Constraints on Oscillation Parameters from ν_{e} Appearance and ν_{µ} Disappearance in NOvA.

Results are reported from an improved measurement of ν_{µ}→ν_{e} transitions by the NOvA experiment. Using an exposure equivalent to 6.05×10^{20} protons on target, 33 ν_{e} candidates are observed with a background of 8.2±0.8 (syst.). Combined with the latest NOvA ν_{µ} disappearance data and external constraints from reactor experiments on sin^{2}2θ_{13}, the hypothesis of inverted mass hierarchy with θ_{23} in the lower octant is disfavored at greater than 93% C.L. for all values of δ_{CP}.

Measurement of the Neutrino Mixing Angle θ_{23} in NOvA.

This Letter reports new results on muon neutrino disappearance from NOvA, using a 14 kton detector equivalent exposure of 6.05×10^{20} protons on target from the NuMI beam at the Fermi National Accelerator Laboratory. The measurement probes the muon-tau symmetry hypothesis that requires maximal θ_{23} mixing (θ_{23}=π/4). Assuming the normal mass hierarchy, we find Δm_{32}^{2}=(2.67±0.11)×10^{-3} eV^{2} and sin^{2}θ_{23} at the two statistically degenerate values 0.404_{-0.022}^{+0.030} and 0.624_{-0.030}^{+0.022}, both at the 68% confidence level. Our data disfavor the maximal mixing scenario with 2.6σ significance.

StreetWise: developing a serious game to support forensic mental health service users' preparation for discharge: a feasibility study.

WHAT IS KNOWN ON THE SUBJECT?: Serious gaming can support learning and development. The use of serious games for skills development and the rehearsal of the management of events that cannot be replicated in real life is well established. Few serious games have been used in mental health services, and none in forensic mental health care. WHAT THIS PAPER ADDS TO EXISTING KNOWLEDGE?: How a serious game may be coproduced by forensic mental health service users and game developers The acceptability of the therapeutic use of serious gaming by forensic mental health service users and providers. WHAT ARE THE IMPLICATIONS FOR PRACTICE?: Computer games may be used by practitioners in their therapeutic work with forensic mental health service users. Mental health nurses to use serious games to creatively and safely bridge the gap for service users between receiving care in controlled environments and living more independent in the community. ABSTRACT: Introduction Assessment of users' skills and confidence to safely respond to risky community-based situations underpins discharge planning. Serious games have been used for skills development, and this study trialled their use in forensic mental health services. Aim The aim was to develop and test the acceptability and usability of an innovative serious game to support forensic mental health service users' preparation for discharge. Method A prototype serious game was developed by service users and researchers. Acceptability and usability testing was undertaken and service providers interviewed about the acceptability of serious gaming for forensic mental health services. Result A prototype game was produced and successfully trialled by service users. However, both service users and providers identified that work needed to be done to develop and test a game with greater complexity. Discussion The acceptability and usability of using serious games to support service users to develop skills needed for successful discharge was demonstrated. Implications for practice Mental health practitioners may use gaming to support their practice and work innovatively with other professions such as game developers to create new ways of working in forensic mental health services.

First Measurement of Electron Neutrino Appearance in NOvA.

We report results from the first search for ν_{µ}→ν_{e} transitions by the NOvA experiment. In an exposure equivalent to 2.74×10^{20} protons on target in the upgraded NuMI beam at Fermilab, we observe 6 events in the Far Detector, compared to a background expectation of 0.99±0.11(syst) events based on the Near Detector measurement. A secondary analysis observes 11 events with a background of 1.07±0.14(syst). The 3.3σ excess of events observed in the primary analysis disfavors 0.1π<δ_{CP}<0.5π in the inverted mass hierarchy at the 90% C.L.

Accumulation of cholera toxin and GM1 ganglioside in the early endosome of Niemann-Pick C1-deficient cells.

We investigated intracellular trafficking of GM1 ganglioside in Niemann-Pick C1 (NPC1)-deficient Chinese hamster ovary cells [NPC1(-) cells] by using cholera toxin (CT) as a probe. Both the holotoxin and the B subunit (CTB) accumulated in GM1-enriched intracellular vesicles of NPC1(-) cells. CTB-labeled vesicles contained the early endosome marker Rab5 but not lysosome-associated membrane protein 2 and were not labeled with either Texas red-transferrin or Lysotracker, indicating that they represent early endosomes. Similarly, CT accumulated in intracellular vesicles of human NPC fibroblasts that contained both Rab5 and early endosomal antigen 1. CTB accumulation in NPC1(-) cells was abolished by expression of wild-type NPC1 but not by mutant proteins with a mutation either in the NPC domain or the sterol-sensing domain. A part of these mutant NPC1 proteins expressed in NPC1(-) cells was localized on CTB-labeled vesicles. U18666A treatment of "knock in" cells [NPC1(-) cells that stably expressed wild-type NPC1] caused CTB accumulation similar to that in NPC1(-) cells, and a part of wild-type NPC1was localized on CTB-labeled vesicles in drug-treated cells. Finally, CT tracer experiments in NPC1(-) cells revealed retarded excretion of internalized toxin into the culture medium and an increase in the intracellular release of A subunits. In accordance with the latter result, CT was more effective in stimulating cAMP formation in NPC1(-) than in wild-type cells. These results suggest that transport of CT/GM1 complexes from the early endosome to the plasma membrane depends on the function of NPC1, whereas transport to the Golgi apparatus/endoplasmic reticulum does not.

Transmembrane molecular pump activity of Niemann-Pick C1 protein.

Niemann-Pick C1 (NPC1) disease is characterized by cholesterol accumulation in lysosomes and aberrant feedback regulation of cellular cholesterol homeostasis. We provide evidence that the NPC1 protein has homology with the resistance-nodulation-division (RND) family of prokaryotic permeases and may normally function as a transmembrane efflux pump. Studies of acriflavine loading in normal and NPC1 fibroblasts indicated that NPC1 uses a proton motive force to remove accumulated acriflavine from the endosomal/lysosomal system. Expression of NPC1 in Escherichia coli (i) facilitated the transport of acriflavine across the plasma membrane, causing cytosolic accumulation, and (ii) resulted in transport of oleic acid but not cholesterol or cholesterol-oleate across the plasma membrane. These studies establish NPC1 as a eukaryotic member of the RND permease family.

Topological analysis of Niemann-Pick C1 protein reveals that the membrane orientation of the putative sterol-sensing domain is identical to those of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein cleavage-activating protein.

The Niemann-Pick C1 (NPC1) protein is predicted to be a polytopic glycoprotein, and it contains a region with extensive homology to the sterol-sensing domains (SSD) of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-R) and sterol regulatory element binding protein cleavage-activating protein (SCAP). To aid the functional characterization of NPC1, a model of NPC1 topology was evaluated by expression of epitope-tagged NPC1 proteins and investigation of epitope accessibility in selectively permeabilized cells. These results were further confirmed by expression of NPC1 and identification of glycosylated domains that are located in the lumen of the endoplasmic reticulum. Our data indicate that this glycoprotein contains 13 transmembrane domains, 3 large and 4 small luminal loops, 6 small cytoplasmic loops, and a cytoplasmic tail. Furthermore, our data show that the putative SSD of NPC1 is oriented in the same manner as those of HMG-R and SCAP, providing strong evidence that this domain is functionally important.

Evidence for a Niemann-pick C (NPC) gene family: identification and characterization of NPC1L1.

Niemann-Pick type C1 (NPC1) disease is caused by defects in the NPC1 protein, which result in perturbation of subcellular cholesterol transport. To identify related proteins that may be involved in subcellular cholesterol trafficking, the expressed sequence tag (EST) database was searched to find homologues of human NPC1. A short, weakly similar EST was identified and used to obtain a full-length human cDNA of about 5 kb and two alternatively spliced transcripts. The gene, named NPC1L1, was mapped to chromosome 7p13, contained 20 exons, including an unusually large 1526-bp exon 2, and spanned approximately 29 kb. In contrast to NPC1, the NPC1L1 putative promoter region contained a sterol-regulatory element. The predicted protein shared 42% identity and 51% similarity with NPC1. Interestingly, NPC1L1 contains the conserved amino-terminal "NPC1 domain" and the putative sterol-sensing domain, providing strong evidence that it is related to human NPC1 and suggesting that these may comprise a new family of NPC1-related proteins. However, the two differ with respect to their putative intracellular targeting signals. Collectively, these data suggest that NPC1L1 and NPC1 form part of a family of related proteins that may have similar functions at different subcellular locations, perhaps at sequential steps of the same cholesterol transport pathway.

Niemann-Pick C1 is a late endosome-resident protein that transiently associates with lysosomes and the trans-Golgi network.

Niemann-Pick type C (NPC) disease is a severe cell lipidosis characterized by the accumulation of unesterified cholesterol in the endosomal/lysosomal system. Recently the primary disease-causing gene, NPC1, was identified, but few clues regarding its potential function(s) could be derived from its predicted amino acid sequence. Therefore, efforts were directed at characterizing the subcellular location of the NPC1 protein. Initial studies with a FLAG-tagged NPC1 cDNA demonstrated that NPC1 is a glycoprotein that associates with the membranes of a population of cytoplasmic vesicles. Immunofluorescence microscopy using anti-NPC1 polyclonal antibodies confirmed this analysis. Double-label immunofluorescence microscopy and subcellular fractionation studies indicated that NPC1 associates predominantly with late endosomes (Rab9 GTPase-positive vesicles) and, to a lesser extent, with lysosomes and the trans-Golgi network. When cholesterol egress from lysosomes was blocked by treatment of cells with U18666A, the NPC1 location shifted from late endosomes to the trans-Golgi network and lysosomes. Subcellular fractionation of liver homogenates from U18666A-treated mice confirmed these observations. These data suggest that U18666A may inhibit the retrograde transport of NPC1 from lysosomes to late endosomes for subsequent transfer to the trans-Golgi network.

Sac3, an Snf1-like serine/threonine kinase that positively and negatively regulates the responses of Chlamydomonas to sulfur limitation.

The Sac3 gene product of Chlamydomonas positively and negatively regulates the responses of the cell to sulfur limitation. In wild-type cells, arylsulfatase activity is detected only during sulfur limitation. The sac3 mutant expresses arylsulfatase activity even when grown in nutrient-replete medium, which suggests that the Sac3 protein has a negative effect on the induction of arylsulfatase activity. In contrast to its effect on arylsulfatase activity, Sac3 positively regulates the high-affinity sulfate transport system-the sac3 mutant is unable to fully induce high-affinity sulfate transport during sulfur limitation. We have complemented the sac3 mutant and cloned a cDNA copy of the Sac3 gene. The deduced amino acid sequence of the Sac3 gene product is similar to the catalytic domain of the yeast Snf1 family of serine/threonine kinases and is therefore classified as a Snf1-related kinase (SnRK). Specifically, Sac3 falls within the SnRK2 subfamily of kinases from vascular plants. In addition to the 11 subdomains common to Snf1-like serine/threonine kinases, Sac3 and the plant kinases have two additional subdomains and a highly acidic C-terminal region. The role of Sac3 in the signal transduction system that regulates the responses of Chlamydomonas to sulfur limitation is discussed.

Differential gene expression in apoptosis: identification of ribosomal protein 23K, a cell proliferation inhibitor.

Gene expression during the camptothecin-induced apoptotic death of human leukemic U937 cells and mouse T-cell hybridoma QW4.1 cells was studied by the mRNA differential display technique. Ten clones were confirmed to be differentially expressed, nine of which encoded novel sequences. One clone, U3.2, was induced approximately 10-fold in camptothecin-treated cells and was found to be identical to a highly basic 23-kDa human protein which contains basic leucine zipper-like motifs and has recently been identified as the human homologue of the rat ribosomal protein L13a. Northern blot analysis revealed a major mRNA of approximately 0.9 kb and a minor mRNA of approximately 1.3 kb. Overexpression of a full-length 23K cDNA, tagged with a FLAG sequence, in COS-7 cells revealed a predominantly nucleolar localization and the absence of any 23K protein from the cytoplasm. Subsequent transfection studies, using antisense phosphorothioate-modified oligonucleotides, revealed that inhibition of 23K expression results in an increased cell proliferation and greater sensitivity of U937 cells to the effects of camptothecin-induced cell death. Upregulation of 23K expression using a cDNA construct resulted in a decrease in cell proliferation and growth arrest, suggesting a role for 23K protein as a proliferation checkpoint following a cellular insult.

The regulation of photosynthetic electron transport during nutrient deprivation in Chlamydomonas reinhardtii.

The light-saturated rate of photosynthetic O2 evolution in Chlamydomonas reinhardtii declined by approximately 75% on a per-cell basis after 4 d of P starvation or 1 d of S starvation. Quantitation of the partial reactions of photosynthetic electron transport demonstrated that the light-saturated rate of photosystem (PS) I activity was unaffected by P or S limitation, whereas light-saturated PSII activity was reduced by more than 50%. This decline in PSII activity correlated with a decline in both the maximal quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers (PSII centers capable of performing a charge separation but unable to reduce the plastoquinone pool). In addition to a decline in the light-saturated rate of O2 evolution, there was reduced efficiency of excitation energy transfer to the reaction centers of PSII (because of dissipation of absorbed light energy as heat and because of a transition to state 2). These findings establish a common suite of alterations in photosynthetic electron transport that results in decreased linear electron flow when C. reinhardtii is limited for either P or S. It was interesting that the decline in the maximum quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers were regulated specifically during S-limited growth by the SacI gene product, which was previously shown to be critical for the acclimation of C. reinhardtii to S limitation (J.P. Davies, F.H. Yildiz, and A.R. Grossman [1996] EMBO J 15: 2150-2159).

Cloning and mapping of human Rab7 and Rab9 cDNA sequences and identification of a Rab9 pseudogene.

Rab GTPases reside in specific intracellular compartments and are key regulators of vesicular transport. To facilitate studies of the mechanism of lysosomal integral membrane protein (LAMP-1) transport, cDNAs for human Rab7 and Rab9 were isolated, and their nucleotide sequences were determined. During isolation and characterization of these cDNAs a Rab9 pseudogene was identified. The sequences are highly homologous to other mammalian Rab proteins and also share homology with proteins of the Rab GTPase family. Rab7 and the Rab9 pseudogene were mapped to chromosomes 3 and 5, respectively, by amplification of their sequences from human monochromosomal somatic cell hybrids. In addition, preliminary studies using antisense expression indicate that down-regulation of either Rab7 or Rab9 proteins induces severe cell vacuolation that resembles the phenotype seen in fibroblasts from patients with Chediak-Higashi syndrome.

Sulfur availability and the SAC1 gene control adenosine triphosphate sulfurylase gene expression in Chlamydomonas reinhardtii.

A Chlamydomonas reinhardtii adenosine triphosphate (ATP) sulfurylase cDNA clone (pATS1) was selected by complementing a mutation in the ATP sulfurylase gene (cysD) of Escherichia coli. E. coli cysD strains harboring pATS1 grow on medium containing sulfate as the sole sulfur source and exhibit ATP sulfurylase activity. The amino acid sequence of the C. reinhardtii ATP sulfurylase, derived from the nucleotide sequence of the complementing gene (ATS1), is 25 to 40% identical to that of ATP sulfurylases in other eukaryotic organisms and has a putative transit peptide at its amino terminus. ATP sulfurylase mRNA was present when cells were grown in sulfur-replete medium, but accumulated to higher levels when the cells were exposed to sulfur-limiting conditions. Furthermore, sulfur-stress-induced accumulation of the ATS1 transcript was reduced in a strain defective in SAC1, a gene that is critical for acclimation to sulfur-limited growth.

Monitoring the integrity of the cement-metal interface of total joint components in vitro using acoustic emission and ultrasound.

Debonding of the cement-metal interface of cemented femoral components of total hip arthroplasty has been shown from clinical and autopsy material to be a common occurrence. Experimentally, debonding has been shown to increase markedly the strains in the adjacent cement mantle. Studies of autopsy-retrieved specimens demonstrate that debonding of the cement-metal interface is a key initiating event in loosening of cemented femoral components of total hip arthroplasty. However, both the radiographic and autopsy evidence of cement-metal interfacial debonding exist after the fact, that is, after debonding has occurred. The lack of prospective data showing that debonding does indeed occur under physiologic loading and occurs prior to other forms of failure of fixation leaves uncertain the issue of debonding and its role in initiating loosening of cemented femoral components. Knowing when, where, and to what extent the cement-metal interface debonds is critical information in understanding the process of loosening of cemented femoral components. Such information would contribute to improving the durability of stems and improving cementing techniques. In this study, the two nondestructive techniques of acoustic emission and ultrasonic evaluation of the cement-metal interface of cemented femoral stems of total hip arthroplasty were combined to investigate when, where, and to what extent cement-metal debonding occurred in vitro in simulated femurs loaded physiologically in fatigue in simulated single-leg stance. Debonding of the cement-metal interface of a cemented femoral component in this model was both an initiating event and a major mechanism of compromise of the cement-metal interface. Additional acoustic emission signals arose from cracks that developed in the cement.

Sac1, a putative regulator that is critical for survival of Chlamydomonas reinhardtii during sulfur deprivation.

The sac1 mutant of Chlamydomonas reinhardtii is aberrant in most of the normal responses to sulfur limitation; it cannot synthesize arylsulfatase, does not take up sulfate as rapidly as wild-type cells, and does not synthesize periplasmic proteins that normally accumulate during sulfur-limited growth. Here, we show that the sac1 mutant dies much more rapidly than wild-type cells during sulfur deprivation; this emphasizes the vital role of the acclimation process. The loss of viability of the sac1 mutant during sulfur deprivation is only observed in the light and is mostly inhibited by DCMU. During sulfur-stress, wild-type cells, but not the sac1 mutant, downregulate photosynthesis. Thus, death of the sac1 mutant during sulfur deprivation is probably a consequence of its inability to downregulate photosynthesis. Furthermore, since SAC1 is necessary for the downregulation of photosynthesis, the process must be highly controlled and not simply the result of a general decrease in protein synthesis due to sulfur limitation. Genomic and cDNA copies of the SAC1 gene have been cloned. The deduced amino acid sequence of Sac1 is similar to an Escherichia coli gene that may involved in the response of E.coli to nutrient deprivation.

Fabry disease: fourteen alpha-galactosidase A mutations in unrelated families from the United Kingdom and other European countries.

The nature of the molecular lesions in the alpha-galactosidase A gene causing Fabry disease in 12 unrelated families from the United Kingdom and 4 from other European countries was determined in order to provide precise heterozygote detection and prenatal diagnosis for these families. The entire alpha-galactosidase A coding region and flanking intronic sequences were analyzed by amplification of genomic DNA and solid-phase direct sequencing or by SSCP analysis followed by solid-phase direct sequencing. Fourteen new mutations were identified including 10 missense mutations (M42V, R49S, C56Y, D92H, D93G, P205T, W236C, W287G, N298H, and W340R), 2 nonsense mutations (Q107X and Q119X) and 2 small deletions (257del18 and 1087del1). Together with the previously reported mutations, a total of 33 lesions in the alpha-galactosidase A gene have been identified in unrelated British families, further documenting the molecular genetic heterogeneity of this disease.