Adjuvant radiotherapy and chemoradiation with gemcitabine after R1 resection in patients with pancreatic adenocarcinoma.

BACKGROUND: The purpose of the study was to evaluate the effect of radiation therapy and chemoradiation with gemcitabine (GEM) after R1 resection in patients with pancreatic adenocarcinoma (PAC). METHODS: We performed a retrospective analysis of 25 patients who were treated with postoperative radiotherapy (RT) or chemoradiation (CRT) after surgery with microscopically positive resection margins for primary pancreatic cancer (PAC). Median age was 60 years (range 34 to 74 years), and there were 17 male and 8 female patients. Fractionated RT was applied with a median dose of 49.6 Gy (range 36 to 54 Gy). Eight patients received additional intraoperative radiotherapy (IORT) with a median dose of 12 Gy. RESULTS: Median overall survival (mOS) of all treated patients was 22 months (95% confidence interval (CI) 7.9 to 36.1 months) after date of resection and 21.1 months (95% CI 7.6 to 34.6 months) after start of (C)RT. Median progression-free survival (mPFS) was 13.0 months (95% CI 0.93 to 25 months). Grading (G2 vs. G3, P = 0.005) and gender (female vs. male, P = 0.01) were significantly correlated with OS. There was a significant difference in mPFS between male and female patients (P = 0.008). A total of 11 from 25 patients experienced local tumour progression, and 19 patients were diagnosed with either locoregional or distant failure. CONCLUSIONS: We demonstrated that GEM-based CRT can be applied in analogy to neoadjuvant protocols in the adjuvant setting for PAC patients at high risk for disease recurrence after incomplete resection. Patients undergoing additive CRT have a rather good OS and PFS compared to historical control patient groups.

Uncovering bifurcation patterns in cortical synapses.

Individual cortical synapses are known to exhibit a very complex short-time dynamic behaviour in response to simple "naturalistic" stimulation. We describe a computational study of the experimentally obtained excitatory post-synaptic potential trains of individual cortical synapses. By adopting a new nonlinear modelling scheme we construct robust and repeatable models of the underlying dynamics. These models suggest that cortical synapses exhibit a wide range of either periodic or chaotic dynamics. For stimulus at a fixed rate our models predict that the response of the individual synapse will vary from a fixed point to periodic and chaotic, depending on the frequency of stimulus. Dynamics for individual synapses vary widely, suggesting that the individual behaviour of synapses is highly tuned and that the dynamic behaviour of even a small network of synapse-coupled neurons could be extremely varied.

Diffusion Properties of Normal-Appearing White Matter Microstructure and Severity of Motor Impairment in Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: The effect of white matter hyperintensities as measured by FLAIR MR imaging on functional impairment and recovery after ischemic stroke has been investigated thoroughly. However, there has been growing interest in investigating normal-appearing white matter microstructural integrity following ischemic stroke onset with techniques such as DTI. MATERIALS AND METHODS: Fifty-two patients with acute ischemic stroke and 36 without stroke were evaluated with a DTI and FLAIR imaging protocol and clinically assessed for the severity of motor impairment using the Motricity Index within 72 hours of suspected symptom onset. RESULTS: There were widespread decreases in fractional anisotropy and increases in mean diffusivity and radial diffusivity for the acute stroke group compared with the nonstroke group. There was a significant positive association between fractional anisotropy and motor function and a significant negative association between mean diffusivity/radial diffusivity and motor function. The normal-appearing white matter ROIs that were most sensitive to the Motricity Index were the anterior/posterior limb of the internal capsule in the infarcted hemisphere and the splenium of the corpus callosum, external capsule, posterior limb/retrolenticular part of the internal capsule, superior longitudinal fasciculus, and cingulum (hippocampus) of the intrahemisphere/contralateral hemisphere. CONCLUSIONS: The microstructural integrity of normal-appearing white matter is a significant parameter to identify neural differences not only between those individuals with and without acute ischemic stroke but also correlated with the severity of acute motor impairment.

Correlation entropy of synaptic input-output dynamics.

The responses of synapses in the neocortex show highly stochastic and nonlinear behavior. The microscopic dynamics underlying this behavior, and its computational consequences during natural patterns of synaptic input, are not explained by conventional macroscopic models of deterministic ensemble mean dynamics. Here, we introduce the correlation entropy of the synaptic input-output map as a measure of synaptic reliability which explicitly includes the microscopic dynamics. Applying this to experimental data, we find that cortical synapses show a low-dimensional chaos driven by the natural input pattern.

High dietary zinc supplementation increases the occurrence of tetracycline and sulfonamide resistance genes in the intestine of weaned pigs.

BACKGROUND: Dietary zinc oxide is used in pig nutrition to combat post weaning diarrhoea. Recent data suggests that high doses (2.5 g/kg feed) increase the bacterial antibiotic resistance development in weaned pigs. Therefore, the aim of this study was to investigate the development of enterobacterial antibiotic resistance genes in the intestinal tract of weaned pigs. FINDINGS: Weaned pigs were fed diets for 4 weeks containing 57 (low), 164 (intermediate) or 2425 (high) mg kg(-1) analytical grade ZnO. DNA extracts from stomach, mid-jejunum, terminal ileum and colon ascendens were amplified by qPCR assays to quantify copy numbers for the tetracycline (tetA) and sulfonamide (sul1) resistance genes in Gram-negative bacteria. Overall, the combined data (n = 336) showed that copy numbers for tetracycline and sulfonamide resistance genes were significantly increased in the high zinc treatment compared to the low (tetA: p value < 10(-6); sul1: p value = 1 × 10(-5)) or intermediate (tetA: P < 1.6 × 10(-4); sul1: P = 3.2 × 10(-4)) zinc treatment. Regarding the time dependent development, no treatment effects were seen 1 week after weaning, but significant differences between high and low/intermediate zinc treatments evolved 2 weeks after weaning. The increased number of tetA and sul1 copies was not confined to the hind gut, but was already present in stomach contents. CONCLUSIONS: The results of this study suggest that the use of high doses of dietary zinc beyond 2 weeks after weaning should be avoided in pigs due to the possible increase of antibiotic resistance in Gram-negative bacteria.

Palliative radiation therapy in patients with metastasized pancreatic cancer - description of a rare patient group.

BACKGROUND: Pancreatic cancer (PAC) patients experience a high rate of locoregional recurrences and distant metastasis finally leading to their demise even after curatively-intended multidisciplinary treatment approaches including surgery, chemotherapy and radiotherapy. However, clinical reports on bone and brain metastases in PAC patients are extremely rare and thus timing and dose description are not well defined. Our work therefore summarizes a mono-institutional experience on the use of radiotherapy (RT) for PAC patients with metastatic disease with the aim of identifying overall survival and treatment response in this rarely reported patient group. METHOD: Forty-four PAC patients with 66 metastatic lesions were treated with palliative radiotherapy (RT). Thirty-three patients (48 lesions), 7 patients (11 lesions) and 5 patients (7 lesions) with bone, liver and brain metastases analyzed respectively were analyzed; one patient had both bone and cerebral metastases and was treated for the lesions, thus including him in both subgroups. Indications for RT were pain, neurological impairment, risk of pathological fracture or imminent danger for development of any of these conditions in case of tumor progression. Median age was 64 years (range 38 to 78 years) and there were 27 male (61%) and 17 (39%) female patients. Analyses of overall survival (OS) and local control were performed. OS was calculated from the first day of RT. RESULTS: Median overall survival (mOS) of all patients after start of RT was 4.2 months. Survival rates after 1, 3 and 6 months were 79.3%, 55.3% and 30.3% respectively. Patients presenting with bone metastasis had a mOS of 3.1 months and after 1, 3 and 6 months, survival rates were 75.3%, 46.5% and 19.9% respectively. Symptomatic response to therapy was recorded in 85% of all evaluated patients with bone metastasis. Patients undergoing radiosurgery because of liver metastasis were locally controlled in all but one patient after a median follow-up of 8.3 months. CONCLUSION: Overall survival of all patients with metastatic disease was considerably worse. A major goal for the future must be the selection of an appropriate RT treatment in terms of duration and technique for these PAC patients.

The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons.

In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2 × 10(5) sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.

The impact of high dietary zinc oxide on the development of the intestinal microbiota in weaned piglets.

Weaned piglets were fed diets containing 57 (low) or 2425 (high) mg kg(-1) analytical grade ZnO for a period of 5 weeks. Intestinal contents were sampled in weekly intervals and analyzed for bacterial cell numbers and main bacterial metabolites. The most severe effects of high dietary zinc were observed 1 week after weaning in the stomach and small intestine. Pronounced reductions were observed for Enterobacteriaceae and the Escherichia group as well as for Lactobacillus spp. and for three of five studied Lactobacillus species. The impact of high dietary zinc diminished for enterobacteria with increasing age, but was permanent for Lactobacillus species. Bifidobacteria, enterococci, streptococci, Weissella spp. and Leuconostoc spp. as well as the Bacteroides-Prevotella-Porphyromonas group were not influenced by high dietary zinc throughout the trial. High dietary zinc reduced bacterial metabolite concentrations and increased molar acetate ratios at the expense of propionate in the proximal intestine, but differences diminished in older animals. Lower lactate concentrations were observed in the high dietary zinc group throughout the feeding trial. This study has shown that the application of dietary zinc at high concentrations leads to transient and lasting effects during the development of the intestinal microbiota, affecting composition as well as metabolic activity.

Ex vivo-growth response of porcine small intestinal bacterial communities to pharmacological doses of dietary zinc oxide.

Piglets were fed diets containing 57 (low) or 2425 (high) mg zinc from analytical grade zinc oxide (ZnO) ·kg(-1) feed. Digesta samples from the stomach and jejuna of 32, 39, 46 and 53 d old animals (n = 6 per group) were incubated in media containing 80, 40, 20 and 0 µg·mL(-1) soluble zinc from ZnO. Turbidity was recorded for 16 h and growth parameters were calculated. Additionally, DNA extracts of selected samples were analyzed via qPCR for different bacterial groups. Samples from animals fed the low dietary zinc concentration always showed highest rate of growth and lowest lag times in media without added zinc. However, media supplemented with zinc displayed highest growth rates and lowest lag time in the high dietary zinc group. Specific growth rates and lag time showed significant differences on day 32 and 39 of age, but rarely on days 46 and 53 of age. Bacterial growth in digesta samples from the high dietary zinc group was less influenced by zinc and recovered growth more rapidly than in the low dietary zinc group. Specific growth rates and bacterial cell numbers from qPCR results showed that lactobacilli were most susceptible to zinc, while bifidobacteria, enterobacteria and enterococci exhibited increased growth rates in samples of animals from the high dietary zinc treatment. No treatment related differences were observed for clostridial cluster IV and the Bacteroides-Prevotella-Porphyromonas cluster. The diversity of enterobacteria after incubation was always higher in the high dietary zinc treatment or in medium supplemented with 80 µg·mL(-1) soluble ZnO. This study has shown that a pharmacological dosage of ZnO leads to a reduced ex vivo-bacterial growth rate of bacteria from the stomach and jejunum of weaned piglets. In view of the rapid bacterial adaptation to dietary zinc, the administration of ZnO in feeds for weaned piglets might only be beneficial in a short period after weaning.

Chemoradiation in patients with isolated recurrent pancreatic cancer - therapeutical efficacy and probability of re-resection.

BACKGROUND: In the present retrospective analysis we analysed the therapeutic outcome of a set of patients, who were treated with chemoradiation (CRT) for recurrent pancreatic cancer (RPC) in a single institution. PATIENTS AND METHODS: Forty-one patients had a history of primary resection for pancreatic cancer. In case of an unresectable recurrency patients were treated with CRT at our institution between 2002 and 2010 with a median dose of 48.4 Gy (range 39.6-54 Gy). Concurrent chemotherapy regimes included Gemcitabine (GEM) in 37/41 patients (90%) and Fluorouracil (FU) or Capecitabine (CAP) in 4/41 patients (10%). Patients were re-evaluated after CRT with computed tomography and/or explorative laparotomy. During re-resection or laparotomy 15 patients received an additional intraoperative radiotherapy (IORT) with a median dose of 15 Gy (range 12-15 Gy). Median age was 65 years (range 39-76 years) and there were 26 male and 15 female patients. RESULTS: The median overall survival (mOS), local control (LC) and progression-free survival (PFS) were 16.1, 13.8 and 6.9 months respectively for all patients after the first day of CRT. Re-resection was possible in five patients (12%) and a complete remission (CR) as defined by tumor-free biopsy was seen in 6 patients (15%). When re-resection could be achieved after CRT mOS was improved to 28.3 months (n = 5 patients, 95%-CI 10.2 - 46.3 months). Patients receiving IORT had a significantly improved mOS compared to no IORT (p = 0.034). Fifteen patients (37%) experienced a local tumour progression and main site of distant metastasis was the liver (11 patients, 27%).Overall treatment-related toxicity was mild, grade III hematologic toxicity was observed in 11 patients (27%). CONCLUSION: In summary we observed a good therapeutic response with mild to moderate toxicity levels for CRT in RPC. Overall survival and PFS were clearly improved in case of induction of a complete remission (tumor-free biopsies) or after achieving a re-resection, thus providing a curative intended therapy even in case of disease recurrence.

Hypofractionated carbon ion therapy delivered with scanned ion beams for patients with hepatocellular carcinoma - feasibility and clinical response.

PURPOSE: Photon-based radiation therapy does currently not play a major role as local ablative treatment for hepatocellular carcinoma (HCC). Carbon ions offer distinct physical and biological advantages. Due to their inverted dose profile and the high local dose deposition within the Bragg peak, precise dose application and sparing of normal tissue is possible. Furthermore, carbon ions have an increased relative biological effectiveness (RBE) compared to photons. METHODS AND MATERIALS: A total of six patients with one or more HCC-lesions were treated with carbon ions delivered by the raster-scanning technique according to our clinical trial protocol. Diagnosis of HCC was confirmed by histology or two different imaging modalities (CT and MRI) according to the AASLD-guidelines. Applied fractionation scheme was 4 × 10 Gy(RBE). Correct dose application was controlled by in-vivo PET measurement of ß + -activity in the irradiated tissue shortly after treatment. RESULTS: Patients were observed for a median time period of 11.0 months (range, 3.4 - 12.7 months). Imaging studies showed a partial response in 4/7 lesions and a stable disease in 3/7 lesions in follow-up CT- and MRI scans. Local control was 100%. One patient with multifocal intrahepatic disease underwent liver transplantation 3 months after carbon ion therapy. During radiotherapy and the follow-up period no severe adverse events have occurred. CONCLUSIONS: We report the first clinical results of patients with HCC undergoing carbon ion therapy using the rasterscanning technique at our institution. All patients are locally controlled and experienced no higher toxicities in a short follow-up period. Further patients will be included in our prospective Phase-I clinical trial PROMETHEUS-01 (NCT01167374).

G protein activation by uncaging of GTP-gamma-S in the leech giant glial cell.

Glial cells can be activated by neurotransmitters via metabotropic, G protein-coupled receptors. We have studied the effects of 'global' G protein activation by GTP-gamma-S on the membrane potential, membrane conductance, intracellular Ca(2+) and Na(+) of the giant glial cell in isolated ganglia of the leech Hirudo medicinalis. Uncaging GTP-gamma-S (injected into a giant glial cell as caged compound) by moderate UV illumination hyperpolarized the membrane due to an increase in K+ conductance. Uncaging GTP-gamma-S also evoked rises in cytosolic Ca(2+) and Na+, both of which were suppressed after depleting the intracellular Ca(2+) stores with cyclopiazonic acid (20 micromol l(-1)). Uncaging inositol-trisphosphate evoked a transient rise in cytosolic Ca(2+) and Na+ but no change in membrane potential. Injection of the fast Ca(2+) chelator BAPTA or depletion of intracellular Ca(2+) stores did not suppress the membrane hyperpolarization induced by uncaging GTP-gamma-S. Our results suggest that global activation of G proteins in the leech giant glial cell results in a rise of Ca(2+)-independent membrane K+ conductance, a rise of cytosolic Ca(2+), due to release from intracellular stores, and a rise of cytosolic Na+, presumably due to increased Na+/Ca(2+) exchange.

Recapture after exocytosis causes differential retention of protein in granules of bovine chromaffin cells.

After exocytosis, chromaffin granules release essentially all their catecholamines in small fractions of a second, but it is unknown how fast they release stored peptides and proteins. Here we compare the exocytic release of fluorescently labelled neuropeptide Y (NPY) and tissue plasminogen activator from single granules. Exocytosis was tracked by measuring the membrane capacitance, and single granules in live cells were imaged by evanescent field microscopy. Neuropeptide Y left most granules in small fractions of a second, while tissue plasminogen activator remained in open granules for minutes. Taking advantage of the dependence on pH of the fluorescence of green fluorescent protein, we used rhythmic external acidification to determine whether and when granules re-sealed. One-third of them re-sealed within 100 s and retained significant levels of tissue plasminogen activator. Re-sealing accounts for only a fraction of the endocytosis monitored in capacitance measurements. When external [Ca2+] was raised, even neuropeptide Y remained in open granules until they re-sealed. It is concluded that a significant fraction of chromaffin granules re-seal after exocytosis, and retain those proteins that leave granules slowly. We suggest that granules vary the stoichiometry of release by varying both granule re-sealing and the association of proteins with the granule matrix.