Experimental Determination of α Widths of

The efficiency of the weak s process in low-metallicity rotating massive stars depends strongly on the rates of the competing ^{17}O(α,n)^{20}Ne and ^{17}O(α,γ)^{21}Ne reactions that determine the potency of the ^{16}O neutron poison. Their reaction rates are poorly known in the astrophysical energy range of interest for core helium burning in massive stars because of the lack of spectroscopic information (partial widths, spin parities) for the relevant states in the compound nucleus ^{21}Ne. In this Letter, we report on the first experimental determination of the α-particle spectroscopic factors and partial widths of these states using the ^{17}O(^{7}Li,t)^{21}Ne α-transfer reaction. With these the ^{17}O(α,n)^{20}Ne and ^{17}O(α,γ)^{21}Ne reaction rates were evaluated with uncertainties reduced by a factor more than 3 with respect to previous evaluations and the present ^{17}O(α,n)^{20}Ne reaction rate is more than 20 times larger. The present (α,n)/(α,γ) rate ratio favors neutron recycling and suggests an enhancement of the weak s process in the Zr-Nd region by more than 1.5 dex in metal-poor rotating massive stars.

Accessing the Single-Particle Structure of the Pygmy Dipole Resonance in

New experimental data on the neutron single-particle character of the Pygmy Dipole Resonance (PDR) in ^{208}Pb are presented. They were obtained from (d,p) and resonant proton scattering experiments performed at the Q3D spectrograph of the Maier-Leibnitz Laboratory in Garching, Germany. The new data are compared to the large suite of complementary, experimental data available for ^{208}Pb and establish (d,p) as an additional, valuable, experimental probe to study the PDR and its collectivity. Besides the single-particle character of the states, different features of the strength distributions are discussed and compared to large-scale shell model (LSSM) and energy-density functional plus quasiparticle-phonon model theoretical approaches to elucidate the microscopic structure of the PDR in ^{208}Pb.

Is γ-ray emission from novae affected by interference effects in the 18F(p,α)15O reaction?

The (18)F(p,α)(15)O reaction rate is crucial for constraining model predictions of the γ-ray observable radioisotope (18)F produced in novae. The determination of this rate is challenging due to particular features of the level scheme of the compound nucleus, (19)Ne, which result in interference effects potentially playing a significant role. The dominant uncertainty in this rate arises from interference between J(π)=3/2(+) states near the proton threshold (S(p)=6.411 MeV) and a broad J(π)=3/2(+) state at 665 keV above threshold. This unknown interference term results in up to a factor of 40 uncertainty in the astrophysical S-factor at nova temperatures. Here we report a new measurement of states in this energy region using the (19)F((3)He,t)(19)Ne reaction. In stark contrast to previous assumptions we find at least 3 resonances between the proton threshold and E(cm)=50 keV, all with different angular distributions. None of these are consistent with J(π)=3/2(+) angular distributions. We find that the main uncertainty now arises from the unknown proton width of the 48 keV resonance, not from possible interference effects. Hydrodynamic nova model calculations performed indicate that this unknown width affects (18)F production by at least a factor of two in the model considered.

DNA methylation, transcriptome and genetic copy number signatures of diffuse cerebral WHO grade II/III gliomas resolve cancer heterogeneity and development.

BACKGROUND: Diffuse lower WHO grade II and III gliomas (LGG) are slowly progressing brain tumors, many of which eventually transform into a more aggressive type. LGG is characterized by widespread genetic and transcriptional heterogeneity, yet little is known about the heterogeneity of the DNA methylome, its function in tumor biology, coupling with the transcriptome and tumor microenvironment and its possible impact for tumor development. METHODS: We here present novel DNA methylation data of an LGG-cohort collected in the German Glioma Network containing about 85% isocitrate dehydrogenase (IDH) mutated tumors and performed a combined bioinformatics analysis using patient-matched genome and transcriptome data. RESULTS: Stratification of LGG based on gene expression and DNA-methylation provided four consensus subtypes. We characterized them in terms of genetic alterations, functional context, cellular composition, tumor microenvironment and their possible impact for treatment resistance and prognosis. Glioma with astrocytoma-resembling phenotypes constitute the largest fraction of nearly 60%. They revealed largest diversity and were divided into four expression and three methylation groups which only partly match each other thus reflecting largely decoupled expression and methylation patterns. We identified a novel G-protein coupled receptor and a cancer-related 'keratinization' methylation signature in in addition to the glioma-CpG island methylator phenotype (G-CIMP) signature. These different signatures overlap and combine in various ways giving rise to diverse methylation and expression patterns that shape the glioma phenotypes. The decrease of global methylation in astrocytoma-like LGG associates with higher WHO grade, age at diagnosis and inferior prognosis. We found analogies between astrocytoma-like LGG with grade IV IDH-wild type tumors regarding possible worsening of treatment resistance along a proneural-to-mesenchymal axis. Using gene signature-based inference we elucidated the impact of cellular composition of the tumors including immune cell bystanders such as macrophages. CONCLUSIONS: Genomic, epigenomic and transcriptomic factors act in concert but partly also in a decoupled fashion what underpins the need for integrative, multidimensional stratification of LGG by combining these data on gene and cellular levels to delineate mechanisms of gene (de-)regulation and to enable better patient stratification and individualization of treatment.

Long-term outcome in patients with short bowel syndrome after longitudinal intestinal lengthening and tailoring.

OBJECTIVES: Longitudinal intestinal lengthening and tailoring (LILT) is a well-established surgical treatment for short bowel syndrome. It has been shown to enhance peristalsis, decrease bacterial overgrowth, and extend mucosal contact time for nutrients. We present the results of a long-term follow-up of patients who underwent LILT and define prognostic parameters for the survival of these patients. PATIENTS AND METHODS: Between 1987 and 2006, 53 patients underwent LILT in our institution. The main diagnoses were gastroschisis, intestinal volvulus, intestinal atresias, and necrotizing enterocolitis. LILT was performed at a mean age of 24 months (range 4144 months). The follow-up time was 79.76 months (range 6234 months). RESULTS: After LILT, 41 of 53 patients survived, and 36 of 41 surviving patients were successfully weaned from parenteral nutrition (PN). In long-term follow-up 79% stayed free of PN. The overall survival rate was 77.36%. Weight gain occurred in 58% of the patients after LILT. The quality of life after LILT is on a high level, with most patients having normal physical strength and participating in normal social life and education. Prognostic factors for survival after LILT in short bowel syndrome are length of small intestine (0.06582 + 0.0131 x bowel cm), length of large bowel (P = 0.039), preoperative liver function, and successful weaning from PN within 18 months postoperatively (P = 0.0032). CONCLUSIONS: Patients undergoing LILT in short bowel syndrome have a high survival rate, weight gain, and a high quality of life. Autologous gastrointestinal reconstruction remains therefore the first choice in the treatment of patients with short bowel syndrome.

Helicobacter pylori strain-specific differences in genetic content, identified by microarray, influence host inflammatory responses.

Helicobacter pylori enhances the risk for ulcer disease and gastric cancer, yet only a minority of H. pylori-colonized individuals develop disease. We examined the ability of two H. pylori isolates to induce differential host responses in vivo or in vitro, and then used an H. pylori whole genome microarray to identify bacterial determinants related to pathogenesis. Gastric ulcer strain B128 induced more severe gastritis, proliferation, and apoptosis in gerbil mucosa than did duodenal ulcer strain G1.1, and gastric ulceration and atrophy occurred only in B128+ gerbils. In vitro, gerbil-passaged B128 derivatives significantly increased IL-8 secretion and apoptosis compared with G1.1 strains. DNA hybridization to the microarray identified several strain-specific differences in gene composition including a large deletion of the cag pathogenicity island in strain G1.1. Partial and complete disruption of the cag island in strain B128 attenuated induction of IL-8 in vitro and significantly decreased gastric inflammation in vivo. These results indicate that the ability of H. pylori to regulate epithelial cell responses related to inflammation depends on the presence of an intact cag pathogenicity island. Use of an H pylori whole genome microarray is an effective method to identify differences in gene content between H. pylori strains that induce distinct pathological outcomes in a rodent model of H. pylori infection.

Different in vivo and in vitro transformation of intestinal stem cells in mismatch repair deficiency.

Mutations in mismatch repair (MMR) genes result in microsatellite instability (MSI) and early onset of colorectal cancer. To get mechanistic insights into the time scale, sequence and frequency of intestinal stem cell (ISC) transformation, we quantified MSI and growth characteristics of organoids of Msh2-deficient and control mice from birth until tumor formation and related them to tissue gene expression. Although in Msh2-deficient organoids MSI continuously increased from birth, growth characteristics remained stable at first. Months before tumor onset, normal Msh2-deficient tissue contained tumor precursor cells forming organoids with higher MSI, cystic growth and growth rates resembling temporarily those of tumor organoids. Consistently, Msh2-deficient tissue exhibited a tumor-like gene signature. Normal Msh2-deficient organoids showed increased inheritable transient cyst-like growth, which became independent of R-spondin. ISC transformation proceeded faster in vitro than in vivo independent of the underlying genotype but more under MMR deficiency. Transient cyst-like growth but not MSI was suppressed by aspirin. In summary, as highlighted by organoids, molecular alterations continuously proceeded long before tumor onset in MMR-deficient intestine, thus increasing its susceptibility for ISC transformation.

Dual function of membrane-bound heat shock protein 70 (Hsp70), Bag-4, and Hsp40: protection against radiation-induced effects and target structure for natural killer cells.

CX+/CX- and Colo+/Colo- tumor sublines with stable heat shock protein 70 (Hsp70) high and low membrane expression were generated by fluorescence activated cell sorting of the parental human colon (CX2) and pancreas (Colo357) carcinoma cell lines, using an Hsp70-specific antibody. Two-parameter flow cytometry revealed that Hsp70 colocalizes with Bag-4, also termed silencer of death domain, not only in the cytosol but also on the plasma membrane. After nonlethal gamma-irradiation, the percentage of membrane-positive cells and the protein density of Hsp70 and Bag-4 were found to be strongly upregulated in carcinoma sublines with initially low expression levels (CX-, Colo-). Membrane expression of Hsp70 was also elevated in Bag-4 overexpressing HeLa cervix carcinoma cells when compared to neo-transfected cells. In response to gamma-irradiation, neo-transfected HeLa cells behaved like Hsp70/Bag-4 low-expressing CX- and Colo-, and Bag-4-transfected HeLa cells like Hsp70/Bag-4 high-expressing carcinoma sublines CX+ and Colo+. Immunoprecipitation studies further confirmed colocalization of Hsp70 and Bag-4 but also point to an association of Hsp70 and Hsp40 on the plasma membrane of CX+ and Colo+ cells; on CX- and Colo- tumor sublines, Hsp40 was detectable in the absence of Hsp70 and Bag-4. Other co-chaperones including Hsp60 and Hsp90 were neither found on the cell surface of CX+/CX-, Colo+/Colo- nor on HeLa neo-/HeLa Bag-4-transfected tumor cells. Functionally, Hsp70/Bag-4 and Hsp70/Hsp40 membrane-positive tumor cells appeared to be better protected against radiation-induced effects, including G2/M arrest and growth inhibition, on the one hand. On the other hand, membrane-bound Hsp70, but neither Bag-4 nor Hsp40, served as a recognition site for the cytolytic attack mediated by natural killer cells.

Experience of 49 longitudinal intestinal lengthening procedures for short bowel syndrome.

PATIENTS, METHODS AND RESULTS: Forty-nine patients with a mean age of 25 months underwent a longitudinal intestinal lengthening procedure for short bowel syndrome (SBS) in our institution. Indications for the operation were dependence on parenteral nutrition in spite of adequate conservative management. The small bowel was lengthened from a mean of 27 cm to a mean of 51 cm. There was no intraoperative mortality. The following early complications occurred in our early series: ischemia of a short bowel segment of 2 cm, requiring resection in two patients, insufficiency of the longitudinal anastomosis in two patients and an intra-abdominal abscess in one. Four of 9 non-survivors died of liver failure and 3 of sepsis. Follow-up showed that 19 patients were weaned from parenteral nutrition after a mean of 9.1 months. Long-term complications encountered were dismotility with malabsorption due to bacterial overgrowth caused by progressive dilatation of the bowel, d-lactic acidosis, cholelithiasis and urolithiasis. CONCLUSIONS: A longitudinal intestinal lengthening procedure is an effective and safe surgical approach for SBS, provided it is performed in time, the patient's preoperative condition is optimized and technical surgical details are taken into account.

Generation of human induced pluripotent stem cells using non-synthetic mRNA.

Here we describe some of the crucial steps to generate induced pluripotent stem cells (iPSCs) using mRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribed mRNA. V. virus' 2'-O-Methyltransferase enzyme creates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach.

Carbonyl reductase from human testis: purification and comparison with carbonyl reductase from human brain and rat testis.

Carbonyl reductase (EC 1.1.1.184) is a cytosolic, monomeric, NADPH-dependent oxidoreductase with broad specificity for carbonyl compounds and a general distribution in human tissues. A carbonyl reductase closely resembling the human enzyme is exclusively expressed in rat reproductive tissues and adrenals (Iwata, N., Inazu, N. and Satoh, T. (1989) J. Biochem. 105, 556-564). In order to investigate the relationship between the human and rat enzyme, carbonyl reductase from human testis was purified to homogeneity. The enzyme was indistinguishable from carbonyl reductase from other human tissues on the basis of physicochemical properties, substrate specificity, inhibitor sensitivity and immunological reactivity. Likewise, the human and rat testis enzymes exhibited greatly overlapping substrate specificities for prostaglandins, steroids as well as many xenobiotic carbonyl compounds, and showed the same susceptibility to inhibition by flavonoids and sulfhydryl-blocking agents. Structural homology between the two enzymes was indicated by the mutual cross-reactivity of antibodies against carbonyl reductase from one species and the enzyme protein from the other species. Unlike the rat enzyme, which is confined to Leydig cells, the human enzyme was detectable in Leydig cells as well as Sertoli and spermatogenic cells.

Analysis of antimicrobial peptide interactions with hybrid bilayer membrane systems using surface plasmon resonance.

The lipid binding behaviour of the antimicrobial peptides magainin 1, melittin and the C-terminally truncated analogue of melittin (21Q) was studied with a hybrid bilayer membrane system using surface plasmon resonance. In particular, the hydrophobic association chip was used which is composed of long chain alkanethiol molecules upon which liposomes adsorb spontaneously to create a hybrid bilayer membrane surface. Multiple sets of sensorgrams with different peptide concentrations were generated. Linearisation analysis and curve fitting using numerical integration analysis were performed to derive estimates for the association (k(a)) and dissociation (k(d)) rate constants. The results demonstrated that magainin 1 preferentially interacted with negatively charged dimyristoyl-L-alpha-phosphatidyl-DL-glycerol (DMPG), while melittin interacted with both zwitterionic dimyristoyl-L-alpha-phosphatidylcholine and anionic DMPG. In contrast, the C-terminally truncated melittin analogue, 21Q, exhibited lower binding affinity for both lipids, showing that the positively charged C-terminus of melittin greatly influences its membrane binding properties. Furthermore the results also demonstrated that these antimicrobial peptides bind to the lipids initially via electrostatic interactions which then enhances the subsequent hydrophobic binding. The biosensor results were correlated with the conformation of the peptides determined by circular dichroism analysis, which indicated that high alpha-helicity was associated with high binding affinity. Overall, the results demonstrated that biosensor technology provides a new experimental approach to the study of peptide-membrane interactions through the rapid determination of the binding affinity of bioactive peptides for phospholipids.

Immunochemical characterization of aldo-keto reductases from human tissues.

Aldose reductase, aldehyde reductase and carbonyl reductase constitute a family of monomeric NADPH-dependent oxidoreductases with similar physical and chemical properties. Characterization of the enzymes from human tissues by immunotitration and an enzyme immunoassay indicated that, despite their apparent likeness, the three reductases do not cross-react immunochemically.

Immunohistochemical localization of carbonyl reductase in human tissues.

Carbonyl reductase, an NADPH-dependent oxidoreductase of broad specificity, is present in many human tissues. Its precise localization, however, has remained unclear, as well as its physiological and possible pathophysiological significance. The present study reports the immunohistochemical localization of the enzyme in normal human tissues. Immunostaining was detectable in all organs investigated. The highest concentrations were found in the parenchymal cells of the liver, the epithelial cells of the stomach and small intestine, the epidermis, the proximal tubules of the kidney, neuronal and glial cells of the central nervous system, and certain cells of the anterior lobe of the pituitary gland. Consistently pronounced staining was also observed in smooth muscle fibers and the endothelium of blood vessels. The results are in agreement with a housekeeping function of carbonyl reductase in the elimination of reactive carbonyl compounds.

Transient cisplatin-resistant murine fibrosarcoma cell characterized by increased metallothionein content.

Cisplatin-resistant mouse fibrosarcoma cells, SSK-R, were isolated after short and low-dose drug treatment of the sensitive SSK cells in vitro. These SSK-R sublines exhibit up to sevenfold cisplatin resistance and are characterized mainly by an increased metallothionein content. Loss of drug resistance after about 140-180 cell divisions in drug-free medium coincides with loss of metallothionein content. The glutathione level is the same in the sensitive and resistant sublines; inhibition of glutathione synthesis by buthionine sulphoximine enhances the sensitivity in both cells lines by a factor of 2.7. The resistant sublines are not cross-resistant to radiation; a radiation exposure followed immediately by cisplatin treatment results in an additive effect. The cellular cisplatin content is slightly reduced in SSK-R2 cells and it remains at this level also upon loss of drug sensitivity.

Trypsinogen gene mutations in patients with chronic or recurrent acute pancreatitis.

Three-point mutations (R117H, N211, A16V) within the cationic trypsinogen gene have been identified in patients with hereditary pancreatitis (HP). A genetic background has also been discussed for idiopathic juvenile chronic pancreatitis (IJCP), which closely mimicks the clinical pattern of HP, and alcoholic chronic pancreatitis because only a small number of heavy drinkers develop pancreatitis. This prompted us to screen 104 patients in our well-defined pancreatitis cohort for the currently known cationic trypsinogen gene mutations. The R117H mutation was detected in seven patients (six patients of two clinically classified HP families, one patient with clinically classified IJCP) and the A16V mutation in one IJCP patient. No cationic trypsinogen gene mutations were found in the remaining 96 patients with chronic and recurrent acute pancreatitis of various etiologies. Our results demonstrate the need for genetic testing to exclude HP, particularly in the presence of an atypical or unknown family history. In addition, cationic trypsinogen gene mutations are no predisposing factor in patients with chronic and recurrent acute pancreatitis of different etiologies.

Characterisation by immobilised metal ion affinity chromatographic procedures of the binding behaviour of several synthetic peptides designed to have high affinity for Cu(II) ions.

In this investigation, several peptides containing an increasing number of histidine residues have been designed and synthesised. The peptides involved repeat units of either the pentameric EAEHA or the tetrameric HLLH sequence motifs. Adsorption isotherms for these synthetic peptides and hexahistidine (hexa-His) as a control substance were measured under batch equilibrium binding conditions with an immobilised Cu(II)-iminodiacetic acid (IDA) sorbent. The experimental data were analysed in terms of Langmuirean binding behaviour. In common with previous studies with synthetic peptides, these investigations have demonstrate that the sequential organisation of the histidine side chains in these peptides can affect the selectivity of the coordination interactions with borderline metal ions in immobilised metal ion affinity chromatographic systems. The results also confirm that peptides selected on the basis of their potential to form amphipathic secondary structures with their histidine residues presented on one face of the molecule can exhibit equivalent or higher affinity constants towards copper ions than hexa-His, although they contain fewer histidine residues. These findings are thus relevant to the selection of peptides produced inter alia by combinatorial synthetic procedures to have enhanced binding properties for Cu(II) or Ni(II) ions, or intended for use as peptide tags in the fusion handle approach for the affinity chromatographic purification of recombinant proteins.

Altered signaling of TNFalpha-TNFR1 and SODD/BAG4 is responsible for radioresistance in human HT-R15 cells.

Radioresistance in pre-irradiated human HT-R15 cells is associated with changes of the TNFR1-dependent death pathway. HT-R15 cells are characterized by elevated protein levels of TNFR1 and TNF and by increased sensitivity to exogenous TNFalpha compared to their parental HT-29 cells. Alterations are also observed downstream in the signaling cascade, such as the activation of NF-kappaB or the overexpression of the death domain kinase RIP and reduced caspase 8 activity. However, these changes seem to be a consequence of defective upstream TNFalpha signaling rather than the primary cause of cellular resistance in HT-R15 cells. Of major importance for resistance in HT-R15 cells is the silencer of death domain, SODD/BAG4. Following gamma-irradiation, the membrane-associated 49 kDa SODD decreases in the parental but not in the resistant cells, whereas after TNFalpha treatment, SODD expression declines only in the resistant cells. A 42 kDa cytoplasmic SODD protein is detected, which is elevated only in the resistant cells. This SODD protein is not involved in the regulation of cell survival after radiation or TNFalpha treatment but rather in altered TNFR1 shedding. Inhibition of TNFR1 release by the metalloprotease inhibitor BB-2516 results in a significant increase of the 42 kDa SODD protein without affecting cell survival in sensitive or resistant HT cells. Moreover, TNFR1 release into the culture medium is augmented in the resistant cells. These results suggest that defective TNFalpha signaling and/or altered silencing by SODD/BAG4 in HT-R15 cells are involved in the radiation resistance of HT-R15 cells and also affect the paracrine functions of TNFR1. Resistance is circumvented by TNFalpha treatment, independent of cytoplasmic TNFalpha/TNFR1 functions.

Clonal variability of radiation-induced cisplatin resistant HeLa cells.

Low-dose fractionated gamma-irradiation (3 cycles of 5x2 Gy) induces moderate cisplatin resistance in HeLa cells which is associated with alterations of the caspase-dependent apoptotic pathway (2). There is a considerable heterogeneity in cell survival among isolated resistant clones (Rf values between 1.4 to 2.4) and in the propensity of the cells to enter apoptosis. These variations are associated with altered activation of the apoptotic pathway by members of the TNF family. The membrane receptor CD95 (Apo-1/Fas), which is upregulated immediately after cisplatin exposure in parental HeLa cells, is expressed at various levels in the resistant clones. There are also changes in the formation of the inhibitor protein I kappa B, which regulates the antiapoptotic transcription factor NF kappa B. Since the response to radiation is unchanged, the results collectively suggest that changes in the activation of the caspase-dependent signalling cascade are involved in the death pathway initiated by cisplatin but not by radiation damage.