Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins

We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3 + 2] azide−alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct in-gel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of O-GlcNAc

Regulation of elongation factor-1α expression by growth factors and anti-receptor blocking antibodies

The Epidermal Growth Factor (EGF) family and its receptors regulate normal and cancerous epithelial cell proliferation, a process that could be suppressed by anti-receptor blocking antibodies. Polypeptide elongation factor-1α (EF-1α) is a multifunctional protein whose levels are positively correlated with the proliferative state of cells. To identify genes, whose expression may be modulated by anti-receptor blocking antibodies, we performed a differential display screening and isolated differentially expressed cDNAs. Isolates from one clone were 100% identical to human EF-1α. Both EGF and heregulin-β1 (HRG) induced EF-1α promoter activity and mRNA and protein expression. Growth factor-mediated EF-1α expression was effectively blocked by pretreatment with humanized anti-EGF receptor antibody C225 or anti-human epidermal growth factor receptor-2 (HER2) antibody herceptin. Mutants and pharmacological inhibitors of p38MAPK and MEK, but not phosphatidylinositol 3-kinase, suppressed both constitutive and HRG-induced stimulation of EF-1α promoter activity in MCF-7 cells. Deletion analysis of the promoter suggested the requirement of the −393 to −204 region for growth factor-mediated transcription of EF-1α. Fine mapping and point mutation studies revealed a role of the SP1 site in the observed HRG-mediated regulation of the EF-1α promoter. In addition, we also provide new evidence to suggest that HRG stimulation of the EF-1α promoter involves increased physical interactions with acetylated histone H3 and histone H4. These results suggest that regulation of EF-1α expression by extracellular signals that function through human EGF receptor family members that are widely deregulated in human cancers and that growth factor regulation of EF-1α expression involve histone acetylation

Learning deterministic probabilistic automata from a model checking perspective

Probabilistic automata models play an important role in the formal design and analysis of hard- and software systems. In this area of applications, one is often interested in formal model-checking procedures for verifying critical system properties. Since adequate system models are often difficult to design manually, we are interested in learning models from observed system behaviors. To this end we adopt techniques for learning finite probabilistic automata, notably the Alergia algorithm. In this paper we show how to extend the basic algorithm to also learn automata models for both reactive and timed systems. A key question of our investigation is to what extent one can expect a learned model to be a good approximation for the kind of probabilistic properties one wants to verify by model checking. We establish theoretical convergence properties for the learning algorithm as well as for probability estimates of system properties expressed in linear time temporal logic and linear continuous stochastic logic. We empirically compare the learning algorithm with statistical model checking and demonstrate the feasibility of the approach for practical system verification

Ecosystem Stewardship: Sustainability Strategies for a Rapidly Changing Planet

Ecosystem stewardship is an action-oriented framework intended to foster social-ecological sustainability of a rapidly changing planet. Recent developments identify three strategies that make optimal use of current understanding in an environment of inevitable uncertainty and abrupt change: reducing the magnitude of, and exposure and sensitivity to, known stresses; focusing on proactive policies that shape change; and avoiding or escaping unsustainable social-ecological traps. All social-ecological systems are vulnerable to recent and projected changes but have sources of adaptive capacity and resilience that can sustain ecosystem services and human well-being through active ecosystem stewardship

Cytoplasmic p53 couples oncogene-driven glucose metabolism to apoptosis and is a therapeutic target in glioblastoma.

Cross-talk among oncogenic signaling and metabolic pathways may create opportunities for new therapeutic strategies in cancer. Here we show that although acute inhibition of EGFR-driven glucose metabolism induces only minimal cell death, it lowers the apoptotic threshold in a subset of patient-derived glioblastoma (GBM) cells. Mechanistic studies revealed that after attenuated glucose consumption, Bcl-xL blocks cytoplasmic p53 from triggering intrinsic apoptosis. Consequently, targeting of EGFR-driven glucose metabolism in combination with pharmacological stabilization of p53 with the brain-penetrant small molecule idasanutlin resulted in synthetic lethality in orthotopic glioblastoma xenograft models. Notably, neither the degree of EGFR-signaling inhibition nor genetic analysis of EGFR was sufficient to predict sensitivity to this therapeutic combination. However, detection of rapid inhibitory effects on [18F]fluorodeoxyglucose uptake, assessed through noninvasive positron emission tomography, was an effective predictive biomarker of response in vivo. Together, these studies identify a crucial link among oncogene signaling, glucose metabolism, and cytoplasmic p53, which may potentially be exploited for combination therapy in GBM and possibly other malignancies

Traumatic subaxial cervical facet subluxation and dislocation: epidemiology, radiographic analyses, and risk factors for spinal cord injury

© 2017 Elsevier Inc. This manuscript version is made available under the CC-BY-NC-ND 4.0 license: http://creativecommons.org/licenses/by-nc-nd/4.0/ This author accepted manuscript is made available following 12 month embargo from date of publication (July 2017) in accordance with the publisher’s archiving policyBackground Context Distractive flexion injuries (DFIs) of the subaxial cervical spine are major contributors to spinal cord injury (SCI). Prompt assessment and early intervention of DFIs associated with SCI are crucial to optimize patient outcome; however, neurologic examination of patients with subaxial cervical injury is often difficult, as patients commonly present with reduced levels of consciousness. Therefore, it is important to establish potential associations between injury epidemiology and radiographic features, and neurologic involvement. Purpose The aims of this study were to describe the epidemiology and radiographic features of DFIs presenting to a major Australian tertiary hospital and to identify those factors predictive of SCI. The agreement and repeatability of radiographic measures of DFI severity were also investigated. Study Design/Setting This is a combined retrospective case-control and reliability-agreement study. Patient Sample Two hundred twenty-six patients (median age 40 years [interquartile range = 34]; 72.1% male) who presented with a DFI of the subaxial cervical spine between 2003 and 2013 were reviewed. Outcome Measures The epidemiology and radiographic features of DFI, and risk factors for SCI were identified. Inter- and intraobserver agreement of radiographic measurements was evaluated. Methods Medical records, radiographs, and computed tomography and magnetic resonance imaging scans were examined, and the presence of SCI was evaluated. Radiographic images were analyzed by two consultant spinal surgeons, and the degree of vertebral translation, facet apposition, spinal canal occlusion, and spinal cord compression were documented. Multivariable logistic regression models identified epidemiology and radiographic features predictive of SCI. Intraclass correlation coefficients (ICCs) examined inter- and intraobserver agreement of radiographic measurements. Results The majority of patients (56.2%) sustained a unilateral (51.2%) or a bilateral facet (48.8%) dislocation. The C6–C7 vertebral level was most commonly involved (38.5%). Younger adults were over-represented among motor-vehicle accidents, whereas falls contributed to a majority of DFIs sustained by older adults. Greater vertebral translation, together with lower facet apposition, distinguished facet dislocation from subluxation. Dislocation, bilateral facet injury, reduced Glasgow Coma Scale, spinal canal occlusion, and spinal cord compression were predictive of neurologic deficit. Radiographic measurements demonstrated at least a “moderate” agreement (ICC>0.4), with most demonstrating an “almost perfect” reproducibility. Conclusions This large-scale cohort investigation of DFIs in the cervical spine describes radiographic features that distinguish facet dislocation from subluxation, and associates highly reproducible anatomical and clinical indices to the occurrence of concomitant SCI

CIViCdb 2022: Evolution of an open-access cancer variant interpretation knowledgebase

CIViC (Clinical Interpretation of Variants in Cancer; civicdb.org) is a crowd-sourced, public domain knowledgebase composed of literature-derived evidence characterizing the clinical utility of cancer variants. As clinical sequencing becomes more prevalent in cancer management, the need for cancer variant interpretation has grown beyond the capability of any single institution. CIViC contains peer-reviewed, published literature curated and expertly-moderated into structured data units (Evidence Items) that can be accessed globally and in real time, reducing barriers to clinical variant knowledge sharing. We have extended CIViC\u27s functionality to support emergent variant interpretation guidelines, increase interoperability with other variant resources, and promote widespread dissemination of structured curated data. To support the full breadth of variant interpretation from basic to translational, including integration of somatic and germline variant knowledge and inference of drug response, we have enabled curation of three new Evidence Types (Predisposing, Oncogenic and Functional). The growing CIViC knowledgebase has over 300 contributors and distributes clinically-relevant cancer variant data currently representing \u3e3200 variants in \u3e470 genes from \u3e3100 publications

Tumor-induced STAT3 activation in monocytic myeloid-derived suppressor cells enhances stemness and mesenchymal properties in human pancreatic cancer

Pancreatic cancer (PC) mobilizes myeloid cells from the bone marrow to the tumor where they promote tumor growth and proliferation. Cancer stem cells (CSCs) are a population of tumor cells that are responsible for tumor initiation. Aldehyde dehydrogenase-1 activity in PC identifies CSCs, and its activity has been correlated with poor overall prognosis in human PC. Myeloid cells have been shown to impact tumor stemness, but the impact of immunosuppressive tumor-infiltrating granulocytic and monocytic myeloid-derived suppressor cells (Mo-MDSC) on ALDH1(Bright) CSCs and epithelial to mesenchymal transition is not well understood. In this study, we demonstrate that Mo-MDSC (CD11b(+)/Gr1(+)/Ly6G(−)/Ly6C(hi)) significantly increase the frequency of ALDH1(Bright) CSCs in a mouse model of PC. Additionally, there was significant upregulation of genes associated with epithelial to mesenchymal transition. We also found that human PC converts CD14(+) peripheral blood monocytes into Mo-MDSC (CD14(+)/HLA-DR(low/−)) in vitro, and this transformation is dependent on the activation of the STAT3 pathway. In turn, these Mo-MDSC increase the frequency of ALDH1(Bright) CSCs and promote mesenchymal features of tumor cells. Finally, blockade of STAT3 activation reversed the increase in ALDH1(Bright) CSCs. These data suggest that the PC tumor microenvironment transforms monocytes to Mo-MDSC by STAT3 activation, and these cells increase the frequency of ALDH1(Bright) CSCs. Therefore, targeting STAT3 activation may be an effective therapeutic strategy in targeting CSCs in PC. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00262-014-1527-x) contains supplementary material, which is available to authorized users

The safety of artemisinins during pregnancy: a pressing question

BACKGROUND: An increasing number of countries in sub-Saharan Africa are changing to artemisinins combination therapy (ACT) as first or second line treatment for malaria. There is an urgent need to assess the safety of these drugs in pregnant women who may be inadvertently exposed to or actively treated with ACTs. OBJECTIVES: To examine existing published evidence on the relationship between artemisinin compounds and adverse pregnancy outcomes and consider the published evidence with regard to the safety of these compounds when administered during pregnancy. METHODS: Studies on ACT use in pregnancy were identified via searches of MEDLINE, EMBASE, Cochrane and Current Contents databases. Data on study characteristics, maternal adverse events, pregnancy outcomes and infant follow up were extracted. RESULTS: Fourteen relevant studies (nine descriptive/case reports and five controlled trials) were identified. Numbers of participants in these studies ranged from six to 461. Overall there were reports on 945 women exposed to an artemisinin during pregnancy, 123 in the 1st trimester and 822 in 2nd or 3rd trimesters. The primary end points for these studies were drug efficacy and parasite clearance. Secondary endpoints were birth outcomes including low birth weight, pre-term birth, pregnancy loss, congenital anomalies and developmental milestones. While none of the studies found evidence for an association between the use of artemisinin compounds and increased risk of adverse pregnancy outcomes, none were of sufficient size to detect small differences in event rates that could be of public health importance. Heterogeneity between studies in the artemisinin and comparator drugs used, and in definitions of adverse pregnancy outcomes, limited any pooled analysis. CONCLUSION: The limited data available suggest that artemisinins are effective and unlikely to be cause of foetal loss or abnormalities, when used in late pregnancy. However, none of these studies had adequate power to rule out rare serious adverse events, even in 2(nd )and 3(rd )trimesters and there is not enough evidence to effectively assess the risk-benefit profile of artemisinin compounds for pregnant women particularly for 1(st )trimester exposure. Methodologically rigorous, larger studies and post-marketing pharmacovigilance are urgently required

Reconnecting a stream channel to its floodplain: implications for benthic diatoms and macroinvertebrate trophic structure

Streams systems draining upland landscapes provide valuable ecosystem services, but they are vulnerable to incision and channelization caused by anthropogenic disturbance. Restoring a degraded stream to its pre‐disturbance condition by reconnecting the channel to its historical floodplain aims to recover lost hydro‐morphological processes and functions. Seeking evidence to indicate whether that aim is met in practice, we examined diatoms and the stream macroinvertebrate trophic structures in three reaches of Whychus Creek, Oregon, United States. Two reaches were reconnected to their pre‐disturbance floodplains in 2012 and 2016. The third, control reach, was not restored and was selected to represent the degraded stream condition prior to restoration. Ordinations showed that benthic diatom species composition shifted from the control reach to the restored reaches. Compared to the control reach, reconnection decreased the percentages of diatoms with nitrogen (N)‐fixing cyanobacterial endosymbionts in the 2012 restored reach and decreased diatoms tolerant to low N conditions in both the restored reaches. δ15N values in both stream macroinvertebrates and tree leaves in the riparian zone were higher in the restored reaches. These findings suggest that floodplain reconnection may modify hydro‐morphological processes and ecosystem functions in ways that enhance organic matter retention and hyporheic exchange, resulting in increased nutrient availability, improved nutrient cycling, and greater primary productivity. More generally, our results suggest that characterizing diatom species composition and trophic interactions using stable isotopes provides the basis for identifying and evaluating the beneficial effects of stream restoration on ecosystem functions and the food‐web