Manipulating cellular microRNAs and analyzing high-dimensional gene expression data using machine learning workflows.

MicroRNAs (miRNAs) are elements of the gene regulatory network and manipulating their abundance is essential toward elucidating their role in patho-physiological conditions. We present a detailed workflow that identifies important miRNAs using a machine learning algorithm. We then provide optimized techniques to validate the identified miRNAs through over-expression/loss-of-function studies. Overall, these protocols apply to any field in biology where high-dimensional data are produced. For complete details on the use and execution of this protocol, please refer to Wong et al. (2021a)

Nitrogen addition alters composition, diversity, and functioning of microbial communities in mangrove soils : an incubation experiment

Mangrove ecosystems are important for carbon storage due to their high productivity and low decomposition rates. Waterways have experienced increased nutrient loads as a result of anthropogenic activities and it is unclear how this may affect carbon and nutrient cycles in downstream mangroves that receive these nutrient-rich waters. Using a laboratory-based incubation experiment, this study aimed to assess the effects of nutrient addition on the diversity and structure of mangrove soil bacterial communities, as well as biomass and activity of the soil microbial community, under different oxygen conditions. Bacterial community diversity and composition was characterised using 16S rRNA gene sequencing and microbial activity was examined through the measurement of microbial respiration and the activities of enzymes associated with organic matter decomposition. Nitrogen addition caused clear shifts in bacterial community composition, with decreases in bacterial diversity and the abundance of sulfate-reducing bacteria. Microbial biomass also decreased with nitrogen addition under reduced oxygen incubations. Changes in bacterial community structure were accompanied by changes in the activity of some enzymes involved in carbon, nitrogen and phosphorus cycling. Under reduced oxygen conditions, nitrogen addition resulted in a significant increase in the microbial metabolic quotient but no accompanying change in microbial respiration, which was explained by a decrease in microbial biomass. The findings of this study indicate that nitrogen loading has potential implications for microbial communities and carbon and nutrient cycling in mangrove environments that warrant further investigation under field conditions

Transglutaminase 2 limits the extravasation and the resultant myocardial fibrosis associated with factor XIII-A deficiency

Background and aims Transglutaminase (TG) 2 and Factor (F) XIII-A have both been implicated in cardiovascular protection and repair. This study was designed to differentiate between two competing hypotheses: that TG2 and FXIII-A mediate these functions in mice by fulfilling separate roles, or that they act redundantly in this respect. Methods Atherosclerosis was assessed in brachiocephalic artery plaques of fat-fed mixed strain apolipoprotein (Apo)e deficient mice that lacked either or both transglutaminases. Cardiac fibrosis was assessed both in the mixed strain mice and also in C57BL/6J Apoe expressing mice lacking either or both transglutaminases. Results No difference was found in the density of buried fibrous caps within brachiocephalic plaques from mice expressing or lacking these transglutaminases. Cardiac fibrosis developed in both Apoe/F13a1 double knockout and F13a1 single knockout mice, but not in Tgm2 knockout mice. However, concomitant Tgm2 knockout markedly increased fibrosis, as apparent in both Apoe/Tgm2/F13a1 knockout and Tgm2/F13a1 knockout mice. Amongst F13a1 knockout and Tgm2/F13a1 knockout mice, the extent of fibrosis correlated with hemosiderin deposition, suggesting that TG2 limits the extravasation of blood in the myocardium, which in turn reduces the pro-fibrotic stimulus. The resulting fibrosis was interstitial in nature and caused only minor changes in cardiac function. Conclusions These studies confirm that FXIII-A and TG2 fulfil different roles in the mouse myocardium. FXIII-A protects against vascular leakage while TG2 contributes to the stability or repair of the vasculature. The protective function of TG2 must be considered when designing clinical anti-fibrotic therapies based upon FXIII-A or TG2 inhibition

Cre/lox Studies Identify Resident Macrophages as the Major Source of Circulating Coagulation Factor XIII-A

Objective— To establish the cellular source of plasma factor (F)XIII-A. Approach and Results— A novel mouse floxed for the F13a1 gene, FXIII-Aflox/flox (Flox), was crossed with myeloid- and platelet-cre–expressing mice, and cellular FXIII-A mRNA expression and plasma and platelet FXIII-A levels were measured. The platelet factor 4-cre.Flox cross abolished platelet FXIII-A and reduced plasma FXIII-A to 23±3% (P<0.001). However, the effect of platelet factor 4-cre on plasma FXIII-A was exerted outside of the megakaryocyte lineage because plasma FXIII-A was not reduced in the Mpl−/− mouse, despite marked thrombocytopenia. In support of this, platelet factor 4-cre depleted FXIII-A mRNA in brain, aorta, and heart of floxed mice, where FXIII-Apos cells were identified as macrophages as they costained with CD163. In the integrin αM-cre.Flox and the double copy lysozyme 2-cre.cre.Flox crosses, plasma FXIII-A was reduced to, respectively, 75±5% (P=0.003) and 30±7% (P<0.001), with no change in FXIII-A content per platelet, further consistent with a macrophage origin of plasma FXIII-A. The change in plasma FXIII-A levels across the various mouse genotypes mirrored the change in FXIII-A mRNA expression in aorta. Bone marrow transplantation of FXIII-A+/+ bone marrow into FXIII-A−/− mice both restored plasma FXIII-A to normal levels and replaced aortic and cardiac FXIII-A mRNA, while its transplantation into FXIII-A+/+ mice did not increase plasma FXIII-A levels, suggesting that a limited population of niches exists that support FXIII-A-releasing cells. Conclusions— This work suggests that resident macrophages maintain plasma FXIII-A and exclude the platelet lineage as a major contributor

Dietary Supplementation with Soluble Plantain Non-Starch Polysaccharides Inhibits Intestinal Invasion of Salmonella Typhimurium in the Chicken

Soluble fibres (non-starch polysaccharides, NSP) from edible plants but particularly plantain banana (Musa spp.), have been shown in vitro and ex vivo to prevent various enteric pathogens from adhering to, or translocating across, the human intestinal epithelium, a property that we have termed contrabiotic. Here we report that dietary plantain fibre prevents invasion of the chicken intestinal mucosa by Salmonella. In vivo experiments were performed with chicks fed from hatch on a pellet diet containing soluble plantain NSP (0 to 200 mg/d) and orally infected with S.Typhimurium 4/74 at 8 d of age. Birds were sacrificed 3, 6 and 10 d post-infection. Bacteria were enumerated from liver, spleen and caecal contents. In vitro studies were performed using chicken caecal crypts and porcine intestinal epithelial cells infected with Salmonella enterica serovars following pre-treatment separately with soluble plantain NSP and acidic or neutral polysaccharide fractions of plantain NSP, each compared with saline vehicle. Bacterial adherence and invasion were assessed by gentamicin protection assay. In vivo dietary supplementation with plantain NSP 50 mg/d reduced invasion by S.Typhimurium, as reflected by viable bacterial counts from splenic tissue, by 98.9% (95% CI, 98.1–99.7; P<0.0001). In vitro studies confirmed that plantain NSP (5–10 mg/ml) inhibited adhesion of S.Typhimurium 4/74 to a porcine epithelial cell-line (73% mean inhibition (95% CI, 64–81); P<0.001) and to primary chick caecal crypts (82% mean inhibition (95% CI, 75–90); P<0.001). Adherence inhibition was shown to be mediated via an effect on the epithelial cells and Ussing chamber experiments with ex-vivo human ileal mucosa showed that this effect was associated with increased short circuit current but no change in electrical resistance. The inhibitory activity of plantain NSP lay mainly within the acidic/pectic (homogalacturonan-rich) component. Supplementation of chick feed with plantain NSP was well tolerated and shows promise as a simple approach for reducing invasive salmonellosis

MAGE-A cancer/testis antigens inhibit MDM2 ubiquitylation function and promote increased levels of MDM4

Melanoma antigen A (MAGE-A) proteins comprise a structurally and biochemically similar sub-family of Cancer/Testis antigens that are expressed in many cancer types and are thought to contribute actively to malignancy. MAGE-A proteins are established regulators of certain cancer-associated transcription factors, including p53, and are activators of several RING finger-dependent ubiquitin E3 ligases. Here, we show that MAGE-A2 associates with MDM2, a ubiquitin E3 ligase that mediates ubiquitylation of more than 20 substrates including mainly p53, MDM2 itself, and MDM4, a potent p53 inhibitor and MDM2 partner that is structurally related to MDM2. We find that MAGE-A2 interacts with MDM2 via the N-terminal p53-binding pocket and the RING finger domain of MDM2 that is required for homo/hetero-dimerization and for E2 ligase interaction. Consistent with these data, we show that MAGE-A2 is a potent inhibitor of the E3 ubiquitin ligase activity of MDM2, yet it does not have any significant effect on p53 turnover mediated by MDM2. Strikingly, however, increased MAGE-A2 expression leads to reduced ubiquitylation and increased levels of MDM4. Similarly, silencing of endogenous MAGE-A expression diminishes MDM4 levels in a manner that can be rescued by the proteasomal inhibitor, bortezomid, and permits increased MDM2/MDM4 association. These data suggest that MAGE-A proteins can: (i) uncouple the ubiquitin ligase and degradation functions of MDM2; (ii) act as potent inhibitors of E3 ligase function; and (iii) regulate the turnover of MDM4. We also find an association between the presence of MAGE-A and increased MDM4 levels in primary breast cancer, suggesting that MAGE-A-dependent control of MDM4 levels has relevance to cancer clinically

A novel asynchronous access method with binary interfaces

© 2008 Silva et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

A retinal code for motion along the gravitational and body axes

Self-motion triggers complementary visual and vestibular reflexes supporting image-stabilization and balance. Translation through space produces one global pattern of retinal image motion (optic flow), rotation another. We examined the direction preferences of direction-sensitive ganglion cells (DSGCs) in flattened mouse retinas in vitro. Here we show that for each subtype of DSGC, direction preference varies topographically so as to align with specific translatory optic flow fields, creating a neural ensemble tuned for a specific direction of motion through space. Four cardinal translatory directions are represented, aligned with two axes of high adaptive relevance: the body and gravitational axes. One subtype maximizes its output when the mouse advances, others when it retreats, rises or falls. Two classes of DSGCs, namely, ON-DSGCs and ON-OFF-DSGCs, share the same spatial geometry but weight the four channels differently. Each subtype ensemble is also tuned for rotation. The relative activation of DSGC channels uniquely encodes every translation and rotation. Although retinal and vestibular systems both encode translatory and rotatory self-motion, their coordinate systems differ

Ethnic variations in asthma hospital admission, readmission and death:a retrospective, national cohort study of 4.62 million people in Scotland

Acknowledgements We thank our SHELS collaborators at NHS Information Services Division and at National Records Scotland and the SHELS Phase 3 Steering Group. We acknowledge input to this report from Jenny Holmes, SHELS Study assistant. AS was supported by The Commonwealth Fund, a private independent foundation based in New York City. The views presented here are those of the authors and not necessarily those of The Commonwealth Fund, its directors, officers, or staff. AS is Director of the Asthma UK Centre for Applied Research; he also acknowledges the support of the Farr Institute. Contributors from the Scottish Health and Ethnicity Linkage Study research team: these contributors served on the Steering Group and some on other important subgroups of SHELS, and therefore gave general direction that helped this analysis. Chris Povey was a co-applicant and the originator of the idea of linking the census data to the data held by ISD and he performed most of the linkage work, including developing linkage methods. Prof Jamie Pearce (co-applicant) advised especially on socioeconomic adjustment. Duncan Buchanan (co-applicant) chaired the analysis subgroup. Ganka Mueller (part study), Alex Stannard (part study) and Kirsty MacLachlan advised particularly in relation to National Records of Scotland contributions. These important contributions did not meet ICMJE authorship requirements. Funding Chief Scientist’s Office of the Scottish Government, British Lung Foundation and NHS Health Scotland.Peer reviewedPublisher PD