Technology 10(4), 413-417]. In real-time PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Assuming a certain amplification efficiency, which typically is close to a doubling of the number of molecules per amplification cycle, it is possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays that are available today the number of DNA molecules of a particular sequence in a complex sample can be determined with unprecedented accuracy and sensitivity sufficient to detect a single molecule. Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism (SNP) analysis, analysis of chromosome aberrations, and most recently also protein detection by real-time immuno PCR.

The transcriptional machinery in individual cells is controlled by a relatively small number of molecules, which may result in stochastic behavior in gene activity. Because of technical limitations in current collection and recording methods, most gene expression measurements are carried out on populations of cells and therefore reflect average mRNA levels. The variability of the transcript levels between different cells remains undefined, although it may have profound effects on cellular activities. Here we have measured gene expression levels of the five genes ActB, Ins1, Ins2, Abcc8, and Kcnj11 in individual cells from mouse pancreatic islets. Whereas Ins1 and Ins2 expression show a strong cell–cell correlation, this is not the case for the other genes. We further found that the transcript levels of the different genes are lognormally distributed. Hence, the geometric mean of expression levels provides a better estimate of gene activity of the typical cell than does the arithmetic mean measured on a cell population.

Cellulases hydrolysing the interior parts of cellulose, also called endoglucanases, were immobilised in micro-immobilised enzyme reactors (RIMER) made of porous silicon with the purpose of investigating the use of such mu IMERs for hydrolysis of cellodextrins and soluble cellulose derivatives. The endoglucanases Trichoderma reesei Cel 12A (TrCel 12A) and Bacillus agaradhaerens Cel 5A (BaCel 5A) were covalently coupled to the surface of a silicon microchip through Schiff base formation. For characterisation cellohexaose was used as substrate for the immobilised enzymes. The characteristics of the RIMER were investigated by studying the product formation when varying the concentration, flow-rate, temperature and pH of the substrate solution. Hydrolysis was performed in the RIMER connected on-line to a chromatographic system, where the products were separated and detected using high-performance anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (PAD). A comparison of the hydrolytic pattern between BaCel 5A and TrCel 12A was carried out and the results show that the two investigated endoglucanases give specific hydrolytic patterns in the products formed that provide important information about the enzymes. The RIMERs are robust and can be employed continuously over a period of at least several days. Moreover, on appropriate storage, no activity loss is seen after 60 days. The ability of the BaCel 5A containing RIMER to perform hydrolysis of derivatised cellulose was also investigated using carboxymethyl cellulose (CMC) as substrate. Separation and detection were carried out using size exclusion chromatography (SEC) with refractive index detection (RI). The results show that the RIMERs are robust and can be employed for on-line hydrolysis of both cellodextrins and derivatised cellulose of high molecular weight. (c) 2005 Elsevier B.V. All rights reserved. (Less)

Microchip immobilized enzyme reactors (microIMERs) with immobilized endoglucanases were applied for the hydrolysis of methyl cellulose (MC). MCs of various molecular weights were hydrolyzed using two microIMERs containing immobilized celloendoglucanase Cel 5A from Bacillus agaradhaerens (BaCel 5A) connected in series. Hydrolysis by the microIMER could be confirmed from the average molar masses and molar mass distributions measured by size exclusion chromatography (SEC) with online multiangle light scattering and refractive index detection. Methylated cellooligosaccharides with degrees of polymerization (DP) between 1 and 6 formed during hydrolysis were analyzed by direct infusion electrospray ionization ion-trap mass spectrometry (ESI-ITMS). Mass spectra of microIMER- and batch-hydrolyzed samples were compared and no significant differences were found, indicating that microIMER hydrolysis was as efficient as conventional batch hydrolysis. A fast and automated hydrolysis with online MS detection was achieved by connecting the microIMER to high-performance liquid chromatography and ESI-ITMS. This online separation reduced the relative intensities of interfering signals and increased the signal-to-noise ratios in MS. The microIMER hydrolysates were also subjected to SEC interfaced with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. With this technique, oligomers with DP 3-30 could be detected. The hydrolysis by the microIMER was performed within 60 min, i.e. significantly faster compared with batch hydrolysis usually performed for at least 24 h. The microIMER also allowed hydrolysis after 10 days of continuous use. The method presented in this work offers new approaches for the analysis of derivatized cellulose and provides the possibility of convenient online, fast, and more versatile analysis compared with the traditional batch method.

A microfluidic system for long-term real-time monitoring of dynamic cellular events of immobilized human cells was investigated. The luciferase reporter gene activity in the reporter cell line HFF11, based on HeLa cells, was used as the model system. The cells were immobilized on silicon flow-through microchips and continuously supplied with a cell medium at 2 microL/min while maintaining the chip at 37 degrees C. The HFF11 cell line was designed for high-throughput screening of ligands for seven-transmembrane receptors. When a ligand binds, the receptor is activated and a cascade of intracellular reactions starts, ending with the synthesis of the reporter protein Photinus luciferase. The major goal was to develop a microfluidic system for continuous long-term assaying of the intracellular reporter gene activity in real time and determine the conditions, which could minimize cells stress and hence unspecific expression of the reporter gene. In the resulting microfluidic system and assay protocol, the cell microchip could be kept and assayed for a period up to 30 h. The developed system and data outcome was compared with a corresponding microtiter plate performed with the same cell line to highlight the advantages obtained in the microfluidic format.

Chemiluminescent (CL) enzyme-based flow-through microchip biosensors (micro-biosensors) for detection of glucose and ethanol were developed for the purpose of monitoring real-time production and release of glucose and ethanol from microchip immobilised yeast cells. Part I of this study focuses on the development and optimisation of the micro-biosensors in a microfluidic sequential injection analysis (microSIA) system. Glucose oxidase (GOX) or alcohol oxidase (AOX) was co-immobilised with horseradish peroxidase (HRP) on porous silicon flow through microchips. The hydrogen peroxide produced from oxidation of the corresponding analyte (glucose or ethanol) took part in the chemiluminescent (CL) oxidation of luminol catalysed by HRP enhanced by addition of p-iodophenol (PIP). All steps in the microSIA system, including control of syringe pump, multiposition valve (MPV) and data readout, were computer controlled. The influence of flow rate and luminol- and PIP concentration were investigated using a 2(3)-factor experiment using the GOX-HRP sensor. It was found that all estimated single factors and the highest order of interaction were significant. The optimum was found at 250 microM luminol and 150 microM PIP at a flow rate of 18 microl min(-1), the latter as a compromise between signal intensity and analysis time. Using the optimised system settings one sample was processed within 5 min. Two different immobilisation chemistries were investigated for both micro-biosensors based on 3-aminopropyltriethoxsilane (APTS)- or polyethylenimine (PEI) functionalisation followed by glutaraldehyde (GA) activation. GOX-HRP micro-biosensors responded linear in a log-log format within the range 10-1000 microM glucose. Both had an operational stability of at least 8 days, but the PEI-GOX-HRP sensor was more sensitive. The AOX-HRP micro-biosensors responded linear (log-log) in the range between 1 and 10 mM ethanol, but the PEI-AOX-HRP sensor was in general more sensitive. Both sensors had an operational stability of at least 8 h, but with a half-life of 2-3 days.

A microfluidic flow injection (microFIA) system was employed for handling and monitoring of cell-released products from living cells immobilised on silicon microchips. The dynamic release of glucose and ethanol produced from sucrose by immobilised Saccharomyces cerevisiae cells was determined using microchip biosensors (micro-biosensors) with either co-immobilised glucose oxidase-horseradish peroxidase (GOX-HRP), or alcohol oxidase-horseradish peroxidase (AOX-HRP), catalysing a series of reactions ending up with chemiluminescence (CL) generated from HRP-catalysed oxidation of luminol in presence of p-iodophenol (PIP). The yeast cells were attached by first treating them with polyethylenimine (PEI) followed by adsorption to the microchip surface. The cell loss during assaying was evaluated qualitatively using scanning electron microscopy (SEM), showing that no cells were lost after 35 min liquid handling of the cell chip at 10 microl min(-1). The enzymes were immobilised on microchips via PEI-treatment followed by glutaraldehyde (GA) activation. The GOX-HRP micro-biosensors could be used during five days without any noticeable decrease in response, while the AOX-HRP micro-biosensors showed continuously decreasing activity, but could still be used employing calibration correction. The glucose and ethanol released from the immobilised yeast chips were quantitatively monitored, by varying the incubation time with sucrose, showing the possibilities and advantages of using a microfluidic system set-up for cell-based assays.

Affinity proteins were covalently immobilised on silicon microchips with overall dimensions of 13.1 x 3.2 mm, comprising 42 porous flow channels of 235 microm depth and 25 microm width, and used to develop microfluidic immunosensors based on horseradish peroxidase (HRP), catalysing the chemiluminescent oxidation of luminol/p-iodophenol (PIP). Different hydrophilic polymers with long flexible chains (polyethylenimine (PEI), dextran (DEX), polyvinyl alcohol, aminodextran) and 3-aminopropyltriethoxysilane (APTS) were employed for modification of the silica surfaces followed by attachment of protein A or G. The resulting immunosensors were compared in an affinity capture assay format, where the competition between the labelled antigen and the analyte for antibody-binding sites took place in the bulk of the solution. The formed immunocomplexes were then trapped by the microchip affinity capture support and the amount of bound tracer was monitored by injection of luminol, PIP and H2O2. All immunosensors were capable of detecting atrazine at the sub-microg l(-1) level. The most sensitive assays were obtained with PEI and DEX polymer modified supports and immobilised protein G, with limits of detection of 0.006 and 0.010 microg l(-1), and IC50 values of 0.096 and 0.130 microg l(-1), respectively. The protein G based immunosensors were regenerated with 0.4 M glycine-HCl buffer pH 2.2, with no loss of activity observed for a storage and operating period of over 8 months. To estimate the applicability of the immunosensors to the analysis of real samples, PEI and DEX based protein G microchips were used to detect atrazine in surface water and fruit juice, spiked with known amounts of the atrazine, giving recovery values of 87-102 and 88-124% at atrazine fortification levels of 0.5-3 and 80-240 microg l(-1), respectively.

Silicon microchips with immobilized antibodies were used to develop microfluidic enzyme immunoassays using chemiluminescence detection and horseradish peroxidase (HRP) as the enzyme label. Polyclonal anti-atrazine antibodies were coupled to the silicon microchip surface with an overall dimension of 13.1 x 3.2 mm, comprising 42 porous flow channels of 235-microm depth and 25-microm width. Different immobilization protocols based on covalent or noncovalent modification of the silica surface with 3-aminopropyltriethoxysilane (APTES) or 3-glycidoxypropyltrimethoxysilane (GOPS), linear polyethylenimine (LPEI, MW 750,000), or branched polyethylenimine (BPEI, MW 25,000), followed by adsorption or covalent attachment of the antibody, were evaluated to reach the best reusability, stability, and sensitivity of the microfluidic enzyme immunoassay (microFEIA). Adsorption of antibodies on a LPEI-modified silica surface and covalent attachment to physically adsorbed BPEI lead to unstable antibody coatings. Covalent coupling of antibodies via glutaraldehyde (GA) to three different functionalized silica surfaces (APTES-GA, LPEI-GA, and GOPS-BPEI-GA) resulted in antibody coatings that could be completely regenerated using 0.4 M glycine/HCl, pH 2.2. The buffer composition was shown to have a dramatic effect on the assay stability, where the commonly used phosphate buffer saline was proved to be the least suitable choice. The best long-term stability was obtained for the LPEI-GA surface with no loss of antibody activity during one month. The detection limits in the microFEIA for the three different immuno surfaces were 45, 3.8, and 0.80 ng/L (209, 17.7, and 3.7 pM) for APTES-GA, LPEI-GA, and GOPS-BPEI-GA, respectively.

We have developed a multichannel electrode array—termed \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}\(\mu \) \end{document}-foil—that comprises ultrathin and flexible electrodes protruding from a thin foil at fixed distances. In addition to allowing some of the active sites to reach less compromised tissue, the barb-like protrusions that also serves the purpose of anchoring the electrode array into the tissue. This paper is an early evaluation of technical aspects and performance of this electrode array in acute in vitro/in vivo experiments. The interface impedance was reduced by up to two decades by electroplating the active sites with platinum black. The platinum black also allowed for a reduced phase lag for higher frequency components. The distance between the protrusions of the electrode array was tailored to match the architecture of the rat cerebral cortex. In vivo acute measurements confirmed a high signal-to-noise ratio for the neural recordings, and no significant crosstalk between recording channels., We have developed a multichannel electrode array, termed m-foil, that comprises ultrathin and flexible electrodes protruding from a thin foil at fixed distances. This study is an early evaluation of technical aspects and performance of this electrode array in acute in vitro/in vivo experiments. The in vivo acute measurements showed a good signal-to-noise ratio for the neural recordings, and no significant cross-talk between recording channels.

Summary Glucagon, secreted by pancreatic islet α cells, is the principal hyperglycemic hormone. In diabetes, glucagon secretion is not suppressed at high glucose, exacerbating the consequences of insufficient insulin secretion, and is inadequate at low glucose, potentially leading to fatal hypoglycemia. The causal mechanisms remain unknown. Here we show that α cell KATP-channel activity is very low under hypoglycemic conditions and that hyperglycemia, via elevated intracellular ATP/ADP, leads to complete inhibition. This produces membrane depolarization and voltage-dependent inactivation of the Na+ channels involved in action potential firing that, via reduced action potential height and Ca2+ entry, suppresses glucagon secretion. Maneuvers that increase KATP channel activity, such as metabolic inhibition, mimic the glucagon secretory defects associated with diabetes. Low concentrations of the KATP channel blocker tolbutamide partially restore glucose-regulated glucagon secretion in islets from type 2 diabetic organ donors. These data suggest that impaired metabolic control of the KATP channels underlies the defective glucose regulation of glucagon secretion in type 2 diabetes., Graphical Abstract, Highlights • KATP channel closure stimulates insulin secretion but inhibits glucagon release • α cell depolarization reduces voltage-gated Ca2+ entry and glucagon release • An activating KATP channel mutation impairs glucagon release in mice • KATP channel closure corrects glucagon secretion defect in type 2 diabetic islets

We have proposed an echo localization estimator which theoretically gives infinite resolution in the axial direction in ultrasonic B-mode images during 1) echo structure tracking and 2) distance measurements of distinct echoes. The performance of the estimator was evaluated using a HDI5000 (Philips Medical Systems, Bothell, WA, USA) with a 35 mm 5-12 MHz linear array transducer. The resulting pixel size in the B-mode image was 100 m. The controlled setup consisted of a small water container lined with ultrasound absorber carpet and placed on a micrometer table, a triangulation laser (M5L/2, MEL Mikroelektronik GmbH, Eching, Germany) connected to a high-performance digital multimeter HP 34401A (Agilent Technologies, USA) and a thermometer TESTO 925 (Testo AG, Germany). In each of the three measurement series the micrometer table was moved approximately 15 m in 10-12 steps. The echo localization estimator succeeded to resolve the minute movement ( 0.998; p

A polymer foil based array with 9 gold electrodes for chronic electrophysiological recordings in the CNS has been developed. A polymer, SU-8, is pattered using photolithography techniques used every day in micro fabrication. This is beneficial if the electrode would be manufactured on a large scale basis. The technique makes it easy to adapt the array to best fit the structure of interest at the time, in this study the rat cerebellar cortex. The use of SU-8 as the base of the array makes the array very flexible, hence lets it stay close to the same cells following the movement of the brain. The electrodes can then be modified with platinum black to lower the impedance of the electrode up to one order of magnitude, making us able to create smaller electrodes but keeping the low impedance necessary to get a get the signal to noise ratio required. Platinum black modified arrays were also implanted chronically and showed excellent signal recording capabilities in rat cerebellum.

OBJECTIVE The aim of the study was to elucidate the cellular mechanism underlying the suppression of glucose-induced insulin secretion in mice fed a high-fat diet (HFD) for 15 weeks. RESEARCH DESIGN AND METHODS C57BL6J mice were fed a HFD or a normal diet (ND) for 3 or 15 weeks. Plasma insulin and glucose levels in vivo were assessed by intraperitoneal glucose tolerance test. Insulin secretion in vitro was studied using static incubations and a perfused pancreas preparation. Membrane currents, electrical activity, and exocytosis were examined by patch-clamp technique measurements. Intracellular calcium concentration ([Ca2+]i) was measured by microfluorimetry. Total internal reflection fluorescence microscope (TIRFM) was used for optical imaging of exocytosis and submembrane depolarization-evoked [Ca2+]i. The functional data were complemented by analyses of histology and gene transcription. RESULTS After 15 weeks, but not 3 weeks, mice on HFD exhibited hyperglycemia and hypoinsulinemia. Pancreatic islet content and β-cell area increased 2- and 1.5-fold, respectively. These changes correlated with a 20–50% reduction of glucose-induced insulin secretion (normalized to insulin content). The latter effect was not associated with impaired electrical activity or [Ca2+]i signaling. Single-cell capacitance and TIRFM measurements of exocytosis revealed a selective suppression (>70%) of exocytosis elicited by short (50 ms) depolarization, whereas the responses to longer depolarizations were (500 ms) less affected. The loss of rapid exocytosis correlated with dispersion of Ca2+ entry in HFD β-cells. No changes in gene transcription of key exocytotic protein were observed. CONCLUSIONS HFD results in reduced insulin secretion by causing the functional dissociation of voltage-gated Ca2+ entry from exocytosis. These observations suggest a novel explanation to the well-established link between obesity and diabetes.

AIMS/HYPOTHESIS: The aim of this study was to characterise electrical activity, ion channels, exocytosis and somatostatin release in human delta cells/pancreatic islets. METHODS: Glucose-stimulated somatostatin release was measured from intact human islets. Membrane potential, currents and changes in membrane capacitance (reflecting exocytosis) were recorded from individual human delta cells identified by immunocytochemistry. RESULTS: Somatostatin secretion from human islets was stimulated by glucose and tolbutamide and inhibited by diazoxide. Human delta cells generated bursting or sporadic electrical activity, which was enhanced by tolbutamide but unaffected by glucose. Delta cells contained a tolbutamide-insensitive, Ba(2+)-sensitive inwardly rectifying K(+) current and two types of voltage-gated K(+) currents, sensitive to tetraethylammonium/stromatoxin (delayed rectifying, Kv2.1/2.2) and 4-aminopyridine (A current). Voltage-gated tetrodotoxin (TTX)-sensitive Na(+) currents contributed to the action potential upstroke but TTX had no effect on somatostatin release. Delta cells are equipped with Ca(2+) channels blocked by isradipine (L), omega-agatoxin (P/Q) and NNC 55-0396 (T). Blockade of any of these channels interferes with delta cell electrical activity and abolishes glucose-stimulated somatostatin release. Capacitance measurements revealed a slow component of depolarisation-evoked exocytosis sensitive to omega-agatoxin. CONCLUSIONS/INTERPRETATION: Action potential firing in delta cells is modulated by ATP-sensitive K(+)-channel activity. The membrane potential is stabilised by Ba(2+)-sensitive inwardly rectifying K(+) channels. Voltage-gated L- and T-type Ca(2+) channels are required for electrical activity, whereas Na(+) currents and P/Q-type Ca(2+) channels contribute to (but are not necessary for) the upstroke of the action potential. Action potential repolarisation is mediated by A-type and Kv2.1/2.2 K(+) channels. Exocytosis is tightly linked to Ca(2+)-influx via P/Q-type Ca(2+) channels. Glucose stimulation of somatostatin secretion involves both K(ATP) channel-dependent and -independent processes.

The neural cell adhesion molecule (NCAM) is required for cell type segregation during pancreatic islet organogenesis. We have investigated the functional consequences of ablating NCAM on pancreatic beta-cell function. In vivo, NCAM(-/-) mice exhibit impaired glucose tolerance and basal hyperinsulinemia. Insulin secretion from isolated NCAM(-/-) islets is enhanced at glucose concentrations below 15 mM but inhibited at higher concentrations. Glucagon secretion from pancreatic alpha-cells evoked by low glucose was also severely impaired in NCAM(-/-) islets. The diminution of insulin secretion is not attributable to defective glucose metabolism or glucose sensing (documented as glucose-induced changes in intracellular Ca(2+) and K(ATP)-channel activity). Resting K(ATP) conductance was lower in NCAM(-/-) beta-cells than wild-type cells, and this difference was abolished when F-actin was disrupted by cytochalasin D (1 muM). In wild-type beta-cells, the submembrane actin network disassembles within 10 min during glucose stimulation (30 mM), an effect not seen in NCAM(-/-) beta-cells. Cytochalasin D eliminated this difference and normalized insulin and glucagon secretion in NCAM(-/-) islets. Capacitance measurements of exocytosis indicate that replenishment of the readily releasable granule pool is suppressed in NCAM(-/-) alpha- and beta-cells. Our data suggest that remodeling of the submembrane actin network is critical to normal glucose regulation of both insulin and glucagon secretion.