mTOR (mammalian target of rapamycin) stimulates cell growth by phosphorylating and promoting activation of AGC (protein kinase A/protein kinase G/protein kinase C) family kinases such as Akt (protein ...kinase B), S6K (p70 ribosomal S6 kinase) and SGK (serum and glucocorticoid protein kinase). mTORC1 (mTOR complex-1) phosphorylates the hydrophobic motif of S6K, whereas mTORC2 phosphorylates the hydrophobic motif of Akt and SGK. In the present paper we describe the small molecule Ku-0063794, which inhibits both mTORC1 and mTORC2 with an IC50 of approximately 10 nM, but does not suppress the activity of 76 other protein kinases or seven lipid kinases, including Class 1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher concentrations. Ku-0063794 is cell permeant, suppresses activation and hydrophobic motif phosphorylation of Akt, S6K and SGK, but not RSK (ribosomal S6 kinase), an AGC kinase not regulated by mTOR. Ku-0063794 also inhibited phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3-phosphoinositide-dependent protein kinase-1). We interpret this as implying phosphorylation of Ser473 promotes phosphorylation of Thr308 and/or induces a conformational change that protects Thr308 from dephosphorylation. In contrast, Ku-0063794 does not affect Thr308 phosphorylation in fibroblasts lacking essential mTORC2 subunits, suggesting that signalling processes have adapted to enable Thr308 phosphorylation to occur in the absence of Ser473 phosphorylation. We found that Ku-0063794 induced a much greater dephosphorylation of the mTORC1 substrate 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) than rapamycin, even in mTORC2-deficient cells, suggesting a form of mTOR distinct from mTORC1, or mTORC2 phosphorylates 4E-BP1. Ku-0063794 also suppressed cell growth and induced a G1-cell-cycle arrest. Our results indicate that Ku-0063794 will be useful in delineating the physiological roles of mTOR and may have utility in treatment of cancers in which this pathway is inappropriately activated.
Substantial amounts of nutrients are lost from soils through leaching. These losses can be environmentally damaging, causing groundwater eutrophication and also comprise an economic burden in terms ...of lost agricultural production. More intense precipitation events caused by climate change will likely aggravate this problem. So far it is unresolved to which extent soil biota can make ecosystems more resilient to climate change and reduce nutrient leaching losses when rainfall intensity increases. In this study, we focused on arbuscular mycorrhizal (AM) fungi, common soil fungi that form symbiotic associations with most land plants and which increase plant nutrient uptake. We hypothesized that AM fungi mitigate nutrient losses following intensive precipitation events (higher amount of precipitation and rain events frequency). To test this, we manipulated the presence of AM fungi in model grassland communities subjected to two rainfall scenarios: moderate and high rainfall intensity. The total amount of nutrients lost through leaching increased substantially with higher rainfall intensity. The presence of AM fungi reduced phosphorus losses by 50% under both rainfall scenarios and nitrogen losses by 40% under high rainfall intensity. Thus, the presence of AM fungi enhanced the nutrient interception ability of soils, and AM fungi reduced the nutrient leaching risk when rainfall intensity increases. These findings are especially relevant in areas with high rainfall intensity (e.g., such as the tropics) and for ecosystems that will experience increased rainfall due to climate change. Overall, this work demonstrates that soil biota such as AM fungi can enhance ecosystem resilience and reduce the negative impact of increased precipitation on nutrient losses.
Arbuscular mycorrhizal (AM) fungi enhance ecosystem resilience to climate change by reducing the negative impact of increased precipitation on nutrient losses. AM fungi enhanced the nutrient perception ability of soils and reduced the nutrient leaching losses. The effects of mycorrhizal fungi were more pronounced under the high rainfall scenario, indicating that these soil fungi could play a role in mitigating the negative consequences of climate change.
Worldwide outbreaks of enterovirus D68 (EV-D68) in 2014 and 2016 have caused serious respiratory and neurological disease. We collected samples from several European countries during the 2018 ...outbreak and determined 53 near full-length genome ('whole genome') sequences. These sequences were combined with 718 whole genome and 1,987 VP1-gene publicly available sequences. In 2018, circulating strains clustered into multiple subgroups in the B3 and A2 subclades, with different phylogenetic origins. Clusters in subclade B3 emerged from strains circulating primarily in the US and Europe in 2016, though some had deeper roots linking to Asian strains, while clusters in A2 traced back to strains detected in East Asia in 2015-2016. In 2018, all sequences from the USA formed a distinct subgroup, containing only three non-US samples. Alongside the varied origins of seasonal strains, we found that diversification of these variants begins up to 18 months prior to the first diagnostic detection during a EV-D68 season. EV-D68 displays strong signs of continuous antigenic evolution and all 2018 A2 strains had novel patterns in the putative neutralizing epitopes in the BC- and DE-loops. The pattern in the BC-loop of the USA B3 subgroup had not been detected on that continent before. Patients with EV-D68 in subclade A2 were significantly older than patients with a B3 subclade virus. In contrast to other subclades, the age distribution of A2 is distinctly bimodal and was found primarily among children and in the elderly. We hypothesize that EV-D68's rapid evolution of surface proteins, extensive diversity, and high rate of geographic mixing could be explained by substantial reinfection of adults. Better understanding of evolution and immunity across diverse viral pathogens, including EV-D68 and SARS-CoV-2, is critical to pandemic preparedness in the future.
Toll-like receptor (TLR) agonists demonstrate therapeutic promise as immunological adjuvants for anticancer immunotherapy. To date, three TLR agonists have been approved by US regulatory agencies for ...use in cancer patients. Additionally, the potential of hitherto experimental TLR ligands to mediate clinically useful immunostimulatory effects has been extensively investigated over the past few years. Here, we summarize recent preclinical and clinical advances in the development of TLR agonists for cancer therapy.
Litter decomposition and nutrient mineralization are crucial in agricultural systems to maintain soil fertility and plant growth. Given that these processes are governed by soil microbial activity, ...agricultural management that affects soil microbial communities may significantly alter rates of decomposition and N release of the same litter. We hypothesized that organic compared to conventional management enhances litter decomposition and litter N release, and that this effect is stronger for litter of low quality (high C:N ratio).
We tested these hypotheses using litter from 4 maize cultivars with varying initial litter quality (different C:N ratios and lignocellulose index). These litters were left to decompose in soil with different management history, yet in the same experimental field site. The field experiment consisted of randomized plots with 11 years of organic or conventional agricultural management (organic vs. mineral fertilization). During the 11 years, in year 3 and 4, two specific organic amendments were applied as soil health treatments (SHT: chitin or compost, and a control without SHT). The maize litter was contained in litter bags, buried in the top 10–15 cm soil and collected after 1, 2 and 3 months. We quantified the litter carbon (C) and nitrogen (N) loss, and soil dissolved organic carbon (DOC), mineral and dissolved organic nitrogen (DON) at each sampling time. We also determined the fungal biomass in the decomposing litter after 3 months of decomposition.
Litter C loss was higher in soil under organic compared to conventional management, irrespective of litter quality. In contrast, the rate of N release from the litter was determined by initial litter quality (higher N release from low C:N litter) and not by agricultural management. In soil under organic management the concentrations of DOC, mineral N and DON were larger than in conventional managed soil, which may have stimulated microbial activity and therefore, litter decomposition. Fungal biomass in the decomposing litter negatively correlated with the amount of N in the decomposing litter, but was not affected by management system or litter cultivar.
Overall, we found that in agroecosystems initial litter quality (C:N) is a main driver of litter N release, whereas soil management is a main driver of decomposing litter C loss. Our results show the importance of integrating both litter quality and soil management to enhance our understanding of litter decomposition and N release, and to harness the ecosystem services provided by crop litter in agricultural fields.
In agroecosystems, the main driver of litter C loss is agricultural practice (conventional vs. organic) while litter quality of the cultivar is the main driver of N loss. Agricultural practice and litter quality also influence the microbial community and the availability of soil nutrients which has an effect on the decomposition process. Display omitted
•Management is a main driver of litter decomposition regardless of litter quality.•Litter quality, not management, is a main driver of litter nitrogen release.•Organic management enhances litter decomposition.•Cultivar selection based on decomposability traits can be used to enhance C cycling.•No 7 year legacy effects of compost and chitin on litter C and N loss.
Antimicrobial drug resistance is a serious challenge in clinical settings worldwide, with biofilm formation having been associated with this problem. In the present study, the synergism of ...oligostyrylbenzene (OSB) compounds in combination with amphotericin B (AmB) against Candida tropicalis biofilms was investigated. In addition, the toxicity in human blood cells was determined. Synergistic combinations of OSBs and AmB were evaluated to consider future effects of OSBs in vivo. The checkerboard microdilution method was used to study the interactions of one anionic (1) and two cationic (2 and 3) OSBs with AmB. We investigated the effects of OSBs on reactive oxygen species (ROS) and the levels of the reactive nitrogen intermediates (RNIs). The cellular stress affected biofilm growth through an accumulation of ROS and RNI, at synergistic concentrations of OSBs and AmB. Furthermore, significant surface topography differences were noted upon treatment with the OSB 2/AmB combination, using confocal laser scanning microscopy in conjunction with the image analysis software COMSTAT. The results revealed a low toxicity to leukocytes and red blood cells at synergistic combinations of cationic OSBs with AmB. These findings demonstrated the antibiofilm effects of OSBs and the synergism of AmB with cationic OSBs against biofilms of C. tropicalis for the first time.
Take Away
A significant reduction of mature biofilms was observed for OSB compounds.
Synergistic activity was observed when OSBs 2 and 3 were combined with AmB against mature biofilms of C. tropicalis.
OSBs promoted oxidative and nitrosative stress in C. tropicalis.
The synergistic combination produced alterations on the surface of the biofilms.
The architecture and topography of the surface revealed different diffusion distances that changed the flow over the interface of the surface and the biofilms.
An epidemiological study was carried out to investigate asymptomatic
Leishmania infantum infection by PCR and ELISA in wild rabbits, humans and domestic dogs in southeastern Spain. Seroprevalence was ...0% (0/36) in rabbits, 2% (13/657) in humans and 7% (14/208) in dogs. The prevalence of PCR-positives was 0.6% (1/162) in rabbits tested in a wide range of tissue samples, 2% (8/392) in humans analysed in blood samples and 10% (20/193) and 67% (29/43) in dogs analysed in blood and lymphoid tissue samples, respectively. Results suggest that wild rabbits have a very low risk of becoming chronically infected with
L. infantum, and provide further evidence that cryptic
L. infantum infection is widespread in the domestic dog population and is also present in a comparatively smaller proportion of healthy humans. The epidemiological and clinical implications of these findings are discussed.
Uncontrolled hemorrhagic shock is a leading cause of early death after injury. Resuscitative endovascular balloon occlusion of the aorta (REBOA) represents a paradigm shift in achieving hemodynamic ...stability and its implementation still remain controversial in different settings. The recently published UK-REBOA Randomized Clinical Trial aimed to determine the effectiveness of REBOA in patients with hemorrhagic shock, concluding its increased mortality compared with standard care alone.
An adjustment of the statistical analysis was performed and a comprehensive analysis was proposed to address the study's limitations and demonstrate that these conclusions cannot be considered as benchmarks.
Primary and secondary outcomes were analyzed using Bayesian logistic regression and generalized linear models suitable for the outcome distribution. No statistically significant differences were observed between the two groups for the primary outcome (p-value 0.3341) nor in most of the secondary outcomes. The results of the principal stratum analyses (to account for intercurrent events) also did not show significant differences after the statistical analysis tests.
It cannot be stated that REBOA increases mortality compared with standard care alone in trauma patients with exsanguinating hemorrhage. Further studies and adequate simulation training programs in REBOA are critical to its successful implementation within a trauma system and to identify the optimum settings and patients.
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•REBOA represents a paradigm shift as a bridge to definitive hemostasis•The UK REBOA-trial concluded that in trauma exsanguinating patients a strategy of REBOA may increase mortality compared with standard care alone.•The present comprehensive analysis and the adjusted statistical analysis reflect that these conclusions cannot be considered benchmarks.•Further studies and adequate simulation training programs in REBOA are critical to its successful implementation within a trauma system.
Texturization of surfaces is usually advantageous in biomaterial engineering. However, the details of the textured surfaces can be more determining on cell adhesion and proliferation, rather than ...their roughness degree. Titanium is extensively used as a dental implant material in the human body. In this paper, the effect of four surface treatments on commercially pure titanium has been evaluated. These treatments were polishing (pTi); hydrofluoric acid (HF) etching (eTi); Al
2O
3 blasting (bTi); Al
2O
3 blasting
+
HF etching (beTi). Roughness and fractal dimensions were obtained from atomic force microscopy. Wettability was measured using water sessile drops. Morphology and surface chemical composition were analyzed with scanning electron microscopy and energy dispersive X-ray (EDX). MG-63 cell cultures were performed at different times (180
min, 24
h, 48
h, 72
h). Lowest roughness was found in pTi samples followed by eTi, bTi and beTi samples. Etching generated surfaces with the highest fractal dimension and negative skewness. Young contact angles were similar except for pTi and bTi surfaces. Silicon and aluminum traces were found in pTi and bTi samples, respectively. Cell adhesion (≤24
h) was greater on bTi and beTi surfaces. After 48
h, cell proliferation, mediated by specific morphologies, was improved in eTi samples followed by beTi surfaces. For the same surface chemistry, cell growth was driven by topography features.
Simultaneous determination of fertility hormones in saliva using disposable immunosensing platforms and a custom designed field-portable dual potentiostat.
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•Simultaneous determination ...of progesterone and luteinizing hormone in saliva samples.•Disposable immunoplatforms and custom designed field-portable dual potentiostat.•LODs of 1.7 pg mL−1 and 0.10 mIU mL-1 for P4 and LH, respectively, in less than 1 h.•Accurate determination of both target hormones in saliva samples.
This work describes a new electroanalytical device for the simultaneous and reliable determination of two fertility relevant hormones (luteinizing hormone, LH, and progesterone, P4) in saliva. The device is constructed using a custom designed and field-portable potentiostat where dual disposable immunosensing platform are connected. The immunosensors are based on sandwich-type and competitive immunoassays implemented onto magnetic microbeads (MBs) functionalized with Neutravidin and Protein G for the determination of LH and P4, respectively. Amperometric detection performed at −0.20 V vs the Ag pseudo-reference electrode using the H2O2/hydroquinone (HQ) system was employed as the transduction technique after placing the MBs with immunocomplexes for each target hormone on the appropriate working electrode of screen-printed dual carbon electrodes (SPdCEs). The method exhibits high sensitivity and selectivity for the target hormones providing detection limits of 1.7 pg mL−1 and 0.10 mIU mL−1 for P4 and LH, respectively, with a 1 h test time. The applicability of the method was confirmed by determining both hormones in saliva samples from different volunteers providing results comparable to those obtained using amperometric transduction with a commercial potentiostat and with ELISA methodologies involving the same immunoreagents for each target hormone.
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