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Autophagy is a housekeeping mechanism tasked with eliminating misfolded proteins and damaged organelles to maintain cellular homeostasis. Autophagy deficiency results in increased oxidative stress, DNA damage and chronic cellular injury. Among the core genes in the autophagy machinery, ATG7 is required for autophagy initiation and autophagosome formation. Based on the analysis of an extended pedigree of familial cholangiocarcinoma, we determined that all affected family members had a novel germline mutation (c.2000C>T p.Arg659* (p.R659*)) in ATG7. Somatic deletions of ATG7 were identified in the tumors of affected individuals. We applied linked-read sequencing to one tumor sample and demonstrated that the ATG7 somatic deletion and germline mutation were located on distinct alleles, resulting in two hits to ATG7. From a parallel population genetic study, we identified a germline polymorphism of ATG7 (c.1591C>G p.Asp522Glu (p.D522E)) associated with increased risk of cholangiocarcinoma. To characterize the impact of these germline ATG7 variants on autophagy activity, we developed an ATG7-null cell line derived from the human bile duct. The mutant p.R659* ATG7 protein lacked the ability to lipidate its LC3 substrate, leading to complete loss of autophagy and increased p62 levels. Our findings indicate that germline ATG7 variants have the potential to impact autophagy function with implications for cholangiocarcinoma development.

Phenotype-based screening of a fungal extract library yielded an active sample from a Penicillium sp. isolate that impaired zebrafish motility. Bioassay-guided purification led to the identification of 14 meroterpenoids including six new metabolites, arisugacins L-Q (4, 5, 8, and 12-14), seven known arisugacins (1-3, 6, 7, 9, and 10), and one known terreulactone (11). Their structures were determined using a combination of NMR and HRESIMS data, evidence secured from theoretical and experimental ECD spectra, and the modified Mosher's method. The purified compounds were tested in zebrafish embryos, as well as in vitro for cholinesterase inhibition activities. Compound 12 produced defects in myotome structure (metameric muscle, which is critical for locomotion) in vivo and showed the most potent and selective acetylcholinesterase inhibitory activity with an IC50 of 191 nM in vitro. The phenotype assay was also used to reveal bioactivities for several previously reported arisugacins, which had failed to show activity in prior cell-based and in vitro testing. This study demonstrates that utilization of the zebrafish phenotype assay is an effective approach for the identification of bioactive extracts, is compatible with the bioassay-guided compound purification strategies, and offers a valuable tool for probing complex natural product sources to detect bioactive small molecules with potential therapeutic or other commercial applications.

Germline mutations in the tumor suppressor Adenomatous Polyposis Coli (APC) define Familial Adenomatous Polyposis (FAP), the genetic predisposition to developing adenomatous polyps. Recent sequencing of FAP adenomas have challenged established dogma that APC mutations alone represent the adenoma mutational landscape because recurrent somatic mutations in non-WNT pathway genes were also discovered. In particular, one of these novel genes, CNOT3, presented E20K and E70K mutations that are predicted to be deleterious in silico. We utilized zebrafish embryos to determine if these mutations affect CNOT3 function and perform novel biology in an APC-dependent pathway in vivo. Human CNOT3 (hCNOT3) and E20K mRNA injection rescued zebrafish cnot3a knockdown lordosis phenotype while E70K did not. In the FAP apcmcr zebrafish model, we show that ctbp1, but not retinoic acid, regulates cnot3a expression. Injection of hCNOT3 and E20K, but not E70K, to homozygous apcmcr zebrafish initiated gut differentiation while cnot3a knockdown in wildtype embryos led to decreased intestinal development and differentiation. Finally, targeted sequencing of 37 additional FAP adenomas revealed CNOT3 mutations in 20% of these samples. Overall, our work supports a mechanism where CTBP1 regulates CNOT3 and that overall CNOT3 perturbation could work in concert with germline APC mutations in advancing adenomas to a more transformed state prior to progression to adenocarcinoma.

Introduction Sleep disordered breathing (SDB) is highly prevalent in tetraplegia, yet most remain undiagnosed and untreated for the disorder. We have described the care models of three international SCI rehabilitation centres that are independently diagnosing and treating SDB, and distilled their common pathways into the SCISTAR (SCI Sleep disordered breathing Treatment & Assessment in Rehabilitation) model. The aim of this project was to adapt the SCISTAR model to suit the Royal Rehab Spinal Injuries Unit, and to prepare for the implementation of this new model of care. Methods A clinical advisory group, consisting of researchers, rehabilitation and respiratory health professionals, met six times over eight months to: • identify the multidisciplinary SDB clinical team, • adapt the SCISTAR model, • develop parameters for safe clinical practice, • identify education needs, • identify and acquire the necessary equipment. Results The SDB clinical team consists of two rehabilitation consultants, two physiotherapists and four nurses. All patients with SCI will be assessed for SDB following admission. Assessments will include symptoms, spirometry and sniff nasal inspiratory pressure, and a Level 3 overnight sleep study (ResMed ApneaLink). Patients diagnosed with un-complicated SDB will be prescribed CPAP, which will be initiated and progressed on the ward. Screening for hypoventilation and predominant central sleep apnoea will occur throughout the pathway and if identified, patients will be referred to a specialist respiratory service. Comprehensive education and equipment has been provided. Conclusions The new care model is currently being piloted for 12 months. A comprehensive evaluation has been developed.

Acute pulmonary Exacerbations (AE) are episodes of clinical worsening in cystic fibrosis (CF), often precipitated by infection. Timely detection is critical to minimize the morbidity and lung function decline associated with acute inflammation during AE. We previously demonstrated that the airway protein Short Palate Lung Nasal epithelium Clone 1 (SPLUNC1) is regulated by inflammatory signals. Here, we investigated the use of SPLUNC1 fluctuations to diagnose and predict AE in CF. We enrolled adult CF subjects from two independent cohorts to measure AE markers of inflammation in sputum and recorded clinical outcomes for a 1-year follow-up period. SPLUNC1 levels were high in healthy control sputum (n=9, 10.7μg/mL), and significantly decreased in CF subjects without AE (n=30, 5.7μg/mL, p=0.016). SPLUNC1 levels were 71.9% lower during AE (n=14, 1.6μg/mL, p=0.0034) regardless of age, sex, CF-causing mutation, or microbiology findings. Cytokines Il–1β and TNFα were also increased in AE,whereas lung function did not consistently decrease. Stable CF subjects with lower SPLUNC1 levels were much more likely to have an AE at 60 days (Hazard Ratio: 11.49, Standard Error: 0.83, p=0.0033). Low-SPLUNC1 stable subjects remained at higher AE risk even one year after sputum collection (Hazard Ratio: 3.21, Standard Error: 0.47,p=0.0125). SPLUNC1 was transcriptionally downregulated by inflammatory cytokines and degraded by proteases increased in sputum during AE. Our findings suggest that low sputum SPLUNC1 levels could detect subjects at increased risk of AE in order to guide early therapeutic interventions in CF. TAKE-HOME MESSAGE Sputum concentrations of the secreted airway protein SPLUNC1 decrease during CF exacerbations. Lower SPLUNC1 levels in stable subjects portend a significantly increased risk of exacerbation and could inform therapeutic interventions. PLAIN LANGUAGE SUMMARY SPLUNC1 is an abundant host defense protein found in the respiratory tract that decreases with inflammation. Individuals with cystic fibrosis experiencing clinical worsening (exacerbation) have much lower levels of SPLUNC1 in their sputum. In stable cystic fibrosis patients, lower levels of SPLUNC1 may predict an upcoming respiratory illness. Therefore, SPLUNC1 may serve as a tool for early diagnosis and treatment of cystic fibrosis exacerbations.

Cholangiocarcinoma is an aggressive cancer originating from the bile duct. Although cholangiocarcinoma does occur in families, to date no specific causative gene has been identified. We identified ATG7 as a cancer susceptibility gene using a joint genetic analysis of an extended pedigree with familial cholangiocarcinoma in combination with a population genetic association study. Affected family members had a germline mutation (c.2000C>T [p.Arg659*]) in the autophagy related gene, ATG7, and all of the affected individuals had cholangiocarcinoma tumors harboring somatic genomic deletions of ATG7. From a population genetic study, we identified a germline polymorphism of ATG7 (c.1591C>G [p.Asp522Glu]) associated with increased risk of cholangiocarcinoma. The autophagy substrate p62 demonstrated a higher accumulation in tumors of p.Asp522Glu carriers compared with non-carriers indicating defective autophagy. To determine whether the germline ATG7 mutation had functional consequences, we developed an ATG7-deficient cholangiocyte cell line, derived from human bile duct, to test for autophagy-mediated lipidation activity. The germline mutation from the familial cholangiocarcinoma demonstrated a lack of lipidation activity compared to the wildtype ATG7. Moreover, in zebrafish embryos depleted of atg7, a reproducible necrotic head phenotype was rescued by injection of wildtype ATG7 but not mutant ATG7. Our findings point to ATG7 as a causative genetic risk factor for cholangiocarcinoma and implicate autophagy as a novel cancer driver mechanism.

Phenotype-based screening of a fungal extract library yielded an active sample from a

Germline mutations in the tumor suppressor Adenomatous Polyposis Coli (APC) define Familial Adenomatous Polyposis (FAP), the genetic predisposition to developing adenomatous polyps. Recent sequencing of FAP adenomas have challenged established dogma that APC mutations alone represent the adenoma mutational landscape because recurrent somatic mutations in non-WNT pathway genes were also discovered. In particular, one of these novel genes, CNOT3, presented E20K and E70K mutations that are predicted to be deleterious in silico. We utilized zebrafish embryos to determine if these mutations affect CNOT3 function and perform novel biology in an APC-dependent pathway in vivo. Human CNOT3 (hCNOT3) and E20K mRNA injection rescued zebrafish cnot3a knockdown lordosis phenotype while E70K did not. In the FAP apcmcr zebrafish model, we show that ctbp1, but not retinoic acid, regulates cnot3a expression. Injection of hCNOT3 and E20K, but not E70K, to homozygous apcmcr zebrafish initiated gut differentiation while cnot3a knockdown in wildtype embryos led to decreased intestinal development and differentiation. Finally, targeted sequencing of 37 additional FAP adenomas revealed CNOT3 mutations in 20% of these samples. Overall, our work supports a mechanism where CTBP1 regulates CNOT3 and that overall CNOT3 perturbation could work in concert with germline APC mutations in advancing adenomas to a more transformed state prior to progression to adenocarcinoma.

Elucidating signaling pathways that regulate cellular metabolism is essential for a better understanding of normal development and tumorigenesis. Recent studies have shown that mitochondrial pyruvate carrier 1 (MPC1), a crucial player in pyruvate metabolism, is downregulated in colon adenocarcinomas. Utilizing zebrafish to examine the genetic relationship between MPC1 and Adenomatous polyposis coli (APC), a key tumor suppressor in colorectal cancer, we found that apc controls the levels of mpc1 and that knock down of mpc1 recapitulates phenotypes of impaired apc function including failed intestinal differentiation. Exogenous human MPC1 RNA rescued failed intestinal differentiation in zebrafish models of apc deficiency. Our data demonstrate a novel role for apc in pyruvate metabolism and that pyruvate metabolism dictates intestinal cell fate and differentiation decisions downstream of apc. DOI: http://dx.doi.org/10.7554/eLife.22706.001, eLife digest Colon cancer remains an important problem in healthcare. Cancer researchers are looking for new ways to detect the disease earlier and treat it more effectively. This is challenging because many of the genetic and molecular causes of colon cancer are still poorly understood. Mutations in the gene that encodes a protein called APC are one of the major causes of the disease. The APC protein normally keeps cells from growing and dividing too fast or in an uncontrolled way and is hence referred to as a tumor suppressor. For example, APC induces stem cells in the intestine to develop into specialized cells that keep the gut working normally. Mutations in tumor suppressor genes are common in many cancers. Other research has shown that cancer cells must reprogram their own metabolism – in other words, all the chemical processes that keep the cell alive – to meet the demands of proliferating rapidly. In particular, recent studies reveal that colon cancer cells produce less of a protein called mpc1, which is involved in metabolism. These discoveries raised the following questions: does APC have an additional role in maintaining normal metabolism in cells by controlling how much mpc1 is produced? Do mutations in the gene for APC lead to colon cancer because they alter the cell’s metabolism? Sandoval et al. have now discovered a connection between APC and changes in cancer cells that help them to adapt to a new metabolic program. Experiments with zebrafish – a model animal that is now commonly used in the field of cancer biology – showed that APC acts via mpc1 to regulate how the cell uses energy. This regulation goes awry in colon cells that have abnormal APC activity; however, restoring the cell’s metabolism back to normal was enough to induce cells in the intestine to develop properly. Together, these findings suggest that restoring the normal balance of energy production in colon cancer cells may be an effective way to make the cells behave normally. This hypothesis remains to be tested and, if confirmed, further studies will be needed to determine whether it will lead to new treatments for colon cancer in humans. DOI: http://dx.doi.org/10.7554/eLife.22706.002

The purpose of this study was to validate inferences about scores of one task designed to measure subject matter knowledge and three tasks designed to measure aspects of pedagogical content knowledge. Evidence for the validity of inferences was based on two expectations. First, if tasks were sensitive to expertise, we would find group differences. Second, tasks that measured similar types of knowledge would correlate strongly, and tasks that measured different types of knowledge would correlate weakly. We recruited and assessed 4 groups of participants including 46 experienced algebra teachers (2+ years experience), 17 novice algebra teachers (0–2 years experience), 10 teaching experts, and 13 subject matter experts. Results indicate that one task differentiated among levels of expertise and measured several aspects of knowledge needed to teach algebra. Results also highlight that future studies should use a combination of tasks to accurately measure different aspects of teacher knowledge.

The Learning Physics through Play Project (LPP) engaged 6–8 year old students (n = 43) in a series of scientific investigations of Newtonian force and motion including a series of augmented reality activities. We outline the two design principles behind the LPP curriculum: 1) the use of socio-dramatic, embodied play in the form of participatory modeling to support inquiry; and 2) progressive symbolization within rich semiotic ecologies to help students construct meaning. We then present pre- and post-test results to show that young students were able to develop a conceptual understanding of force, net force, friction and two-dimensional motion after participating in the LPP curriculum. Finally, we present two case studies that illustrate the design principles in action. Taken together the cases show some of the strengths and challenges associated with using augmented reality, embodied play, and a student invented semiotic ecology for scientific inquiry.

Hybrid reality games (HRGs) employ mobile technologies and GPS devices as tools for transforming physical spaces into interactive game boards. Rather than situating participants in simulated environments, which mimic the physical world, HRGs make use of physical world immersion by merging physical and digital spaces. Online multiuser environments already connect users who do not share contiguous spaces. With mobile devices, players may additionally incorporate interactions with the surrounding physical space. This article is a speculative study about the potential uses of HRGs in education, as activities responsible for taking learning practices outside the closed classroom environment into open, public spaces. Adopting the framework of sociocultural learning theory, the authors analyze design elements of existing HRGs, such as mobility and location awareness, collaboration/sociability, and the configuration of the game space, with the aim of reframing these games into an educational context to foresee how future games might contribute to discovery and learning.

We propose that future rifle marksmanship research be framed within a phases-of-skill-development model (Ackerman, 1987, 1992; Anderson, 1982; Fitts & Posner, 1967). Prior research on predicting sh...

In this study we examined the role of anxiety on novices' performance on the psychomotor task of rifle marksmanship. The stages-of-skill-development model suggests that aptitude influences performance while learning a skill; however, the model does not address the role of affect. Assessments for state anxiety and state worry were administered to 42 novice shooters. Aptitude and state worry predicted shooting performance (r = .67) comparable to more direct psychomotor measures used in prior research. Incremental validity analyses showed that aptitude accounted for 11% of the variance, and state worry explained an additional 34% above and beyond aptitude. The use of noncognitive assessments can broaden the understanding of skilled performance. Although aptitude influences learning a skill, it appears that novice shooters' performance is also influenced by affective states such as anxiety.

In this chapter we examine cognitive readiness for solving equations in the domain of pre-algebra. We describe the knowledge required to solve a multistep equation, and present a novel technique for developing assessment items and a novel assessment item format, designed to measure both the prerequisites of cognitive readiness for solving equations and the skills themselves. Assessment items were drawn from the solution path of a complex multistep equation. The items used a “next step” format, which asked students to write only the first step of their solution. Using a sample of 42 middle school students, data were gathered on item performance and how performance on the next step item compared to performance on the more traditional “solve for x” format. Unsurprisingly, students performed higher the simpler the equation was but large drops in performance occurred at steps that were essential to isolating a variable (i.e., applying the equality properties of addition and multiplication). Students performed higher on the traditional items compared to next step items and there was some evidence that performance on the next step item predicted performance on the traditional items. Assessment and instructional implications of this work include diagnosing precisely where in the solution path students have difficulty, which is tantamount to identifying students’ cognitive readiness for solving equations.

Mutation in the tumor suppressor Adenomatous Polyposis Coli (APC) is the primary initiating mutation in a majority of colon tumors. Recent studies have shown that APC positively correlates with Mitochondrial Pyruvate Carrier 1 (MPC1), a crucial player in pyruvate metabolism and has been reported to repress the Warburg effect and growth in colon cancer cells. Utilizing the zebrafish to examine the relationship between apc and mpc1, we found that mpc1 expression is reduced in embryos harboring a genetic mutation (apcmcr) or diminished expression (apc mo) of apc. Antisense morpholino knockdown of mpc1 in wild type embryos (mpc1 mo) resulted in an array of developmental defects that recapitulated phenotypes of impaired apc function including failed intestinal differentiation. This was accompanied by mitochondrial dysfunction in mpc1 mo as evidenced by a decrease in metabolic respiration and triglyceride levels. We also observed altered mitochondrial function and dysregulation of enzymes involved in pyruvate metabolism in apcmcr and apc mo. Moreover, hMPC1 RNA rescued intestinal differentiation in both zebrafish models of apc deficiency. Meta-analyses using TCGA human tumor samples corroborated our findings in the zebrafish. Our data demonstrate a novel role for apc in pyruvate metabolism through regulation of mpc1 that drives normal intestinal differentiation and support the broader idea that metabolic changes can dictate cell fate programs. Citation Format: Imelda T. Sandoval, Richard Glenn C. Delacruz, Braden N. Miller, Christeena Satterfield, Kristophor Olson, Shauna Hill, Holly Van Remmen, Jared Rutter, David A. Jones. A metabolic switch controls cell fate decision-making in intestinal differentiation downstream of the tumor suppressor adenomatous polyposis coli (apc). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 229.

Cynomolgus monkeys, as animal models of scrub typhus, are typically infected with Orientia tsutsugamushi by intradermal inoculation. However, the clinical and histological features at the O. tsutsugamushi inoculation sites, akin to "eschars" at chigger inoculation sites in humans, have not been fully characterized. We intradermally inoculated one medial thigh of six cynomolgus monkeys with semi-purified O. tsutsugamushi (Karp). Within 7 days, two animals developed scrub typhus-like eschars and four had dusky plaques, accompanied by inguinal lymphadenopathy. Biopsies of eschars and an enlarged regional lymph node resembled human disease and stained positively for O. tsutsugamushi by Giemsa, anti-Karp fluorescent antibody, or streptavidin alkaline phosphatase. O. tsutsugamushi-specific IgM and IgG antibody levels measured in both of two monkeys rose steadily after infection. This pilot study shows that cynomolgus intradermally inoculated with O. tsutsugamushi replicate the localized cutaneous pathogenesis of human scrub typhus infections, strengthening the value of this animal model.

The UCLA National Center for Research on Evaluation, Standards, and Student Testing (CRESST) designed an authoring system to support the design of problem-solving assessments. To support assessment design, the system design includes a constraint network, in which nodes are variables that can assume a range of values and the topology specifies how the variables and values are related, to describe the permissible relations and states among assessment and problem-solving variables. This report describes the design for the authoring system and discusses next steps in developing and conducting research on the system.

We developed four types of GaAs PCDs: bremsstrahlung (two types), soft X-ray, and gamma for pulsed reactors. GaAs PCDs are advantageous in applications that require fast response, high dose rate, and/or neutron insensitivity. Desired detector sensitivity and response were obtained through modification of carrier lifetime by neutron irradiation, size of the sensing element, and orientation of the sensing element. The GaAs PCDs were used to characterize pulsed radiation sources in over 10 different tests and the measured data from these tests are presented. GaAs PCDs (E7 - E11 rad(Si)/s) for use with pulsed bremsstrahlung sources were tested at DTRA Radiation sources, Linac (Idaho Accelerator Center), Linac (White Sands), hybrid radiation source (NRL), GAMBLE II (NRL), and HERMES III (Sandia). GaAs PCDs for gamma characterization in pulsed reactors (E6 - E7 rad(Si)/s) were calibrated with a Linac (White Sands), checked for high dose rate (E12 rad(Si)/s) response at HERMES III, checked for low dose rate response (5-500 rad(Si)/s) and stability at a Co-60 source (Sandia), and checked for stability, sensitivity, and residual radioactivity at the ACCR reactor (Sandia). Soft X-ray (1 to 15 keV) GaAs PCDs (E2-E6 Watts/cm2) were used to measure the argon plasma free-bound continuum temperature from a plasma radiation source (PRS) and compared with the plasma temperature obtained from line ratio measurements.

The Office of Naval Research (ONR) has funded the UCLA National Center for Research on Evaluation, Standards, and Student Testing (CRESST) and its subcontractor, the University of Southern California Behavioral Technology Laboratories (BTL), to conduct research on the integration of assessment models and tools with instructional simulation authoring and delivery tools designed to support Navy and Marine Corps distance learning (DL). An assessment model is a formal specification of tasks and measures producing data that can be used as evidence to support inferences about what the learner knows. For this project the assessment models and tools provide precision measurement, analysis, and diagnosis of learner performance guiding selection and delivery of remedial training through the instructional simulations. ONR is also funding CHI Systems, Inc. to conduct field-based qualitative research on Navy and Marine Corps DL implementations to develop practical guidelines and procedures to support effective DL employment in the Navy and Marine Corps.

We report the discovery and initial characterization of the T-superfamily of conotoxins. Eight different T-superfamily peptides from five Conus species were identified; they share a consensus signal sequence, and a conserved arrangement of cysteine residues (- -CC- -CC-). T-superfamily peptides were found expressed in venom ducts of all major feeding types of Conus; the results suggest that the T-superfamily will be a large and diverse group of peptides, widely distributed in the 500 different Conus species. These peptides are likely to be functionally diverse; although the peptides are small (11-17 amino acids), their sequences are strikingly divergent, with different peptides of the superfamily exhibiting varying extents of post-translational modification. Of the three peptides tested for in vivo biological activity, only one was active on mice but all three had effects on fish. The peptides that have been extensively characterized are as follows: p5a, GCCPKQMRCCTL*; tx5a, gammaCCgammaDGW(+)CCT( section sign)AAO; and au5a, FCCPFIRYCCW (where gamma = gamma-carboxyglutamate, W(+) = bromotryptophan, O = hydroxyproline, T( section sign) = glycosylated threonine, and * = COOH-terminal amidation). We also demonstrate that the precursor of tx5a contains a functional gamma-carboxylation recognition signal in the -1 to -20 propeptide region, consistent with the presence of gamma-carboxyglutamate residues in this peptide.

The biotin/streptavidin ligand/receptor pair is used extensively in biotechnology. However, less is known about HABA (2-(4-hydroxyphenylazo)benzoic acid), which binds to streptavidin with a bond energy and dissociation constant that more closely mimics antibody/antigen interactions. In this work we demonstrate some of HABA’s useful properties that may make it a good substitute for biotin in a broad range of biochemical research. Specifically, we investigate its ease of conjugation to an anchoring pegylated lipid, characterization with MALDI, NMR, and visible-wavelength spectroscopies, and incorporation into lipid vesicles.