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David R Powell,1 Jean-Pierre Revelli,1 Deon D Doree,1 Christopher M DaCosta,1 Urvi Desai,1 Melanie K Shadoan,1 Lawrence Rodriguez,2 Michael Mullens,2 Qi M Yang,1 Zhi-Ming Ding,1 Laura L Kirkpatrick,3 Peter Vogel,1 Brian Zambrowicz,1– 3 Arthur T Sands,1– 3 Kenneth A Platt,3 Gwenn M Hansen,3 Robert Brommage1 1Department of Pharmaceutical Biology, Lexicon Pharmaceuticals, Inc, The Woodlands, TX, USA; 2Department of Information Technology, Lexicon Pharmaceuticals, Inc, The Woodlands, Tx, USA; 3Department of Molecular Biology, Lexicon Pharmaceuticals, Inc, The Woodlands, Tx, USACorrespondence: David R PowellDepartment of Pharmaceutical Biology, Lexicon Pharmaceuticals, Inc, 2445 Technology Forest Boulevard, The Woodlands, Tx, 77381, USATel +1 713 249 3972Fax +1 281 863 8115Email dpowell@lexpharma.comPurpose: Obesity is a major public health problem. Understanding which genes contribute to obesity may better predict individual risk and allow development of new therapies. Because obesity of a mouse gene knockout (KO) line predicts an association of the orthologous human gene with obesity, we reviewed data from the Lexicon Genome5000TM high throughput phenotypic screen (HTS) of mouse gene KOs to identify KO lines with high body fat.Materials and Methods: KO lines were generated using homologous recombination or gene trapping technologies. HTS body composition analyses were performed on adult wild-type and homozygous KO littermate mice from 3758 druggable mouse genes having a human ortholog. Body composition was measured by either DXA or QMR on chow-fed cohorts from all 3758 KO lines and was measured by QMR on independent high fat diet-fed cohorts from 2488 of these KO lines. Where possible, comparisons were made to HTS data from the International Mouse Phenotyping Consortium (IMPC).Results: Body fat data are presented for 75 KO lines. Of 46 KO lines where independent external published and/or IMPC KO lines are reported as obese, 43 had increased body fat. For the remaining 29 novel high body fat KO lines, Ksr2 and G2e3 are supported by data from additional independent KO cohorts, 6 (Asnsd1, Srpk2, Dpp8, Cxxc4, Tenm3 and Kiss1) are supported by data from additional internal cohorts, and the remaining 21 including Tle4, Ak5, Ntm, Tusc3, Ankk1, Mfap3l, Prok2 and Prokr2 were studied with HTS cohorts only.Conclusion: These data support the finding of high body fat in 43 independent external published and/or IMPC KO lines. A novel obese phenotype was identified in 29 additional KO lines, with 27 still lacking the external confirmation now provided for Ksr2 and G2e3 KO mice. Undoubtedly, many mammalian obesity genes remain to be identified and characterized.Keywords: obesity, druggable, homologous recombination, gene trapping

David R Powell, Deon D Doree, Christopher M DaCosta, Kenneth A Platt, Gwenn M Hansen, Isaac van Sligtenhorst, Zhi-Ming Ding, Jean-Pierre Revelli, Robert Brommage Lexicon Pharmaceuticals Inc, The Woodlands, TX, 77381, USACorrespondence: David R PowellLexicon Pharmaceuticals Inc., 8800 Technology Forest Place, The Woodlands, TX 77381, USATel +1 281 863 3060Fax +1 281 863 8115Email dpowell@lexpharma.comPurpose: In humans, single nucleotide polymorphisms (SNPs) near the adjacent protein kinase D1 (PRKD1) and G2/M-phase-specific E3 ubiquitin protein ligase (G2E3) genes on chromosome 14 are associated with obesity. To date, no published evidence links inactivation of either gene to changes in body fat. These two genes are also adjacent on mouse chromosome 12. Because obesity genes are highly conserved between humans and mice, we analyzed body fat in adult G2e3 and Prkd1 knockout (KO) mice to determine whether inactivating either gene leads to obesity in mice and, by inference, probably in humans.Methods: The G2e3 and Prkd1 KO lines were generated by gene trapping and by homologous recombination methodologies, respectively. Body fat was measured by DEXA in adult mice fed chow from weaning and by QMR in a separate cohort of mice fed high-fat diet (HFD) from weaning. Glucose homeostasis was evaluated with oral glucose tolerance tests (OGTTs) performed on adult mice fed HFD from weaning.Results: Body fat was increased in multiple cohorts of G2e3 KO mice relative to their wild-type (WT) littermates. When data from all G2e3 KO (n=32) and WT (n=31) mice were compared, KO mice showed increases of 11% in body weight (P< 0.01), 65% in body fat (P< 0.001), 48% in % body fat (P< 0.001), and an insignificant 3% decrease in lean body mass. G2e3 KO mice were also glucose intolerant during an OGTT (P< 0.05). In contrast, Prkd1 KO and WT mice had comparable body fat levels and glucose tolerance.Conclusion: Significant obesity and glucose intolerance were observed in G2e3, but not Prkd1, KO mice. The conservation of obesity genes between mice and humans strongly suggests that the obesity-associated SNPs located near the human G2E3 and PRKD1 genes are linked to variants that decrease the amount of functional human G2E3.Keywords: glucose tolerance, gene trap, homologous recombination, Prkd1, PRKD1, SNP

PURPOSE: To document histologic and immunohistochemical changes in the anterior digastric muscle during distraction osteogenesis (DO). MATERIALS AND METHODS: Nineteen Yucatan minipigs with mixed dentition were used for these experiments. Group A (n = 16) underwent unilateral mandibular distraction at a rate of 1 mm/day (no latency) for 12 days. Animals were killed at mid-DO (n = 5), end-DO (n = 5), mid-fixation (n = 4), and end-fixation (n = 2). Group B (n = 2) underwent acute 12-mm advancement, and group C (n = 1) dissection and osteotomy. Animals from groups B and C were killed at the end-DO time point. Digastric muscles from treatment and contralateral sides of all animals were harvested and embedded in paraffin. Specimens were stained with hematoxylin/eosin or immunohistochemically for proliferating cell nuclear antigen (PCNA; total cell proliferation), paired Box-7 gene protein (Pax7; satellite cells), or myogenic differentiation 1 protein (MyoD; differentiating myoblasts). Descriptive and bivariate statistics were computed to compare groups (P <= .05 statistically significant). RESULTS: All animals survived the operation and observation period; there were no device failures. Two animals (1 at mid-DO, 1 at mid-fixation) were eliminated from the study because of postoperative infection. There was minimal digastric inflammation, fibrosis, and muscle fiber size variability during active DO. Immunohistochemical analysis showed statistically significant increases in PCNA (cellular proliferation), Pax7 (satellite cells), and MyoD (differentiating myoblasts) positive nuclei in digastrics at mid-DO and end-DO. CONCLUSIONS: Results of this study indicate that there are minimal pathologic changes but significant increases in PCNA, Pax7, and MyoD positive nuclei during active distraction. This supports the hypothesis that the digastric muscle response to DO consists of proliferation and hypertrophy. [ABSTRACT FROM AUTHOR]

The aim of this study was to describe the extent and application of patient diaries in Danish intensive care units (ICUs) in 2006. Following critical illness, many patients experience disturbed and disconnected memories. Patient diaries in the ICU have been introduced locally by nurses in the Scandinavian countries and the UK as a means to improve cognitive recovery and prevent psychological trauma following critical illness. Descriptive design, using qualitative key informant telephone interviews (n= 19) was used as the source of data. A semi-structured interview guide was used and field notes from the interviews were mailed to the informants for verification and additional information. Nineteen out of 48 Danish ICUs use patient diaries. Patient diaries are mainly used for sedated, ventilated patients during critical illness. The purpose of diaries was mainly to assist memory due to post-ICU amnesia. There was no systematic follow-up after the ICU stay. The study showed a number of legal and ethical issues that still need to be resolved. Patient diaries are ambiguous documents that are neither the property of the hospital nor the patient. The diaries have been implemented at Danish ICUs without a plan for the assessment of their effect, thus bypassing established control mechanisms that evaluate the usefulness of an intervention, such as Health Technology Assessment or nursing research. [ABSTRACT FROM AUTHOR]

AIMS: This paper presents a survey of evidence-based practice among cardiac nurses exploring nurses' attitudes towards evidence-based practice and the types of knowledge they employ in clinical practice. BACKGROUND: Research utilization and evidence-based practice are required at hospitals around the world, although the definition of evidence-based practice is still unclear. An ongoing debate exists about the sources of knowledge and the hierarchy of research evidence in clinical practice, and nursing research has been threatened by the dominance of randomized controlled trials. Evidence-based practice has been described as a new paradigm, which promotes patient-centred care by integrating external evidence and patient preferences. METHODS: The study was a cross-sectional survey with a descriptive and comparative design, using self-administered postal questionnaires. The questionnaires were sent to 33 head nurses and 51 bedside nurses representing one or two units in each cardiac department in Denmark (n = 28). The final response rate was 81%. The study was carried out in 2004. RESULTS: Respondents had a positive attitude towards evidence-based practice, although they relied upon personal clinical experience. Head nurses were statistically significantly more familiar with the concept of evidence-based practice than bedside nurses, and read scientific journals more frequently. Introductory courses to evidence-based practice are rare and seldom mandatory, and the data suggest that respondents lacked knowledge of the finer points of evidence-based practice and equated the concept with research utilization. CONCLUSIONS: Barriers to evidence-based practice are inadequate education, unfamiliarity with English, and low organizational position. Facilitators include the implementation of guidelines, provision of continuing education, and an increase in the accountability of bedside nurses. [ABSTRACT FROM AUTHOR]

Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients.

Background: Materials extracted from atherosclerotic arteries can disclose data about the molecular pathology of cardiovascular disease, but obtaining such samples is complex and requires invasive surgery. To overcome this barrier, this study investigated whether angioplasty balloons inflated during standard percutaneous coronary interventions retain proteins from treated (dilated) atherosclerotic lesions and whether proteomic analysis of this material could provide data on lesion protein profiles and distinguish between patients with stable and unstable coronary artery disease., Methods: Patients with ST-segment-elevation myocardial infarction and stable angina pectoris were subjected to routine percutaneous coronary interventions. All angioplasty balloons inflated in a coronary artery were collected. Proteins retained on the balloons were extracted and analyzed using shotgun proteomic analysis., Results: Proteomics identified and quantified 1365 unique proteins captured on percutaneous coronary intervention balloons. Control balloons inflated in the ascending aorta showed minimal nonspecific protein binding, indicating specificity to the luminal region of atherosclerotic lesions of the diseased artery wall. Clustering and principal component analyses showed that ST-segment-elevation myocardial infarction and stable angina pectoris subjects could be separated by variations in protein content and abundance. We identified 206 proteins as differentially abundant between ST-segment-elevation myocardial infarction and stable angina pectoris subjects. Pathway analysis indicated several enriched processes in the ST-segment-elevation myocardial infarction group involved in neutrophil-mediated immunity and platelet activation., Conclusions: Disease-related proteins from coronary artery lesions adhere to angioplasty balloons and constitute a source of material for proteomic analysis. This approach can identify proteins and processes occurring in unstable coronary atherosclerotic lesions and suggest novel therapeutic approaches.

The development of mouse models that replicate the genetic and pathological features of human disease is important in preclinical research because these types of models enable the completion of meaningful pharmacokinetic, safety, and efficacy studies. Numerous relevant mouse models of human disease have been discovered in high-throughput screening programs, but there are important specific phenotypes revealed by histopathology that are not reliably detected by any other physiological or behavioral screening tests. As part of comprehensive phenotypic analyses of over 4000 knockout (KO) mice, histopathology identified 12 lines of KO mice with lesions indicative of an autosomal recessive myopathy. This report includes a brief summary of histological and other findings in these 12 lines. Notably, the inverted screen test detected muscle weakness in only 4 of these 12 lines ( Scyl1 , Plpp7 , Chkb , and Asnsd1 ), all 4 of which have been previously recognized and published. In contrast, 6 of 8 KO lines showing negative or inconclusive findings on the inverted screen test ( Plppr2 , Pnpla7 , Tenm1 , Srpk3 , Sidt2 , Yif1b , Mrs2 , and Pnpla2 ) had not been previously identified as having myopathies. These findings support the need to include histopathology in phenotype screening protocols in order to identify novel genetic myopathies that are not clinically evident or not detected by the inverted screen test.

Introduction: We hypothesised that reduced shoulder function post stroke improves during constraint-induced movement therapy and that improvement in scapula upward rotation measured with three-dimensional kinematics is associated with improvements in clinical and patient reported outcomes., Methods: Thirty-seven patients were tested pre and post constraint-induced movement therapy and again at three-month follow-up. Kinematic outcome measures - with scapula upward rotation as the primary outcome - during tasks 5 (ReachLow) and 6 (ReachHigh) from the Wolf Motor Function Test were included together with clinical and patient reported outcomes. Changes in outcome measures were analysed with linear mixed models and logistic regression analysis., Findings: Scapula upward rotation was reduced from 16.2° pre intervention through 15.9° post intervention to 15.6° at three-month follow-up during ReachHigh. Statistically significant reductions of <2° were also found for shoulder flexion during ReachLow and trunk lateral flexion during ReachHigh. The clinical and patient reported outcomes showed improvements post constraint-induced movement therapy, and at follow-up, the outcomes resembled post values., Interpretation: The minimal improvements in selected 3D kinematic measures of upper extremity movements did not reflect any clinically meaningful changes. Therefore, the clinical and patient reported improvements could not be related to restitution of shoulder function., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Increased intensity of training in the subacute phase after acquired brain injury facilitates plasticity and enhances better function. Group training can be a motivating factor and an effective means of increasing intensity. Reports on patients' and health care professionals' experiences on increasing the amount of active practice through group training during in-patient rehabilitation after acquired brain injury have been limited., Methods: Two focus groups, patients and health care professionals, participated each in two interviews, before and after implementation of the Activity block, i.e., 2-hour daily intensive group training. The data from the interviews were analyzed from a phenomenological perspective., Results: Three categories emerged from the data analyzes (i) training intensity, (ii) motivation and meaningfulness, and (iii) expectations and concerns. Both groups experienced that the training after implementation of the Activity block had become more intense and that motivation was increased induced by the group setting. Also, both groups found self-management enhanced. Some challenges were also reported. Patients expressed concerns to finding a balance between rest and activity, while the health professionals mentioned practical challenges, i.e., planning the content of the day and finding their role in the Activity block., Conclusion: Activity block benefitted a heterogeneous group of patients with acquired brain injury and was perceived as an overall positive experience by patients and health personnel. Matching the training to the individuals' need for support, finding a balance between rest and activity and using tasks that support patients' motivation, appeared important., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Gunhild Mo Hansen et al.)

Aims: To investigate the association between measures of peripheral neuropathy (PN) and impaired left ventricular diastolic function, and the prognosis in patients with type 1 diabetes (T1DM) and no known cardiovascular disease (CVD), and to test the incremental prognostic value of including measures of PN and diastolic function to the established Steno T1 Risk Engine., Methods: Echocardiography and quantitative biothesiometry was performed to evaluate diastolic function and PN. The participants were categorized according to severity of diastolic function and PN. The study endpoint was combined cardiovascular (CV) events and all-cause death. Associations were analysed using multivariable regression models. The prognostic capability was assessed with Harrell's C-statistics and tested against the Steno T1 Risk Engine., Results: A total of 946 individuals (51.5% men) were included. The mean (SD) follow-up was 6 (1.3) years. The total number of CV events and all-cause death were 100. In the multi-adjusted analysis, both PN and impaired diastolic function were associated with increased risk of CV events and all-cause death: severe PN versus no PN: hazard ratio (HR) 2.23 (95% confidence interval [CI] 1.06-4.68; P = 0.035); severe diastolic impairment versus normal function: HR 2.27 (95% CI 1.16-4.44; P = 0.016). Measures of diastolic function improved prognostic capability when added to the Steno T1 Risk Engine: C-statistic 0.797 (95% CI 0.793-0.817) versus 0.785 (95% CI 0.744-0.825; P = 0.006)., Conclusion: Peripheral neuropathy and impaired diastolic function are associated with an increased risk of CV events and all-cause death in patients with T1DM. Measures of diastolic function improved prediction of prognosis by the Steno T1 Risk Engine., (© 2020 John Wiley & Sons Ltd.)

Pneumopericardium is a potential complication to frequent cannabis smoking due to barotrauma of the airways caused by forceful coughing and Valsalva's manoeuvre. This is a case report of a 27-year-old man, who had a daily consumption of cannabis and presented with abdominal pain and nausea consistent with cannabinoid hyperemesis syndrome. A CT scan revealed an asymptomatic isolated minor pneumopericardium. The patient was observed, and no intervention was needed. At an outpatient control ten days later the pneumopericardium had spontaneously resolved.

Background: Good midlife cardiorespiratory fitness (CRF) may reduce the risk of chronic obstructive pulmonary disease (COPD). Reverse causation may play a role if follow-up time is short. We examined the association between CRF and both incident COPD and COPD mortality in employed men with up to 46 years follow-up, which allowed us to account for reverse causality., Methods: Middle-aged men (n=4730) were recruited in 1970-1971. CRF was determined as VO 2 max by ergometer test. Categories of CRF (low, normal, high) were defined as ± 1 Z-score (± 1 SD) above or below the age-adjusted mean. Endpoints were identified through national registers and defined as incident COPD, and death from COPD. Multi-adjusted Cox models and restricted mean survival times (RMST) were performed., Results: Compared with low CRF, the estimated risk of incident COPD was 21% lower in participants with normal CRF (HR 0.79, 95% CI 0.63 to 0.99) and 31 % lower with high CRF (HR 0.69, 95% CI 0.52 to 0.91). Compared with low CRF, the risk of death from COPD was 35% lower in participants with normal CRF (HR 0.65, 95% CI 0.46 to 0.91) and 62% lower in participants with high CRF (HR 0.38, 95% CI 0.23 to 0.61). RMST showed a delay to incident COPD and death from COPD in the magnitude of 1.3-1.8 years in normal and high CRF vs low CRF. Test for reverse causation did not alter the results., Conclusion: In a population of healthy, middle-aged men, higher levels of CRF were associated with a lower long-term risk of incident COPD and death from COPD., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Blood flow restriction training (BFRT) has been proposed for elderly and clinical populations with weakness. Before being used in these populations it is important to understand the neurological effects of, and subject perceptions to, BFRT. Seventeen healthy subjects were recruited and performed 2 experimental sessions, BFRT and training without blood flow restriction (TR-only), on separate days. Four sets of concentric/eccentric dorsiflexion contractions against theraband resistance were performed. Surface electromyography of the tibialis anterior was recorded during exercise and for the electrophysiological measures. At baseline, immediately-post, 10-min-post and 20-min-post exercise, motor evoked potentials (MEPs) from single pulse transcranial magnetic stimulation (TMS), paired-pulse TMS with interstimulus intervals of 2-ms (SICI) and 15-ms (ICF), and the M-max amplitude were recorded in the resting TA. Following training, subjects provided a numerical rating of the levels of pain, discomfort, fatigue, focus and difficulty during training. Muscle activation was higher in the last 20 contractions during BFRT compared to TR. There was no difference (time × condition interaction) between BFRT and TR for single-pulse MEP, SICI, ICF or M-max amplitude. There was a significant main effect of timepoint for single-pulse MEP and M-max amplitudes with both significantly reduced for 20-min-post exercise. No reductions were observed for SICI and ICF amplitudes. Taken together, BFRT and TR-only were only different during exercise and both regimes induced similar significant reductions in M-Max and MEP-amplitude post-training. Due to the lack of changes in SICI and ICF, it is unlikely that changes occurred in cortical sites related to these pathways. The increased surface electromyography activity in the last 20 contractions, indicate that the training regimes are different and that BFRT possibly induces more fatigue than TR. As such, BFRT could be used as an adjunct to conventional training. However, as subjects perceived BFRT as more painful, difficult and uncomfortable than TR-only, people should be selected carefully to undertake BFRT.

Background: It is important to monitor progress during rehabilitation of stroke patients. To that end, clinical function tests may be supported by three-dimensional kinematic measures. The aim of this study was to evaluate the inter- and intra-rater reliability of three-dimensional kinematic measures of shoulder movements in stroke patients with reduced shoulder function., Methods: Seventeen patients were tested in three sessions by two trained raters. Three-dimensional motion capture was performed of the more affected upper extremity and the trunk. Measures of movements of the scapula and humerus related to the trunk, the trunk related to the laboratory, the forearm related to the humerus, and temporospatial measures were obtained during two reach tasks from the Wolf Motor Function Test, ReachLow and ReachHigh. Inter- and intra-rater reliability was quantified with intraclass correlation coefficients (ICC)., Findings: In general, range of movements of scapula, shoulder, trunk and elbow and movement time and reach length showed high inter-rater reliability (ICC ∞ 0.84-0.98) and intra-rater reliability (ICC ∞ 0.75-1.00), A minimum of five trials per task were required to achieve reliable ICC estimates., Interpretation: Selected three-dimensional kinematic measures can be used reliably to evaluate specific movements of the shoulder in stroke patients with reduced shoulder function., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The disability, mortality and costs caused by non-vertebral osteoporotic fractures are enormous. Existing osteoporosis therapies are highly effective at reducing vertebral but not non-vertebral fractures. Cortical bone is a major determinant of non-vertebral bone strength. To identify novel osteoporosis drug targets, we phenotyped cortical bone of 3 366 viable mouse strains with global knockouts of druggable genes. Cortical bone thickness was substantially elevated in Notum -/- mice. NOTUM is a secreted WNT lipase and we observed high NOTUM expression in cortical bone and osteoblasts but not osteoclasts. Three orally active small molecules and a neutralizing antibody inhibiting NOTUM lipase activity were developed. They increased cortical bone thickness and strength at multiple skeletal sites in both gonadal intact and ovariectomized rodents by stimulating endocortical bone formation. Thus, inhibition of NOTUM activity is a potential novel anabolic therapy for strengthening cortical bone and preventing non-vertebral fractures., Competing Interests: The authors declare no competing interests.

Background Several predictors have been associated with upper extremity (UE) recovery after stroke, but characteristics that predict shoulder function after constraint-induced movement therapy (CIMT) have not yet been identified. Objectives To identify predictors associated with satisfactory shoulder function in patients with reduced shoulder function at admission to CIMT. Methods One hundred and seventy five patients were treated using CIMT while in a specialized inpatient hospital. Satisfactory shoulder function was defined according to the functional ability scale of the Wolf Motor Function test. Predictors of satisfactory shoulder function after CIMT were identified using multivariable logistic regression. Results Better distal arm function and good proximal shoulder function on admission to CIMT were strong predictors of satisfactory shoulder function, while age and time of admission to CIMT since stroke were not. Seventeen percent of all CIMT-participants with reduced shoulder function pre-CIMT reached a level of satisfactory shoulder function after CIMT. Discussion A substantial part of patients with reduced shoulder function reached a level of satisfactory shoulder function after CIMT. Intensive CIMT training, comprising tasks that require both distal and proximal UE function, may increase shoulder function in patients with a potential functional reserve.

Background: In recent years, virtual reality (VR) therapy systems for upper limb training after stroke have been increasingly used in clinical practice. Therapy systems employing VR technology can enhance the intensity of training and can also boost patients' motivation by adding a playful element to therapy. However, reports on user experiences are still scarce., Methods: A qualitative investigation of patients' and therapists' perspectives on VR upper limb training. Semistructured face-to-face interviews were conducted with six patients in the final week of the VR intervention. Therapists participated in two focus group interviews after the completion of the intervention. The interviews were analyzed from a phenomenological perspective emphasizing the participants' perceptions and interpretations., Results: Five key themes were identified from the patients' perspectives: (i) motivational factors, (ii) engagement, (iii) perceived improvements, (iv) individualization, and (v) device malfunction. The health professionals described the same themes as the patients but less positively, emphasizing negative technical challenges., Conclusion: Patients and therapists mainly valued the intensive and motivational character of VR training. The playful nature of the training appeared to have a significant influence on the patients' moods and engagement and seemed to promote a "gung-ho" spirit, so they felt that they could perform more repetitions.

The β 2 -adrenergic receptor (β 2 AR) has been a model system for understanding regulatory mechanisms of G-protein-coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known β-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human β 2 AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2 S )-3-((( S )-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the β 2 AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the β 2 AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the β 2 AR. In cell-signaling studies, 15 inhibits cAMP production through the β 2 AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits β-arrestin recruitment to the activated β 2 AR. This study presents an allosteric small-molecule ligand for the β 2 AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects.

Chronic infection is associated with an increased risk of atherothrombotic disease and direct bacterial infection of arteries has been suggested to contribute to the development of unstable atherosclerotic plaques. In this study, we examined coronary thrombi obtained in vivo from patients with ST-segment elevation myocardial infarction (STEMI) for the presence of bacterial DNA and bacteria. Aspirated coronary thrombi from 22 patients with STEMI were collected during primary percutaneous coronary intervention and arterial blood control samples were drawn from radial or femoral artery sheaths. Analyses were performed using 16S polymerase chain reaction and with next-generation sequencing to determine bacterial taxonomic classification. In selected thrombi with the highest relative abundance of Pseudomonas aeruginosa DNA, peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) with universal and species specific probes was performed to visualize bacteria within thrombi. From the taxonomic analysis we identified a total of 55 different bacterial species. DNA from Pseudomonas aeruginosa represented the only species that was significantly associated with either thrombi or blood and was >30 times more abundant in thrombi than in arterial blood (p<0.0001). Whole and intact bacteria present as biofilm microcolonies were detected in selected thrombi using universal and P. aeruginosa-specific PNA-FISH probes. P. aeruginosa and vascular biofilm infection in culprit lesions may play a role in STEMI, but causal relationships remain to be determined., Competing Interests: The authors have declared that no competing interests exist.

Periodontitis and atherosclerosis are highly prevalent chronic inflammatory diseases, and it has been suggested that periodontitis is an independent risk factor of cardiovascular disease (CVD) and that a causal link may exist between the 2 diseases. Using Danish national registers, we identified a nationwide cohort of 17,691 patients who received a hospital diagnosis of periodontitis within a 15-year period and matched them with 83,003 controls from the general population. We performed Poisson regression analysis to determine crude and adjusted incidence rate ratios of myocardial infarction, ischemic stroke, cardiovascular death, major adverse cardiovascular events, and all-cause mortality. The results showed that patients with periodontitis were at higher risk of all examined end points. The findings remained significant after adjustment for increased baseline co-morbidity in periodontitis patients compared with controls, for example, with adjusted incidence rate ratio 2.02 (95% CI 1.87 to 2.18) for cardiovascular death and 2.70 (95% CI 2.60 to 2.81) for all-cause mortality. Patients with a hospital diagnosis of periodontitis have a high burden of co-morbidity and an increased risk of CVD and all-cause mortality. In conclusion, our results support that periodontitis may be an independent risk factor for CVD., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Secreted WNT proteins control cell differentiation and proliferation in many tissues, and NOTUM is a secreted enzyme that modulates WNT morphogens by removing a palmitoleoylate moiety that is essential for their activity. To better understand the role this enzyme in development, the authors produced NOTUM-deficient mice by targeted insertional disruption of the Notum gene. The authors discovered a critical role for NOTUM in dentin morphogenesis suggesting that increased WNT activity can disrupt odontoblast differentiation and orientation in both incisor and molar teeth. Although molars in Notum(-/-) mice had normal-shaped crowns and normal mantle dentin, the defective crown dentin resulted in enamel prone to fracture during mastication and made teeth more susceptible to endodontal inflammation and necrosis. The dentin dysplasia and short roots contributed to tooth hypermobility and to the spread of periodontal inflammation, which often progressed to periapical abscess formation. The additional incidental finding of renal agenesis in some Notum (-/-) mice indicated that NOTUM also has a role in kidney development, with undiagnosed bilateral renal agenesis most likely responsible for the observed decreased perinatal viability of Notum(-/-) mice. The findings support a significant role for NOTUM in modulating WNT signaling pathways that have pleiotropic effects on tooth and kidney development., (© The Author(s) 2016.)

Periodontitis is a chronic, bacterially-induced inflammatory disease of the tooth-supporting tissues, which may result in transient bacteremia and a systemic inflammatory response. Periodontitis is associated with coronary artery disease independently of established cardiovascular risk factors, and translocation of bacteria from the oral cavity to the coronary arteries may play a role in the development of coronary artery disease. Very few studies have used angioplasty balloons for in vivo sampling from diseased coronary arteries, and with varying results. Therefore, the aim of this study was to assess if bacterial DNA from primarily oral bacteria could be detected on coronary angioplasty balloons by use of an optimized sampling process combined with an internally validated sensitive polymerase chain reaction (PCR) assay. Coronary angioplasty balloons and control samples from a total of 45 unselected patients with stable angina, unstable angina/non-ST elevation myocardial infarction, and ST-elevation myocardial infarction (n = 15 in each group) were collected and analyzed using a PCR assay with high sensitivity and specificity for 16S rRNA genes of the oral microbiome. Despite elimination of extraction and purification steps, and demonstration of sensitivity levels of 25-125 colony forming units (CFU), we did not detect bacterial DNA from any of the coronary angioplasty balloons. A subsequent questionnaire indicated that the prevalence of periodontitis in the study cohort was at least 39.5%. Although coronary angioplasty balloons are unlikely to be useful for detection of bacteria with current PCR techniques in unselected patients with coronary artery disease, more studies are warranted to determine the extent to which bacteria contribute to atherosclerosis and its clinical manifestations and whether the presence of bacteria in the arteries is a transient phenomenon.

Background: The Global Burden of Disease Study 2013 (GBD 2013) aims to bring together all available epidemiological data using a coherent measurement framework, standardised estimation methods, and transparent data sources to enable comparisons of health loss over time and across causes, age-sex groups, and countries. The GBD can be used to generate summary measures such as disability-adjusted life-years (DALYs) and healthy life expectancy (HALE) that make possible comparative assessments of broad epidemiological patterns across countries and time. These summary measures can also be used to quantify the component of variation in epidemiology that is related to sociodemographic development., Methods: We used the published GBD 2013 data for age-specific mortality, years of life lost due to premature mortality (YLLs), and years lived with disability (YLDs) to calculate DALYs and HALE for 1990, 1995, 2000, 2005, 2010, and 2013 for 188 countries. We calculated HALE using the Sullivan method; 95% uncertainty intervals (UIs) represent uncertainty in age-specific death rates and YLDs per person for each country, age, sex, and year. We estimated DALYs for 306 causes for each country as the sum of YLLs and YLDs; 95% UIs represent uncertainty in YLL and YLD rates. We quantified patterns of the epidemiological transition with a composite indicator of sociodemographic status, which we constructed from income per person, average years of schooling after age 15 years, and the total fertility rate and mean age of the population. We applied hierarchical regression to DALY rates by cause across countries to decompose variance related to the sociodemographic status variable, country, and time., Findings: Worldwide, from 1990 to 2013, life expectancy at birth rose by 6·2 years (95% UI 5·6-6·6), from 65·3 years (65·0-65·6) in 1990 to 71·5 years (71·0-71·9) in 2013, HALE at birth rose by 5·4 years (4·9-5·8), from 56·9 years (54·5-59·1) to 62·3 years (59·7-64·8), total DALYs fell by 3·6% (0·3-7·4), and age-standardised DALY rates per 100 000 people fell by 26·7% (24·6-29·1). For communicable, maternal, neonatal, and nutritional disorders, global DALY numbers, crude rates, and age-standardised rates have all declined between 1990 and 2013, whereas for non-communicable diseases, global DALYs have been increasing, DALY rates have remained nearly constant, and age-standardised DALY rates declined during the same period. From 2005 to 2013, the number of DALYs increased for most specific non-communicable diseases, including cardiovascular diseases and neoplasms, in addition to dengue, food-borne trematodes, and leishmaniasis; DALYs decreased for nearly all other causes. By 2013, the five leading causes of DALYs were ischaemic heart disease, lower respiratory infections, cerebrovascular disease, low back and neck pain, and road injuries. Sociodemographic status explained more than 50% of the variance between countries and over time for diarrhoea, lower respiratory infections, and other common infectious diseases; maternal disorders; neonatal disorders; nutritional deficiencies; other communicable, maternal, neonatal, and nutritional diseases; musculoskeletal disorders; and other non-communicable diseases. However, sociodemographic status explained less than 10% of the variance in DALY rates for cardiovascular diseases; chronic respiratory diseases; cirrhosis; diabetes, urogenital, blood, and endocrine diseases; unintentional injuries; and self-harm and interpersonal violence. Predictably, increased sociodemographic status was associated with a shift in burden from YLLs to YLDs, driven by declines in YLLs and increases in YLDs from musculoskeletal disorders, neurological disorders, and mental and substance use disorders. In most country-specific estimates, the increase in life expectancy was greater than that in HALE. Leading causes of DALYs are highly variable across countries., Interpretation: Global health is improving. Population growth and ageing have driven up numbers of DALYs, but crude rates have remained relatively constant, showing that progress in health does not mean fewer demands on health systems. The notion of an epidemiological transition--in which increasing sociodemographic status brings structured change in disease burden--is useful, but there is tremendous variation in burden of disease that is not associated with sociodemographic status. This further underscores the need for country-specific assessments of DALYs and HALE to appropriately inform health policy decisions and attendant actions., Funding: Bill & Melinda Gates Foundation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Through several observational and mechanistic studies, microbial infection is known to promote cardiovascular disease. Direct infection of the vessel wall, along with the cardiovascular risk factors, is hypothesized to play a key role in the atherogenesis by promoting an inflammatory response leading to endothelial dysfunction and generating a proatherogenic and prothrombotic environment ultimately leading to clinical manifestations of cardiovascular disease, e.g., acute myocardial infarction or stroke. There are many reports of microbial DNA isolation and even a few studies of viable microbes isolated from human atherosclerotic vessels. However, high-resolution investigation of microbial infectious agents from human vessels that may contribute to atherosclerosis is very limited. In spite of the progress in recent sequencing technologies, analyzing host-associated metagenomes remain a challenge., Results: To investigate microbiome diversity within human atherosclerotic tissue samples, we employed high-throughput metagenomic analysis on: (1) atherosclerotic plaques obtained from a group of patients who underwent endarterectomy due to recent transient cerebral ischemia or stroke. (2) Presumed stabile atherosclerotic plaques obtained from autopsy from a control group of patients who all died from causes not related to cardiovascular disease. Our data provides evidence that suggest a wide range of microbial agents in atherosclerotic plaques, and an intriguing new observation that shows these microbiota displayed differences between symptomatic and asymptomatic plaques as judged from the taxonomic profiles in these two groups of patients. Additionally, functional annotations reveal significant differences in basic metabolic and disease pathway signatures between these groups., Conclusions: We demonstrate the feasibility of novel high-resolution techniques aimed at identification and characterization of microbial genomes in human atherosclerotic tissue samples. Our analysis suggests that distinct groups of microbial agents might play different roles during the development of atherosclerotic plaques. These findings may serve as a reference point for future studies in this area of research.

After creating >4,650 knockouts (KOs) of independent mouse genes, we screened them by high-throughput phenotyping and found that cannabinoid receptor 1 (Cnr1) KO mice had the same lean phenotype published by others. We asked if our KOs of DAG lipase α or β (Dagla or Daglb), which catalyze biosynthesis of the endocannabinoid (EC) 2-arachidonoylglycerol (2-AG), or Napepld, which catalyzes biosynthesis of the EC anandamide, shared the lean phenotype of Cnr1 KO mice. We found that Dagla KO mice, but not Daglb or Napepld KO mice, were among the leanest of 3651 chow-fed KO lines screened. In confirmatory studies, chow- or high fat diet-fed Dagla and Cnr1 KO mice were leaner than wild-type (WT) littermates; when data from multiple cohorts of adult mice were combined, body fat was 47 and 45% lower in Dagla and Cnr1 KO mice, respectively, relative to WT values. By contrast, neither Daglb nor Napepld KO mice were lean. Weanling Dagla KO mice ate less than WT mice and had body weight (BW) similar to pair-fed WT mice, and adult Dagla KO mice had normal activity and VO2 levels, similar to Cnr1 KO mice. Our Dagla and Cnr1 KO mice also had low fasting insulin, triglyceride, and total cholesterol levels, and after glucose challenge had normal glucose but very low insulin levels. Dagla and Cnr1 KO mice also showed similar responses to a battery of behavioral tests. These data suggest: (1) the lean phenotype of young Dagla and Cnr1 KO mice is mainly due to hypophagia; (2) in pathways where ECs signal through Cnr1 to regulate food intake and other metabolic and behavioral phenotypes observed in Cnr1 KO mice, Dagla alone provides the 2-AG that serves as the EC signal; and (3) small molecule Dagla inhibitors with a pharmacokinetic profile similar to that of Cnr1 inverse agonists are likely to mirror the ability of these Cnr1 inverse agonists to lower BW and improve glycemic control in obese patients with type 2 diabetes, but may also induce undesirable neuropsychiatric side-effects.

Objectives: We estimated the prevalence of any drinking and binge drinking from 2002 to 2012 and heavy drinking from 2005 to 2012 in every US county., Methods: We applied small area models to Behavioral Risk Factor Surveillance System data. These models incorporated spatial and temporal smoothing and explicitly accounted for methodological changes to the Behavioral Risk Factor Surveillance System during this period., Results: We found large differences between counties in all measures of alcohol use: in 2012, any drinking prevalence ranged from 11.0% to 78.7%, heavy drinking prevalence ranged from 2.4% to 22.4%, and binge drinking prevalence ranged from 5.9% to 36.0%. Moreover, there was wide variation in the proportion of all drinkers who engaged in heavy or binge drinking. Heavy and binge drinking prevalence increased in most counties between 2005 and 2012, but the magnitude of change varied considerably., Conclusions: There are large differences within the United States in levels and recent trends in alcohol use. These estimates should be used as an aid in designing and implementing targeted interventions and to monitor progress toward reducing the burden of excessive alcohol use.

Mice deficient in TMEM218 (Tmem218(-/-) ) were generated as part of an effort to identify and validate pharmaceutically tractable targets for drug development through large-scale phenotypic screening of knockout mice. Routine diagnostics, expression analysis, histopathology, and electroretinogram analyses completed on Tmem218(-/-) mice identified a previously unknown role for TMEM218 in the development and function of the kidney and eye. The major observed phenotypes in Tmem218(-/-) mice were progressive cystic kidney disease and retinal degeneration. The renal lesions were characterized by diffuse renal cyst development with tubulointerstitial nephropathy and disruption of tubular basement membranes in essentially normal-sized kidneys. The retinal lesions were characterized by slow-onset loss of photoreceptors, which resulted in reduced electroretinogram responses. These renal and retinal lesions are most similar to those associated with nephronophthisis (NPHP) and retinitis pigmentosa in humans. At least 10% of NPHP cases present with extrarenal conditions, which most often include retinal degeneration. Senior-Løken syndrome is characterized by the concurrent development of autosomal recessive NPHP and retinitis pigmentosa. Since mutations in the known NPHP genes collectively account for only about 30% of NPHP cases, it is possible that TMEM218 could be involved in the development of similar ciliopathies in humans. In reviewing all other reported mouse models of NPHP, we suggest that Tmem218(-/-) mice could provide a useful model for elucidating the pathogenesis of cilia-associated disease in both the kidney and the retina, as well as in developing and testing novel therapeutic strategies for Senior-Løken syndrome., (© The Author(s) 2014.)

Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening (HTS) data for 3 762 distinct global gene knockout (KO) mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type (WT) cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass: 23 previously reported genes (Calcr, Cebpb, Crtap, Dcstamp, Dkk1, Duoxa2, Enpp1, Fgf23, Kiss1/Kiss1r, Kl (Klotho), Lrp5, Mstn, Neo1, Npr2, Ostm1, Postn, Sfrp4, Slc30a5, Slc39a13, Sost, Sumf1, Src, Wnt10b), five novel genes extensively characterized (Cldn18, Fam20c, Lrrk1, Sgpl1, Wnt16), five novel genes with preliminary characterization (Agpat2, Rassf5, Slc10a7, Slc26a7, Slc30a10) and three novel undisclosed genes coding for potential osteoporosis drug targets.

Periodontitis is a prevalent chronic inflammatory disease induced by bacterial biofilm in the dental pocket resulting in destruction of the periodontal tissues. Periodontitis is associated with ischaemic heart disease and we here provide a summary of the current evidence linking these two disorders.

Background: Cigarette smoking is a leading risk factor for morbidity and premature mortality in the United States, yet information about smoking prevalence and trends is not routinely available below the state level, impeding local-level action., Methods: We used data on 4.7 million adults age 18 and older from the Behavioral Risk Factor Surveillance System (BRFSS) from 1996 to 2012. We derived cigarette smoking status from self-reported data in the BRFSS and applied validated small area estimation methods to generate estimates of current total cigarette smoking prevalence and current daily cigarette smoking prevalence for 3,127 counties and county equivalents annually from 1996 to 2012. We applied a novel method to correct for bias resulting from the exclusion of the wireless-only population in the BRFSS prior to 2011., Results: Total cigarette smoking prevalence varies dramatically between counties, even within states, ranging from 9.9% to 41.5% for males and from 5.8% to 40.8% for females in 2012. Counties in the South, particularly in Kentucky, Tennessee, and West Virginia, as well as those with large Native American populations, have the highest rates of total cigarette smoking, while counties in Utah and other Western states have the lowest. Overall, total cigarette smoking prevalence declined between 1996 and 2012 with a median decline across counties of 0.9% per year for males and 0.6% per year for females, and rates of decline for males and females in some counties exceeded 3% per year. Statistically significant declines were concentrated in a relatively small number of counties, however, and more counties saw statistically significant declines in male cigarette smoking prevalence (39.8% of counties) than in female cigarette smoking prevalence (16.2%). Rates of decline varied by income level: counties in the top quintile in terms of income experienced noticeably faster declines than those in the bottom quintile., Conclusions: County-level estimates of cigarette smoking prevalence provide a unique opportunity to assess where prevalence remains high and where progress has been slow. These estimates provide the data needed to better develop and implement strategies at a local and at a state level to further reduce the burden imposed by cigarette smoking.

Almost all mitochondrial proteins are encoded in the nuclear DNA and synthesized in the cytosol as pre-proteins. There is a protein translocase located in the mitochondrial outer membrane that transports mitochondrial pre-proteins into mitochondria. The central component of this translocase of the outer mitochondrial membrane (TOMM) complex is TOMM40, and TOMM5 is one of three small subunits associated with TOMM40. Translocase of outer mitochondrial membrane 5 homolog (Tomm5(-/-)) knockout mice demonstrated an unexpected lung-specific phenotype characterized by widespread intra-alveolar fibrosis. Although TOMM5-deficient mice tested normal in a very broad range of phenotyping assays, they displayed histopathological lesions in the lung that were consistent with those reported in humans with cryptogenic organizing pneumonia (COP), which is also known as bronchiolitis obliterans organizing pneumonia (BOOP). The lesions had a patchy distribution in the lung and were characterized by the presence of intraluminal fibrogenic buds consisting of fibroblasts and myofibroblasts embedded in a loose connective tissue matrix that occupied the lumina of alveoli and alveolar ducts, with preservation of underlying alveolar architecture. In addition to macrophages, which were numerous in affected and surrounding alveoli, eosinophils comprised the most common and widespread inflammatory cell. Taken together, the findings in Tomm5(-/-) mice provide yet another example of the value of histopathology as a baseline assay in high-throughput phenotyping systems.

The FAM20 family of secreted proteins consists of three members (FAM20A, FAM20B, and FAM20C) recently linked to developmental disorders suggesting roles for FAM20 proteins in modulating biomineralization processes. The authors report here findings in knockout mice having null mutations affecting each of the three FAM20 proteins. Both Fam20a and Fam20c null mice survived to adulthood and showed biomineralization defects. Fam20b (-/-) embryos showed severe stunting and increased mortality at E13.5, although early lethality precluded detailed investigations. Physiologic calcification or biomineralization of extracellular matrices is a normal process in the development and functioning of various tissues (eg, bones and teeth). The lesions that developed in teeth, bones, or blood vessels after functional deletion of either Fam20a or Fam20c support a significant role for their encoded proteins in modulating biomineralization processes. Severe amelogenesis imperfecta (AI) was present in both Fam20a and Fam20c null mice. In addition, Fam20a (-/-) mice developed disseminated calcifications of muscular arteries and intrapulmonary calcifications, similar to those of fetuin-A deficient mice, although they were normocalcemic and normophosphatemic, with normal dentin and bone. Fam20a gene expression was detected in ameloblasts, odontoblasts, and the parathyroid gland, with local and systemic effects suggesting both local and/or systemic effects for FAM20A. In contrast, Fam20c (-/-) mice lacked ectopic calcifications but were severely hypophosphatemic and developed notable lesions in both dentin and bone to accompany the AI. The bone and dentin lesions, plus the marked hypophosphatemia and elevated serum alkaline phosphatase and FGF23 levels, are indicative of autosomal recessive hypophosphatemic rickets/osteomalacia in Fam20c (-/-) mice.

This study characterized sequential molecular and cellular events in the porcine mandibular distraction osteogenesis (DO) wound. Nineteen Yucatan minipigs were divided into three treatment groups: Group A, unilateral mandibular distraction with 0 day latency, 1mm/day rate for 12 days, 24 days fixation (n=16); Group B, acute lengthening 12 mm (n=2); Group C, sham control (n=1). Group A was further divided by death date: mid-DO (n=5), end-DO (n=4), mid-fixation (n=5) and end-fixation (n=2). Groups B and C were killed on postoperative day 36, corresponding to end-fixation. Specimens were stained for proliferating cell nuclear antigen (PCNA) and bone morphogenetic protein-4 (BMP4). Cellular proliferation (PCNA) was assessed quantitatively and BMP4 staining was assessed on a semi-quantitative scale. Progenitor cell proliferation was greatest during mid-DO and decreased from end-DO through end-fixation. Proliferation in the acute lengthening group was elevated relative to sham control and comparable to end-DO. BMP4 staining intensity (localized to the periosteal cambium layer) was greatest during mid- and end-DO, decreased at mid-fixation and was undetectable at end-fixation. Progenitor cell proliferation and BMP4 expression are greatest during mid-DO and decrease progressively thereafter. At the time of death of the acute lengthening group, only increased cell proliferation was demonstrated., (Copyright © 2011 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

The lysosomal storage disorder alpha-mannosidosis is caused by deficiency of the enzyme lysosomal alpha-mannosidase (MAN2B1). In this study, 96 disease-associated sequence variants were identified in 130 unrelated alpha-mannosidosis patients from 30 countries. Eighty-three novel variants were detected, extending the mutation spectrum from 42 to 125. In total, 256 of the 260 mutant alleles (98.5%) were identified. Most of the variants were unique to each family, however, c.2248C>T (p.Arg750Trp) was detected in 50 patients from 16 countries, and accounted for 27.3% of the disease alleles. Haplotype analysis revealed that the c.2248T variant was present on four MAN2B1 haplotype backgrounds, where one major haplotype accounted for 95% of the alleles. The distribution of the c.2248T-associated haplotypes differed remarkably from those of the control populations, suggesting that c.2248C>T has occurred on a few ancestral haplotypes where the major haplotype subsequently has spread by founder effects. The disease-associated missense mutations were introduced into the human MAN2B1 cDNA, expressed in cell culture and assayed for MAN2B1 activity. The majority of the variants were inactive, however, ten showed MAN2B1 activity above background, and more detailed studies are necessary to further evaluate the pathogenicity of these variants., (© 2011 Wiley Periodicals, Inc.)

There is evidence that genetic factors play a role in the complex multifactorial pathogenesis of hydrocephalus. Identification of the genes involved in the development of this neurologic disorder in animal models may elucidate factors responsible for the excessive accumulation of cerebrospinal fluid in hydrocephalic humans. The authors report here a brief summary of findings from 12 lines of genetically engineered mice that presented with autosomal recessive congenital hydrocephalus. This study illustrates the value of knockout mice in identifying genetic factors involved in the development of congenital hydrocephalus. Findings suggest that dysfunctional motile cilia represent the underlying pathogenetic mechanism in 8 of the 12 lines (Ulk4, Nme5, Nme7, Kif27, Stk36, Dpcd, Ak7, and Ak8). The likely underlying cause in the remaining 4 lines (RIKEN 4930444A02, Celsr2, Mboat7, and transgenic FZD3) was not determined, but it is possible that some of these could also have ciliary defects. For example, the cerebellar malformations observed in RIKEN 4930444A02 knockout mice show similarities to a number of developmental disorders, such as Joubert, Meckel-Gruber, and Bardet-Biedl syndromes, which involve mutations in cilia-related genes. Even though the direct relevance of mouse models to hydrocephalus in humans remains uncertain, the high prevalence of familial patterns of inheritance for congenital hydrocephalus in humans suggests that identification of genes responsible for development of hydrocephalus in mice may lead to the identification of homologous modifier genes and susceptibility alleles in humans. Also, characterization of mouse models can enhance understanding of important cell signaling and developmental pathways involved in the pathogenesis of hydrocephalus.

In vitro studies have identified LIMK2 as a key downstream effector of Rho GTPase-induced changes in cytoskeletal organization. LIMK2 is phosphorylated and activated by Rho associated coiled-coil kinases (ROCKs) in response to a variety of growth factors. The biochemical targets of LIMK2 belong to a family of actin binding proteins that are potent modulators of actin assembly and disassembly. Although numerous studies have suggested that LIMK2 regulates cell morphology and motility, evidence supportive of these functions in vivo has remained elusive. In this study, a knockout mouse was created that abolished LIMK2 biochemical activity resulting in a profound inhibition of epithelial sheet migration during eyelid development. In the absence of LIMK2, nascent eyelid keratinocytes differentiate and acquire a pre-migratory phenotype but the leading cells fail to nucleate filamentous actin and remain immobile causing an eyes open at birth (EOB) phenotype. The failed nucleation of actin was associated with significant reductions in phosphorylated cofilin, a major LIMK2 biochemical substrate and potent modulator of actin dynamics. These results demonstrate that LIMK2 activity is required for keratinocyte migration in the developing eyelid.

An early lesion in many kidney diseases is damage to podocytes, which are critical components of the glomerular filtration barrier. A number of proteins are essential for podocyte filtration function, but the signaling events contributing to development of nephrotic syndrome are not well defined. Here we show that class II phosphoinositide 3-kinase C2α (PI3KC2α) is expressed in podocytes and plays a critical role in maintaining normal renal homeostasis. PI3KC2α-deficient mice developed chronic renal failure and exhibited a range of kidney lesions, including glomerular crescent formation and renal tubule defects in early disease, which progressed to diffuse mesangial sclerosis, with reduced podocytes, widespread effacement of foot processes, and modest proteinuria. These findings were associated with altered expression of nephrin, synaptopodin, WT-1, and desmin, indicating that PI3KC2α deficiency specifically impacts podocyte morphology and function. Deposition of glomerular IgA was observed in knockout mice; importantly, however, the development of severe glomerulonephropathy preceded IgA production, indicating that nephropathy was not directly IgA mediated. PI3KC2α deficiency did not affect immune responses, and bone marrow transplantation studies also indicated that the glomerulonephropathy was not the direct consequence of an immune-mediated disease. Thus, PI3KC2α is critical for maintenance of normal glomerular structure and function by supporting normal podocyte function.

Large collections of knockout organisms facilitate the elucidation of gene functions. Here we used retroviral insertion or homologous recombination to disrupt 472 genes encoding secreted and membrane proteins in mice, providing a resource for studying a large fraction of this important class of drug target. The knockout mice were subjected to a systematic phenotypic screen designed to uncover alterations in embryonic development, metabolism, the immune system, the nervous system and the cardiovascular system. The majority of knockout lines exhibited altered phenotypes in at least one of these therapeutic areas. To our knowledge, a comprehensive phenotypic assessment of a large number of mouse mutants generated by a gene-specific approach has not been described previously.

Situs inversus (SI) is a congenital condition characterized by left-right transposition of thoracic and visceral organs and associated vasculature. The usual asymmetrical positioning of organs is established early in development in a transient structure called the embryonic node. The 2-cilia hypothesis proposes that 2 kinds of primary cilia in the embryonic node determine left-right asymmetry: motile cilia that generate a leftward fluid flow, and immotile mechanosensory cilia that respond to the flow. Here, we describe 3 mouse SI models that provide support for the 2-cilia hypothesis. In addition to having SI, Dpcd/Poll(-/-) mice (for: deleted in a mouse model of primary ciliary dyskinesia) and Nme7(-/-) mice (for: nonmetastatic cells 7) had lesions consistent with deficient ciliary motility: Hydrocephalus, sinusitis, and male infertility developed in Dpcd/Poll(-/-) mice, whereas hydrocephalus and excessive nasal exudates were seen in Nme7(-/-) mice. In contrast, the absence of respiratory tract lesions, hydrocephalus, and male infertility in Pkd1l1(-/-) mice (for: polycystic kidney disease 1 like 1) suggested that dysfunction of motile cilia was not involved in the development of SI in this line. Moreover, the gene Pkd1l1 has considerable sequence similarity with Pkd1 (for: polycystic kidney disease 1), which encodes a protein (polycystin-1) that is essential for the mechanosensory function of immotile primary cilia in the kidney. The markedly reduced viability of Pkd1l1(-/-) mice is somewhat surprising given the absence of any detected abnormalities (other than SI) in surviving Pkd1l1(-/-) mice subjected to a comprehensive battery of phenotype-screening exams. However, the heart and great vessels of Pkd1l1(-/-) mice were not examined, and it is possible that the decreased viability of Pkd1l1(-/-) mice is due to undiagnosed cardiovascular defects associated with heterotaxy.

Background: S1PL is an aldehyde-lyase that irreversibly cleaves sphingosine 1-phosphate (S1P) in the terminal step of sphingolipid catabolism. Because S1P modulates a wide range of physiological processes, its concentration must be tightly regulated within both intracellular and extracellular environments., Methodology: In order to better understand the function of S1PL in this regulatory pathway, we assessed the in vivo effects of different levels of S1PL activity using knockout (KO) and humanized mouse models., Principal Findings: Our analysis showed that all S1PL-deficient genetic models in this study displayed lymphopenia, with sequestration of mature T cells in the thymus and lymph nodes. In addition to the lymphoid phenotypes, S1PL KO mice (S1PL(-/-)) also developed myeloid cell hyperplasia and significant lesions in the lung, heart, urinary tract, and bone, and had a markedly reduced life span. The humanized knock-in mice harboring one allele (S1PL(H/-)) or two alleles (S1PL(H/H)) of human S1PL expressed less than 10 and 20% of normal S1PL activity, respectively. This partial restoration of S1PL activity was sufficient to fully protect both humanized mouse lines from the lethal non-lymphoid lesions that developed in S1PL(-/-) mice, but failed to restore normal T-cell development and trafficking. Detailed analysis of T-cell compartments indicated that complete absence of S1PL affected both maturation/development and egress of mature T cells from the thymus, whereas low level S1PL activity affected T-cell egress more than differentiation., Significance: These findings demonstrate that lymphocyte trafficking is particularly sensitive to variations in S1PL activity and suggest that there is a window in which partial inhibition of S1PL could produce therapeutic levels of immunosuppression without causing clinically significant S1P-related lesions in non-lymphoid target organs.

We report the construction and analysis of a mouse gene trap mutant resource created in the C57BL/6N genetic background containing more than 350,000 sequence-tagged embryonic stem (ES) cell clones. We also demonstrate the ability of these ES cell clones to contribute to the germline and produce knockout mice. Each mutant clone is identified by a genomic sequence tag representing the exact insertion location, allowing accurate prediction of mutagenicity and enabling direct genotyping of mutant alleles. Mutations have been identified in more than 10,000 genes and show a bias toward the first intron. The trapped ES cell lines, which can be requested from the Texas A&M Institute for Genomic Medicine, are readily available to the scientific community.

We developed a high-throughput approach to knockout (KO) and phenotype mouse orthologs of the 5,000 potential drug targets in the human genome. As part of the phenotypic screen, dual-energy X-ray absorptiometry (DXA) technology estimates body-fat stores in eight KO and four wild-type (WT) littermate chow-fed mice from each line. Normalized % body fat (nBF) (mean KO % body fat/mean WT littermate % body fat) values from the first 2322 lines with viable KO mice at 14 weeks of age showed a normal distribution. We chose to determine how well this screen identifies body-fat phenotypes by selecting 13 of these 2322 KO lines to serve as benchmarks based on their published lean or obese phenotype on a chow diet. The nBF values for the eight benchmark KO lines with a lean phenotype were > or =1 s.d. below the mean for seven (perilipin, SCD1, CB1, MCH1R, PTP1B, GPAT1, PIP5K2B) but close to the mean for NPY Y4R. The nBF values for the five benchmark KO lines with an obese phenotype were >2 s.d. above the mean for four (MC4R, MC3R, BRS3, translin) but close to the mean for 5HT2cR. This screen also identifies novel body-fat phenotypes as exemplified by the obese kinase suppressor of ras 2 (KSR2) KO mice. These body-fat phenotypes were confirmed upon studying additional cohorts of mice for KSR2 and all 13 benchmark KO lines. This simple and cost-effective screen appears capable of identifying genes with a role in regulating mammalian body fat.

Beta-mannosidosis (OMIM # 248510) is an autosomal-recessive lysosomal storage disorder caused by deficiency of the lysosomal enzyme beta-mannosidase (MANBA, E.C. 3.2.1.25). The disorder has been reported in goat, cattle and man. The human disorder is rare and only 20 cases in 16 families have been reported. We have sequenced the exons and exon-intron borders in a European patient with infantile onset of beta-mannosidosis. The patient was compound heterozygous for a silent mutation (c.375A>G) in exon 3 causing alternative splicing, and a missense mutation (c.1513T>C, p.Ser505Pro) in exon 12. The alternative splicing event deleted four nucleotides from the transcript and was predicted to result in premature termination of translation. In order to evaluate the consequence of the missense mutation, we inserted the human beta-mannosidase gene into an expression vector, performed site-directed mutagenesis and expressed the normal and mutant enzyme in COS-7 cells. We also included the previously reported beta-mannosidosis-associated missense mutations c.544C>T (p.Arg182Trp) and c.1175G>A (p.Gly392Glu), which were found in patients presenting a milder phenotype. Cells transfected with the wild-type construct showed a 33-fold increase in beta-mannosidase activity compared to mock-transfected cells, whereas cells transfected with the mutant constructs showed no detectable increase in activity. We propose that the milder phenotype described in some beta-mannosidosis patients with missense mutations in the MANBA gene is not due to residual beta-mannosidase activity, but rather caused by epigenetic and/or environmental factors.

AKXD recombinant inbred (RI) strains develop a variety of leukemias and lymphomas due to somatically acquired insertions of retroviral DNA into the genome of hematopoetic cells that can mutate cellular proto-oncogenes and tumor suppressor genes. We generated a new set of tumors from nine AKXD RI strains selected for their propensity to develop B-cell tumors, the most common type of human hematopoietic cancers. We employed a PCR technique called viral insertion site amplification (VISA) to rapidly isolate genomic sequence at the site of provirus insertion. Here we describe 550 VISA sequence tags (VSTs) that identify 74 common insertion sites (CISs), of which 21 have not been identified previously. Several suspected proto-oncogenes and tumor suppressor genes lie near CISs, providing supportive evidence for their roles in cancer. Furthermore, numerous previously uncharacterized genes lie near CISs, providing a pool of candidate disease genes for future research. Pathway analysis of candidate genes identified several signaling pathways as common and powerful routes to blood cancer, including Notch, E-protein, NFkappaB, and Ras signaling. Misregulation of several Notch signaling genes was confirmed by quantitative RT-PCR. Our data suggest that analyses of insertional mutagenesis on a single genetic background are biased toward the identification of cooperating mutations. This tumor collection represents the most comprehensive study of the genetics of B-cell leukemia and lymphoma development in mice. We have deposited the VST sequences, CISs in a genome viewer, histopathology, and molecular tumor typing data in a public web database called VISION (Viral Insertion Sites Identifying Oncogenes), which is located at http://www.mouse-genome.bcm.tmc.edu/vision .