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

Minimal Holocene retreat of large tidewater glaciers in Køge Bugt, southeast Greenland

Abstract Køge Bugt, in southeast Greenland, hosts three of the largest glaciers of the Greenland Ice Sheet; these have been major contributors to ice loss in the last two decades. Despite its importance, the Holocene history of this area has not been investigated. We present a 9100 year sediment core record of glaciological and oceanographic changes from analysis of foraminiferal assemblages, the abundance of ice-rafted debris, and sortable silt grain size data. Results show that ice-rafted debris accumulated constantly throughout the core; this demonstrates that glaciers in Køge Bugt remained in tidewater settings throughout the last 9100 years. This observation constrains maximum Holocene glacier retreat here to less than 6 km from present-day positions. Retreat was minimal despite oceanic and climatic conditions during the early-Holocene that were at least as warm as the present-day. The limited Holocene retreat of glaciers in Køge Bugt was controlled by the subglacial topography of the area; the steeply sloping bed allowed glaciers here to stabilise during retreat. These findings underscore the need to account for individual glacier geometry when predicting future behaviour. We anticipate that glaciers in Køge Bugt will remain in stable configurations in the near-future, despite the predicted continuation of atmospheric and oceanic warming

Distinct cardioprotective mechanisms of immediate, early and delayed ischaemic postconditioning

Cardioprotection against ischaemia/reperfusion injury in mice can be achieved by delayed ischaemic postconditioning (IPost) applied as late as 30 min after the onset of reperfusion. We determined the efficacy of delayed IPost in a rat model of myocardial infarction (MI) and investigated potential underlying mechanisms of this phenomenon. Rats were subjected to 20, 30 or 45 min of coronary artery occlusion followed by 120 min of reperfusion (I/R). Immediate and early IPost included six cycles of I/R (10/10 s) applied 10 s or 10 min after reperfusion onset. In the second series of experiments, the rats were subjected to 30 min of coronary occlusion followed by IPost applied 10 s, 10, 30, 45 or 60 min after the onset of reperfusion. Immediate and early IPost (applied 10 s or 10 min of reperfusion) established cardioprotection only when applied after a period of myocardial ischaemia lasting 30 min. Delayed IPost applied after 30 or 45 min of reperfusion reduced infarct sizes by 36 and 41 %, respectively (both P < 0.01). IPost applied 60 min after reperfusion onset was ineffective. Inhibition of RISK pathway (administration of ERK1/2 inhibitor PD-98059 or PI3K inhibitor LY-294002) abolished cardioprotection established by immediate IPost but had no effect on cardioprotection conferred by early IPost. Blockade of SAFE pathway using JAK/STAT inhibitor AG490 had no effect on the immediate or early IPost cardioprotection. Blockade of mitochondrial KATP (mitoKATP) channels (with 5-Hydroxydecanoate) abolished cardioprotection achieved by immediate and early IPost, but had no effect on cardioprotection when IPost was applied 30 or 45 min into the reperfusion period. Immediate IPost increased phosphorylation of PI3K-AKT and ERK1/2. Early or delayed IPost had no effect on phosphorylation of PI3K-AKT, ERK1/2 or STAT3. These data show that in the rat model, delayed IPost confers significant cardioprotection even if applied 45 min after onset of reperfusion. Cardioprotection induced by immediate and early postconditioning involves recruitment of RISK pathway and/or mitoKATP channels, while delayed postconditioning appears to rely on a different mechanism

Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics

In December 2016, a panel of experts in microbiology, nutrition and clinical research was convened by the International Scientific Association for Probiotics and Prebiotics to review the definition and scope of prebiotics. Consistent with the original embodiment of prebiotics, but aware of the latest scientific and clinical developments, the panel updated the definition of a prebiotic: a substrate that is selectively utilized by host microorganisms conferring a health benefit. This definition expands the concept of prebiotics to possibly include non-carbohydrate substances, applications to body sites other than the gastrointestinal tract, and diverse categories other than food. The requirement for selective microbiota-mediated mechanisms was retained. Beneficial health effects must be documented for a substance to be considered a prebiotic. The consensus definition applies also to prebiotics for use by animals, in which microbiota-focused strategies to maintain health and prevent disease is as relevant as for humans. Ultimately, the goal of this Consensus Statement is to engender appropriate use of the term ‘prebiotic’ by relevant stakeholders so that consistency and clarity can be achieved in research reports, product marketing and regulatory oversight of the category. To this end, we have reviewed several aspects of prebiotic science including its development, health benefits and legislation

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

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Larvicidal effects of Chinaberry (Melia azederach) powder on Anopheles arabiensis in Ethiopia

<p>Abstract</p> <p>Background</p> <p>Synthetic insecticides are employed in the widely-used currently favored malaria control techniques involving indoor residual spraying and treated bednets. These methods have repeatedly proven to be highly effective at reducing malaria incidence and prevalence. However, rapidly emerging mosquito resistance to the chemicals and logistical problems in transporting supplies to remote locations threaten the long-term sustainability of these techniques. Chinaberry (<it>Melia azederach</it>) extracts have been shown to be effective growth-inhibiting larvicides against several insects. Because several active chemicals in the trees' seeds have insecticidal properties, the emergence of resistance is unlikely. Here, we investigate the feasibility of Chinaberry as a locally available, low-cost sustainable insecticide that can aid in controlling malaria. Chinaberry fruits were collected from Asendabo, Ethiopia. The seeds were removed from the fruits, dried and crushed into a powder. From developmental habitats in the same village, <it>Anopheles arabiensis </it>larvae were collected and placed into laboratory containers. Chinaberry seed powder was added to the larval containers at three treatment levels: 5 g m^-2, 10 g m^-2and 20 g m^-2, with 100 individual larvae in each treatment level and a control. The containers were monitored daily and larvae, pupae and adult mosquitoes were counted. This experimental procedure was replicated three times.</p> <p>Results</p> <p>Chinaberry seed powder caused an inhibition of emergence of 93% at the 5 g m^-2treatment level, and 100% inhibition of emergence at the two higher treatment levels. The Chinaberry had a highly statistically significant larvicidal effect at all treatment levels (χ²= 184, 184, and 155 for 5 g m^-2, 10 g m^-2and 20 g m^-2, respectively; p < 0.0001 in all cases). In addition, estimates suggest that sufficient Chinaberry seed exists in Asendabo to treat developmental habitat for the duration of the rainy season and support a field trial.</p> <p>Conclusions</p> <p>Chinaberry seed is a very potent growth-inhibiting larvicide against the major African malaria vector <it>An. arabiensis</it>. The seed could provide a sustainable additional malaria vector control tool that can be used where the tree is abundant and where <it>An. arabiensis </it>is a dominant vector. Based on these results, a future village-scale field trial using the technique is warranted.</p

Dysregulation of MicroRNA-34a Expression in Head and Neck Squamous Cell Carcinoma Promotes Tumor Growth and Tumor Angiogenesis

MicroRNAs (miRs) are small non-coding RNAs that play an important role in cancer development where they can act as oncogenes or as tumor-suppressors. miR-34a is a tumor-suppressor that is frequently downregulated in a number of tumor types. However, little is known about the role of miR-34a in head and neck squamous cell carcinoma (HNSCC).miR-34a expression in tumor samples, HNSCC cell lines and endothelial cells was examined by real time PCR. Lipofectamine-2000 was used to transfect miR-34a in HNSCC cell lines and human endothelial cells. Cell-proliferation, migration and clonogenic survival was examined by MTT, Xcelligence system, scratch assay and colony formation assay. miR-34a effect on tumor growth and tumor angiogenesis was examined by in vivo SCID mouse xenograft model. Our results demonstrate that miR-34a is significantly downregulated in HNSCC tumors and cell lines. Ectopic expression of miR-34a in HNSCC cell lines significantly inhibited tumor cell proliferation, colony formation and migration. miR-34a overexpression also markedly downregulated E2F3 and survivin levels. Rescue experiments using microRNA resistant E2F3 isoforms suggest that miR-34a-mediated inhibition of cell proliferation and colony formation is predominantly mediated by E2F3a isoform. In addition, tumor samples from HNSCC patients showed an inverse relationship between miR-34a and survivin as well as miR-34a and E2F3 levels. Overexpression of E2F3a completely rescued survivin expression in miR-34a expressing cells, thereby suggesting that miR-34a may be regulating survivin expression via E2F3a. Ectopic expression of miR-34a also significantly inhibited tumor growth and tumor angiogenesis in a SCID mouse xenograft model. Interestingly, miR-34a inhibited tumor angiogenesis by blocking VEGF production by tumor cells as well as directly inhibiting endothelial cell functions.Taken together, these findings suggest that dysregulation of miR-34a expression is common in HNSCC and modulation of miR34a activity might represent a novel therapeutic strategy for the treatment of HNSCC