Targeted knock-down of miR21 primary transcripts using snoMEN vectors induces apoptosis in human cancer cell lines

We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2

Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines

The snoMEN (snoRNA Modulator of gene ExpressioN) vector technology was developed from a human box C/D snoRNA, HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets, allowing knock-down of targeted genes. Here we have screened additional human nucleolar snoRNAs and assessed their application for gene specific knock-downs to improve the efficiency of snoMEN vectors. We identify and characterise a new snoMEN vector, termed 47snoMEN, that is derived from box C/D snoRNA U47, demonstrating its use for knock-down of both endogenous cellular proteins and G/YFP-fusion proteins. Using multiplex 47snoMEM vectors that co-express multiple 47snoMEN in a single transcript, each of which can target different sites in the same mRNA, we document >3-fold increase in knock-down efficiency when compared with the original HBII-180C based snoMEN. The multiplex 47snoMEM vector allowed the construction of human protein replacement cell lines with improved efficiency, including the establishment of novel GFP–HIF-1α replacement cells. Quantitative mass spectrometry analysis confirmed the enhanced efficiency and specificity of protein replacement using the 47snoMEN-PR vectors. The 47snoMEN vectors expand the potential applications for snoMEN technology in gene expression studies, target validation and gene therapy

Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCF^Slmb degron

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

Perceived barriers to the regionalization of adult critical care in the United States: a qualitative preliminary study

<p>Abstract</p> <p>Background</p> <p>Regionalization of adult critical care services may improve outcomes for critically ill patients. We sought to develop a framework for understanding clinician attitudes toward regionalization and potential barriers to developing a tiered, regionalized system of care in the United States.</p> <p>Methods</p> <p>We performed a qualitative study using semi-structured interviews of critical care stakeholders in the United States, including physicians, nurses and hospital administrators. Stakeholders were identified from a stratified-random sample of United States general medical and surgical hospitals. Key barriers and potential solutions were identified by performing content analysis of the interview transcriptions.</p> <p>Results</p> <p>We interviewed 30 stakeholders from 24 different hospitals, representing a broad range of hospital locations and sizes. Key barriers to regionalization included personal and economic strain on families, loss of autonomy on the part of referring physicians and hospitals, loss of revenue on the part of referring physicians and hospitals, the potential to worsen outcomes at small hospitals by limiting services, and the potential to overwhelm large hospitals. Improving communication between destination and source hospitals, provider education, instituting voluntary objective criteria to become a designated referral center, and mechanisms to feed back patients and revenue to source hospitals were identified as potential solutions to some of these barriers.</p> <p>Conclusion</p> <p>Regionalization efforts will be met with significant conceptual and structural barriers. These data provide a foundation for future research and can be used to inform policy decisions regarding the design and implementation of a regionalized system of critical care.</p

Magnesium deficiency: effect on bone mineral density in the mouse appendicular skeleton

BACKGROUND: Dietary magnesium (Mg) deficiency in the mouse perturbs bone and mineral homeostasis. The objective of the present study was to evaluate bone mineral density of the femur in control and Mg-deficient mice. METHODS: BALB/c mice aged 28 days at study initiation were maintained on a normal or Mg deficient (0.0002% Mg) diet, and at time points 0, 2, 4 or 6 weeks bones were harvested for bone mineral density analysis. Peripheral quantitative computed tomography (pQCT) was used to assess the trabecular metaphyseal compartment and the cortical midshaft. RESULTS: Although mean total bone density of the femoral midshaft in Mg deficient mice did not differ significantly from controls throughout the study, the trabecular bone compartment showed significantly decreased mineral content after 4 (p < 0.001) and 6 weeks (p < 0.001) of Mg depletion. CONCLUSIONS: This study demonstrates the profound effect of Mg depletion on the trabecular compartment of bone, which, with its greater surface area and turnover, was more responsive to Mg depletion than cortical bone in the appendicular skeleton of the mouse

Depression literacy among Australians of Chinese-speaking background in Melbourne, Australia

<p>Abstract</p> <p>Background</p> <p>This study investigated the knowledge of depression and preference for professional help, medications and treatment methods among Australians of Chinese-speaking background, and the perceptions of this population of the causes of mental illness.</p> <p>Methods</p> <p>Adopting a cluster convenience sampling method, the study recruited 200 Chinese-speaking subjects from four major areas in metropolitan Melbourne where many Chinese live. The respondents were presented with a vignette describing an individual with depression and then asked questions to assess their understanding of depression and preference for professional help, medications and treatment methods. A comparative approach was used to compare the findings with those of a previous study of the mental health literacy of Australian and Japanese adults.</p> <p>Results</p> <p>Compared to the Australian and Japanese samples, a much lower percentage of Chinese-speaking Australians (14%) could correctly identify major depression described in the vignette, and a higher percentage believed that counseling professionals could be helpful. Higher percentages of those who believed that close family members could be helpful were found in the Chinese-speaking Australian and Japanese samples, and these two groups also expressed more uncertainty about the usefulness or harmfulness of certain medications compared to the Australian sample. Higher percentages of respondents in both the Chinese-speaking Australian and the Australian sample considered "lifestyle changes" to be helpful compared to the Japanese sample. In the Chinese-speaking sample, 30%, 17.4%, 33% and 27% of the respondents rated "traditional Chinese medicine doctors," "Chinese herbal medications," "taking Chinese nutritional foods/supplements" and "<it>qiqong</it>" as helpful. Many perceived "changing <it>fungshui</it>" and "traditional Chinese worship" to be harmful. Regarding the perception of causes of mental illness, items related to psychosocial perspectives including "life stress" and "interpersonal conflict" were rated highly by the respondents, whereas traditional beliefs including "punishment for misdeeds conducted by ancestors" and "demon possession" had the lowest ratings.</p> <p>Conclusions</p> <p>Campaigns to increase the mental health literacy of Chinese-speaking Australians are needed. The abovementioned socially and culturally driven beliefs need to be taken into consideration in the development of culturally relevant education programs.</p

Outcome of a risk-related therapeutic strategy used prospectively in a population-based study of Hodgkin's lymphoma in adolescents

The aim was to assess outcome in a population-based cohort of adolescents with Hodgkin's lymphoma (HL) diagnosed in the UK's northern region over a 10-year period. Among a population of 3.09 million, 55 of 676 patients (8%) diagnosed with HL were aged 13–19. Seven had nodular lymphocyte-predominant HL, 48 classical HL (cHL). Of the latter, 36 were ⩾16 years. Application of the Scottish and Newcastle Lymphoma Group (SNLG) prognostic index meant 21 patients were considered high risk (index ⩾0.5). They received PVACEBOP multi-agent chemotherapy as primary therapy. Standard risk patients (SNLG index <0.5) were treated with standard ChlVPP or ABVD chemotherapy±radiotherapy. Scottish and Newcastle Lymphoma Group indexing is not valid for patients under 16. Twelve patients therefore received UKCCSG protocols (n=8), ABVD plus radiotherapy (n=2), or PVACEBOP (n=2). Forty-six patients with cHL (96%) achieved complete remission. Seven patients relapsed but all entered complete remission after salvage therapy. Five patients died: three of HL, one in an accident and one of disseminated varicella complicating cystic fibrosis. Five- and 10-year overall survival was 93 and 86%, respectively; disease-specific survival was 95 and 92%. The data suggest that older adolescents with high-risk HL require intensive protocols as primary therapy to secure optimal outcome

High-mobility group box-1 protein, lipopolysaccharide-binding protein, interleukin-6 and C-reactive protein in children with community acquired infections and bacteraemia: a prospective study

<p>Abstract</p> <p>Introduction</p> <p>Even though sepsis is one of the common causes of children morbidity and mortality, specific inflammatory markers for identifying sepsis are less studied in children. The main aim of this study was to compare the levels of high-mobility group box-1 protein (HMGB1), Lipopolysaccharide-binding protein (LBP), Interleukin-6 (IL-6) and C-reactive protein (CRP) between infected children without systemic inflammatory response syndrome (SIRS) and children with severe and less severe sepsis. The second aim was to examine HMGB1, LBP, IL6 and CRP as markers for of bacteraemia.</p> <p>Methods</p> <p>Totally, 140 children with suspected or proven infections admitted to the Children's Clinical University Hospital of Latvia during 2008 and 2009 were included. Clinical and demographical information as well as infection focus were assessed in all patients. HMGB1, LBP, IL-6 and CRP blood samples were determined. Children with suspected or diagnosed infections were categorized into three groups of severity of infection: (i) infected without SIRS (n = 36), (ii) sepsis (n = 91) and, (iii) severe sepsis (n = 13). They were furthermore classified according bacteraemia into (i) bacteremia (n = 30) and (ii) no bacteraemia (n = 74).</p> <p>Results</p> <p>There was no statistically significant difference in HMGB1 levels between children with different levels of sepsis or with and without bacteraemia. The levels of LBP, IL-6 and CRP were statistically significantly higher among patients with sepsis compared to those infected but without SIRS (<it>p </it>< 0.001). Furthermore, LBP, IL-6 and CRP were significantly higher in children with severe sepsis compared to those ones with less severe sepsis (<it>p </it>< 0.001). Median values of LBP, IL6 and CRP were significantly higher in children with bacteraemia compared to those without bacteraemia. The area under the receiver operating curve (ROC) for detecting bacteraemia was 0.87 for both IL6 and CRP and 0.82 for LBP, respectively.</p> <p>Conclusion</p> <p>Elevated levels of LBP, IL-6 and CRP were associated with a more severe level of infection in children. Whereas LBP, IL-6 and CRP seem to be good markers to detect patients with bacteraemia, HMGB1 seem to be of minor importance. LBP, IL-6 and CRP levels may serve as good biomarkers for identifying children with severe sepsis and bacteraemia and, thus, may be routinely used in clinical practice.</p

Hepatitis C Virus Core-Derived Peptides Inhibit Genotype 1b Viral Genome Replication via Interaction with DDX3X

The protein DDX3X is a DEAD-box RNA helicase that is essential for the hepatitis C virus (HCV) life cycle. The HCV core protein has been shown to bind to DDX3X both in vitro and in vivo. However, the specific interactions between these two proteins and the functional importance of these interactions for the HCV viral life cycle remain unclear. We show that amino acids 16–36 near the N-terminus of the HCV core protein interact specifically with DDX3X both in vitro and in vivo. Replication of HCV replicon NNeo/C-5B RNA (genotype 1b) is significantly suppressed in HuH-7-derived cells expressing green fluorescent protein (GFP) fusions to HCV core protein residues 16–36, but not by GFP fusions to core protein residues 16–35 or 16–34. Notably, the inhibition of HCV replication due to expression of the GFP fusion to HCV core protein residues 16–36 can be reversed by overexpression of DDX3X. These results suggest that the protein interface on DDX3X that binds the HCV core protein is important for replicon maintenance. However, infection of HuH-7 cells by HCV viruses of genotype 2a (JFH1) was not affected by expression of the GFP fusion protein. These results suggest that the role of DDX3X in HCV infection involves aspects of the viral life cycle that vary in importance between HCV genotypes