Final Results of the OPERA Experiment on ν_{τ} Appearance in the CNGS Neutrino Beam.

The OPERA experiment was designed to study ν_{µ}→ν_{τ} oscillations in the appearance mode in the CERN to Gran Sasso Neutrino beam (CNGS). In this Letter, we report the final analysis of the full data sample collected between 2008 and 2012, corresponding to 17.97×10^{19} protons on target. Selection criteria looser than in previous analyses have produced ten ν_{τ} candidate events, thus reducing the statistical uncertainty in the measurement of the oscillation parameters and of ν_{τ} properties. A multivariate approach for event identification has been applied to the candidate events and the discovery of ν_{τ} appearance is confirmed with an improved significance level of 6.1σ. |Δm_{32}^{2}| has been measured, in appearance mode, with an accuracy of 20%. The measurement of the ν_{τ} charged-current cross section, for the first time with a negligible contamination from ν[over ¯]_{τ}, and the first direct evidence for the ν_{τ} lepton number are also reported.

Discovery of τ Neutrino Appearance in the CNGS Neutrino Beam with the OPERA Experiment.

The OPERA experiment was designed to search for ν_{µ}→ν_{τ} oscillations in appearance mode, i.e., by detecting the τ leptons produced in charged current ν_{τ} interactions. The experiment took data from 2008 to 2012 in the CERN Neutrinos to Gran Sasso beam. The observation of the ν_{µ}→ν_{τ} appearance, achieved with four candidate events in a subsample of the data, was previously reported. In this Letter, a fifth ν_{τ} candidate event, found in an enlarged data sample, is described. Together with a further reduction of the expected background, the candidate events detected so far allow us to assess the discovery of ν_{µ}→ν_{τ} oscillations in appearance mode with a significance larger than 5σ.

Development of insulin allergy after bone marrow transplantation.

BACKGROUND: Insulin allergy is a not uncommon condition even though human insulin and insulin analogues are widely used. However, the development of insulin allergy after bone marrow transplantation has not been reported. CASE REPORT: A 44-year-old Japanese woman had aplastic anaemia and secondary haemochromatosis. She was diagnosed with having diabetes at age 32 years and had been treated with human insulin. At age 34 years, bone marrow transplantation was performed. One year later, a rash and urticaria appeared immediately after insulin injections. Intracutaneous tests were positive for both human insulins and analogues, whereas the test for protamine was negative. Furthermore, an IgE-radioallergosorbent test against insulin was positive. Thus, we diagnosed the patient with having an IgE-mediated type I allergy against insulin. Insulin therapy with insulin aspart, which showed the least skin reaction, was continued and the insulin allergy disappeared in 7 years. CONCLUSIONS: This is the first description of insulin allergy after bone marrow transplantation. Our case underscores the effects of bone marrow cells on IgE-mediated type I allergy for insulin.

Does the phenotypic morphology of the human brachial plexus reflect the theoretical development of concomitant regulation in thoracolumbar spines and nerves?

BACKGROUND: Experimental evidence identified that thoracolumbar mutants caused by Hox genes 7-10 mutants also involve a craniocaudal shift and/or the addition or reduction of segments of the limb plexus roots. This study investigated whether the theoretical concomitant shift of the brachial plexus roots in human different thoracolumbar counts is shared as confirmed in those of the human lumbosacral plexus. MATERIALS AND METHODS: The phenotypic morphology of the brachial plexus and its arterial interaction on 20 sides of 10 atypical human thoracolumbar counts out of the 354 sides of the 177 cadavers, were compared with those of 52 sides of 26 cases in a typical human vertebral formula (7C_12T_5L_5S). RESULTS: Regardless of the course and branching patterns of the axillary artery, our results showed that the main brachial plexus roots were composed of only five segments of the 5th-9th spinal nerves, with small contributions from the 4th and/or 10th nerves. This root composition is identical to a typical human thoracolumbar formula, and therefore, neither a craniocaudal shift nor additional/reduced main roots occurred in our thoracolumbar variants. CONCLUSIONS: Unlike the concomitant shift of the lumbosacral plexus roots, our present cases suggest that the phenotypic morphology of the human brachial plexus may be less likely to show theoretical craniocaudal shifts, further data on the root changes in different vertebral formulae are needed for its accurate validation.

Dynamic changes in RAS gene status in circulating tumour DNA: a phase II trial of first-line FOLFOXIRI plus bevacizumab for RAS-mutant metastatic colorectal cancer (JACCRO CC-11).

BACKGROUND: Few prospective studies have used liquid biopsy testing in RAS-mutant metastatic colorectal cancer (mCRC), and its clinical significance remains unknown. Therefore, this study aimed to carry out a biomarker analysis by liquid biopsy using updated data of the phase II trial of FOLFOXIRI plus bevacizumab as first-line chemotherapy for RAS-mutant mCRC. MATERIALS AND METHODS: A total of 64 patients who received modified FOLFOXIRI regimen (irinotecan 150 mg/m2, oxaliplatin 85 mg/m2, levofolinate 200 mg/m2, and fluorouracil 2400 mg/m2) plus bevacizumab biweekly were enrolled. The primary endpoint was the objective response rate (ORR). Plasma samples were collected at pre-treatment, 8 weeks after treatment, and progression in participants included in the biomarker study. The levels of circulating tumour DNA (ctDNA) and specific KRAS and NRAS variants were evaluated using real-time PCR assays. RESULTS: There were 62 patients (median age: 62.5 years, 92% performance status 0, 27% right side) who were assessable for efficacy and 51 for biomarker analysis. ORR was 75.8% (95% confidence interval 65.1% to 86.5%). The median progression-free survival was 12.1 months, and the median overall survival (OS) was 30.2 months. In 78% of patients, RAS mutations disappeared in the ctDNA at 8 weeks after treatment; these patients tended to have better outcomes than those with RAS mutations. Interestingly, RAS mutations remained undetectable during progression in 62% of patients. Survival analysis indicated that the median OS from progression was significantly longer in patients with RAS mutation clearance than in those with RAS mutation in the ctDNA at disease progression (15.1 versus 7.3 months, hazard ratio: 0.21, P = 0.0046). CONCLUSIONS: Our biomarker study demonstrated no RAS mutations in ctDNA at disease progression in 62% of patients with RAS-mutant mCRC. Both OS and post-progression survival were better in patients with clearance of RAS mutations in ctDNA after triplet-based chemotherapy.

OPERA tau neutrino charged current interactions.

The OPERA experiment was designed to discover the vτ appearance in a vµ beam, due to neutrino oscillations. The detector, located in the underground Gran Sasso Laboratory, consisted of a nuclear photographic emulsion/lead target with a mass of about 1.25 kt, complemented by electronic detectors. It was exposed from 2008 to 2012 to the CNGS beam: an almost pure vµ beam with a baseline of 730 km, collecting a total of 1.8·1020 protons on target. The OPERA Collaboration eventually assessed the discovery of vµâ†’vτ oscillations with a statistical significance of 6.1 σ by observing ten vτ CC interaction candidates. These events have been published on the Open Data Portal at CERN. This paper provides a detailed description of the vτ data sample to make it usable by the whole community.

Evidence that cAMP-dependent protein kinase and a protein factor are involved in reactivation of triton X-100 models of sea urchin and starfish spermatozoa.

A fraction obtained from detergent-extract of sea urchin or starfish spermatozoa using DEAE-cellulose chromatography reactivated Triton X-100 models of the spermatozoa in a cAMP-dependent manner. The DEAE fraction contained cAMP-dependent protein kinase with a high level of specific activity. Rabbit muscle inhibitor protein highly specific for cAMP-dependent protein kinases inhibited the ability of the deae fraction to induce reactivation of Triton X-100 models.l This inhibition paralleled inhibition of cAMP-dependent protein kinase activity of the DEAE fraction, suggesting participation of the enzyme in the cAMP-dependent reactivation of Triton X-100 models. However, cAMP-dependent protein kinase further purified from the DEAE fraction was incapable of reactivating these models by itself. A protein factor which was separated from the protein kinase in the course of purification of the enzyme was found to also be necessary for the reactivation. When cAMP-dependent protein kinase was pretreated with protein kinase inhibitor before addition of the protein factor, the reactivation of Triton X-100 models was no longer detected. However, after the protein factor had been incubated with cAMP and cAMP-dependent protein kinase, protein kinase inhibitor did not repress reactivation of Triton X-100 models. We propose that the reactivation needs phosphorylation of the protein factor by cAMP-dependent protein kinase.

Caveolin-2 is targeted to lipid droplets, a new "membrane domain" in the cell.

Caveolin-1 and -2 constitute a framework of caveolae in nonmuscle cells. In the present study, we showed that caveolin-2, especially its beta isoform, is targeted to the surface of lipid droplets (LD) by immunofluorescence and immunoelectron microscopy, and by subcellular fractionation. Brefeldin A treatment induced further accumulation of caveolin-2 along with caveolin-1 in LD. Analysis of mouse caveolin-2 deletion mutants revealed that the central hydrophobic domain (residues 87-119) and the NH(2)-terminal (residues 70-86) and COOH-terminal (residues 120-150) hydrophilic domains are all necessary for the localization in LD. The NH(2)- and COOH-terminal domains appeared to be related to membrane binding and exit from ER, respectively, implying that caveolin-2 is synthesized and transported to LD as a membrane protein. In conjunction with recent findings that LD contain unesterified cholesterol and raft proteins, the result implies that the LD surface may function as a membrane domain. It also suggests that LD is related to trafficking of lipid molecules mediated by caveolins.

The cysteine-rich domain of human ADAM 12 supports cell adhesion through syndecans and triggers signaling events that lead to beta1 integrin-dependent cell spreading.

The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments: the cys-teine-rich domain made in Escherichia coli (rADAM 12-cys), the disintegrin-like and cysteine-rich domain made in insect cells (rADAM 12-DC), and full-length human ADAM 12-S tagged with green fluorescent protein made in mammalian cells (rADAM 12-GFP). Mesenchymal cells specifically and in a dose-dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin beta1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading, and chondroblasts lacking beta1 integrin attached but did not spread. These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain-mediated cell adhesion, and then the beta1 integrin to induce cell spreading. Interestingly, carcinoma cells attached but did not spread on ADAM 12. However, spreading could be efficiently induced by the addition of either 1 mM Mn(2+) or the beta1 integrin-activating monoclonal antibody 12G10, suggesting that in these carcinoma cells, the ADAM 12-syndecan complex fails to modulate the function of beta1 integrin.

The role of silicon in preventing appressorial penetration by the rice blast fungus.

To test the hypothesis that silicon (Si) confers resistance against appressorial penetration of the rice blast fungus, the proportion of appressorial penetration into the leaf epidermis to total appressoria formed was compared among rice plants amended with various rates of silica gel to those plants nonamended. The amounts of Si in the youngest leaves were consistent with the amounts of silica gel applied to the rice plants. Relative Si levels on the adaxial surface of leaves as detected by energy dispersive X-ray analysis also increased with the amounts of silica gel applied. Based on light microscopic observation of the adaxial surface of rice leaves, the proportion of appressorial penetration was reduced by increasing amounts of silica gel applied and increased with the length of period after spray inoculation. Consequently, these results strongly support the hypothesis and suggest that Si in the leaf epidermis may confer resistance against appressorial penetration. Meanwhile, the number of lesions per leaf also decreased with the amount of Si applied, while only a certain part of penetrated appressoria could become sporulating susceptible lesions. This suggests that Si also confers physiological resistance against blast infection after the penetration.

Complete antithrombin deficiency in mice results in embryonic lethality.

Antithrombin is a plasma protease inhibitor that inhibits thrombin and contributes to the maintenance of blood fluidity. Using targeted gene disruption, we investigated the role of antithrombin in embryogenesis. Mating mice heterozygous for antithrombin gene (ATIII) disruption, ATIII(+/-), yielded the expected Mendelian distribution of genotypes until 14.5 gestational days (gd). However, approximately 70% of the ATIII(-/-) embryos at 15.5 gd and 100% at 16.5 gd had died and showed extensive subcutaneous hemorrhage. Histological examination of those embryos revealed extensive fibrin(ogen) deposition in the myocardium and liver, but not in the brain or lung. Furthermore, no apparent fibrin(ogen) deposition was detected in the extensive hemorrhagic region, suggesting that fibrinogen might be decreased due to consumptive coagulopathy and/or liver dysfunction. These findings suggest that antithrombin is essential for embryonic survival and that it plays an important role in regulation of blood coagulation in the myocardium and liver.

Footprint analysis of the RAG protein recombination signal sequence complex for V(D)J type recombination.

We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.

Reduction of mucin-1 gene expression associated with increased Escherichia coli adherence in the canine uterus in the early stage of dioestrus.

The relation between adherence of Escherichia coli and expression of mucin-1 (Muc1: an integral membrane mucin) mRNA in the endometrium was studied in beagle bitches at different stages of the oestrous cycle and in those with cystic endometrial hyperplasia/pyometra complex (pyometra). The number of E. coli adhering to the endometrium was low at pro-oestrus and oestrus and increased at the early stage (day 10) of dioestrus, corresponding to the implantation period; it declined thereafter. Adhesion of the organisms to endometrial epithelial cells collected at day 10 of dioestrus was inhibited by the addition of D-mannose. When endometrial epithelial cells collected at pro-oestrus were treated with hyaluronidase, an enzyme that digests mucins, the numbers of E. coli adhering to the cells tended to increase. With polymerase chain reaction analysis it was possible to detect Muc1 gene transcripts in the endometrium at all stages of the oestrous cycle, although the level of Muc1 mRNA decreased by day 10 of dioestrus. The levels of Muc1 mRNA in bitches with a clinical stage of pyometra were low and comparable to those at day 10 of dioestrus. The number of E. coli adhering to the endometrium and Muc1 mRNA levels in the endometrium were inversely correlated (r=-0.77, P<0.01). Immunohistochemical analysis showed little staining for Muc1 in the endometrial epithelia at day 10 of dioestrus and in bitches with pyometra. These results suggest that reduction of Muc1 expression is associated with increased E. coli adherence in the canine uterus at the early stage of dioestrus, possibly facilitating the development of pyometra.

Effects of Elevated Atmospheric CO(2) Concentration on the Infection of Rice Blast and Sheath Blight.

ABSTRACT The effect of elevated atmospheric CO(2) concentration on rice blast and sheath blight disease severity was studied in the field in northern Japan for 3 years. With free-air CO(2) enrichment (FACE), rice plants were grown in ambient and elevated ( approximately 200 to 280 mumol mol(-1) above ambient) CO(2) concentrations, and were artificially inoculated with consist of Magnaporthe oryzae. Rice plants grown in an elevated CO(2) concentration were more susceptible to leaf blast than those in ambient CO(2) as indicated by the increased number of leaf blast lesions. Plants grown under elevated CO(2) concentration had lower leaf silicon content, which may have contributed to the increased susceptibility to leaf blast under elevated CO(2) concentrations. In contrast to leaf blast, panicle blast severity was unchanged by the CO(2) enrichment under artificial inoculation, whereas it was slightly but significantly higher under elevated CO(2) concentrations in a spontaneous rice blast epidemic. For naturally occurring epidemics of the sheath blight development in rice plants, the percentage of diseased plants was higher under elevated as opposed to ambient CO(2) concentrations. However, the average height of lesions above the soil surface was similar between the treatments. One hypothesis is that the higher number of tillers observed under elevated CO(2) concentrations may have increased the chance for fungal sclerotia to adhere to the leaf sheath at the water surface. Consequently, the potential risks for infection of leaf blast and epidemics of sheath blight would increase in rice grown under elevated CO(2) concentration.

Enhancement of the differentiation-inducing properties of 6-thioguanine by hypoxanthine and its nucleosides in HL-60 promyelocytic leukemia cells.

Previous work has shown that 6-thioguanine (TGua) is an effective inducer of differentiation of Friend and HL-60 leukemia cells which lack hypoxanthine-guanine phosphoribosyltransferase but is at best only weakly active in inducing maturation in parental wild-type cells. Studies in wild-type and mutant HL-60 cells have provided evidence that the free-base TGua is the form of this drug that induces differentiation, while the formation of TGua nucleotides leads to cytotoxicity and inhibits differentiation. To attempt to increase the potential of TGua to serve as an inducer of parental HL-60 leukemia cells, physiological purine and pyrimidine nucleosides were tested for their ability to protect HL-60 cells against TGua-induced cytotoxicity. Adenosine, deoxyadenosine, inosine, and deoxyinosine completely prevented the toxic action of the purinethiol, while guanosine and deoxyguanosine were only partially effective. The capacity of adenosine and deoxyadenosine to prevent the cytotoxicity of TGua was abolished by the inhibitor of adenosine deaminase, deoxycoformycin, implying that inosine and deoxyinosine were the active forms of the protecting agents. The protective activities of inosine and deoxyinosine appeared to depend on phosphorolysis catalyzed by purine nucleoside phosphorylase, since exogenously added hypoxanthine was as effective as inosine in reducing the cytotoxicity of the purine antimetabolite. Accumulation of TGua nucleotides in the acid-soluble fraction of HL-60 cells treated with TGua was significantly decreased by the presence of inosine. Inosine also served under these circumstances as a D-ribose 1-phosphate donor to TGua, as evidenced by its increased conversion to 6-thioguanosine. The prevention of the cytotoxicity of TGua by the simultaneous administration of hypoxanthine or its nucleosides resulted in an expression of the differentiation-inducing properties of TGua in HL-60 cells, as measured by the accumulation of nitroblue tetrazolium-positive cells. These findings support the concept that the processes of cytotoxicity and differentiation are separable events produced by different metabolic forms of the purine antimetabolite.

Altered 5-phosphoribosyl 1-pyrophosphate amidotransferase activity in 6-thioguanine-resistant HL-60 human acute promyelocytic leukemia cells.

A novel mechanism of resistance to the antileukemic agent 6-thioguanine (TGua) was demonstrated in a clone (TGuo-30-2) derived from HL-60 human acute promyelocytic leukemia cells. The clone was isolated by prescreening mutagenized HL-60 cells in hypoxanthine-amethopterin-thymidine medium, followed by selection with 6-thioguanosine. TGuo-30-2 cells were cross-resistant to TGua and beta-2'-deoxythioguanosine. TGuo-30-2 cells exhibited a marked decrease in the capacity to accumulate intracellular TGua nucleotides after treatment with TGua. The decrease in accumulation was not caused by a defect in transport, a lack or alteration of hypoxanthine-guanine phosphoribosyltransferase activity, or enhanced degradation of TGua nucleotides but appeared to be due to the maintenance of a lowered level of 5-phosphoribosyl 1-pyrophosphate (PRPP) in the resistant variant, which corresponded to 20% of the parental concentration. Despite the decrease in PRPP levels, incorporation of glycine into purine nucleotides was greater in TGuo-30-2 than in parental cells. Measurement of PRPP amidotransferase activity using cell homogenates revealed altered kinetics for the enzyme from TGuo-30-2 cells, which included significant loss of sensitivity to feedback inhibition by 6-thioguanosine 5'-phosphate and greater catalytic activity at low concentrations of PRPP.

Regulation of transferrin receptor in myeloid and monocytic differentiation of HL-60 leukemia cells.

Modulation of surface transferrin receptor activity has been associated with leukemia cell differentiation and proliferation. To examine the mechanisms involved in regulating this event, receptor protein and mRNA levels were measured in HL-60 promyelocytic leukemia cells induced to differentiate along the myelocytic and monocytic pathways. Transferrin receptor down-regulation which occurs during granulocytic differentiation by dimethyl sulfoxide, retinoic acid, or aclacinomycin A appears to be kinetically compatible with reduced biosynthesis resulting from reductions in the level of steady-state mRNA. In contrast, genetic modulation does not appear to mediate the initial receptor down-regulation seen during 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiation. However, a reduction in levels of receptor message appears to contribute to the maintenance of diminished transferrin receptor activity in these 12-O-tetradecanoylphorbol-13-acetate-treated cells. A common feature of myelocytic and monocytic differentiating cells is the complete inhibition of cellular proliferation observed within 10 to 16 h following a four-fold reduction in surface transferrin receptor. We conclude that the early decline in transferrin receptor levels precludes its regulation as a consequence of the decrease in proliferation, but rather implicates its role in the programmed cessation of growth which is requisite for the terminal differentiation of these cells.

cDNA cloning of a novel brain-specific protein p25.

A cDNA for a bovine brain-specific protein p25 which had been originally found as a major protein in a partially purified fraction of tau protein kinases was cloned. The deduced amino-acid sequence consists of 218 amino acids (M(r) 23,472) and has no significant homology with previously reported proteins. p25 is a basic protein and has a consensus sequence for ATP-binding in the C-terminal region.

Characterization of tau phosphorylation in glycogen synthase kinase-3beta and cyclin dependent kinase-5 activator (p23) transfected cells.

One of the histopathological markers in Alzheimer's disease is the accumulation of hyperphosphorylated tau in neurons called neurofibrillary tangles (NFT) composing paired helical filaments (PHF). Combined tau protein kinase II (TPK II), which consists of CDK5 and its activator (p23), and glycogen synthase kinase-3beta (GSK-3beta) phosphorylate tau to the PHF-form in vitro. To investigate tau phosphorylation by these kinases in intact cells, the phosphorylation sites were examined in detail using well-characterized phosphorylation-dependent anti-tau antibodies after overexpressing the kinases in COS-7 cells with a human tau isoform. The overexpression of tau in COS-7 cells showed extensive phosphorylation at Ser-202 and Ser-404. The p23 overexpression induced a mobility shift of tau, but most of the phosphorylation sites overlapped the endogenous phosphorylation sites. GSK-3beta transfection showed the phosphorylation at Ser-199, Thr-231, Ser-396, and Ser-413. Triplicated transfection resulted in phosphorylation of tau at 8 observed sites (Ser-199, Ser-202, Thr-205, Thr-231, Ser-235, Ser-396, Ser-404, and Ser-413).

Ultrastructural characterization of cerebellar diffuse plaques in Alzheimer's disease.

We examined the ultrastructural localization of beta/A4 protein in cerebellar diffuse plaques (DP) in three Alzheimer's disease brains by the indirect immunoperoxidase technique. Intense immunoreaction products were scattered in the DP; they were strongly suggested to be located between small cell processes. Reaction products were dot-like and/or amorphous, and occasionally fibrillar. Adjacent semithin sections of regions immunoreactive for beta/A4 protein revealed only very small amounts of amyloid fibrils between cell processes and/or small numbers of degenerating neurites. The small degenerating neurites which appeared in most DP lacked paired helical filaments (PHF) and neurofilaments (NF). These findings suggest that the majority of the beta/A4-immunoreactive substance in cerebellar DP is non-fibrillar pre-amyloid found between cell processes and barely detectable by routine electron microscopy.