Quantifying the Detrimental Impacts of Land-Use and Management Change on European Forest Bird Populations

The ecological impacts of changing forest management practices in Europe are poorly understood despite European forests being highly managed. Furthermore, the effects of potential drivers of forest biodiversity decline are rarely considered in concert, thus limiting effective conservation or sustainable forest management. We present a trait-based framework that we use to assess the detrimental impact of multiple land-use and management changes in forests on bird populations across Europe. Major changes to forest habitats occurring in recent decades, and their impact on resource availability for birds were identified. Risk associated with these changes for 52 species of forest birds, defined as the proportion of each species' key resources detrimentally affected through changes in abundance and/or availability, was quantified and compared to their pan-European population growth rates between 1980 and 2009. Relationships between risk and population growth were found to be significantly negative, indicating that resource loss in European forests is an important driver of decline for both resident and migrant birds. Our results demonstrate that coarse quantification of resource use and ecological change can be valuable in understanding causes of biodiversity decline, and thus in informing conservation strategy and policy. Such an approach has good potential to be extended for predictive use in assessing the impact of possible future changes to forest management and to develop more precise indicators of forest health

Melting and differentiation of early-formed asteroids: The perspective from high precision oxygen isotope studies

A number of distinct methodologies are available for determining the oxygen isotope composition of minerals and rocks, these include laser-assisted fluorination, secondary ion mass spectrometry (SIMS)and UV laser ablation. In this review we focus on laser-assisted fluorination, which currently achieves the highest levels of precision available for oxygen isotope analysis. In particular, we examine how results using this method have furthered our understanding of early-formed differentiated meteorites. Due to its rapid reaction times and low blank levels, laser-assisted fluorination has now largely superseded the conventional externally-heated Ni “bomb” technique for bulk analysis. Unlike UV laser ablation and SIMS analysis, laser-assisted fluorination is not capable of focused spot analysis. While laser fluorination is now a mature technology, further analytical improvements are possible via refinements to the construction of sample chambers, clean-up lines and the use of ultra-high resolution mass spectrometers. High-precision oxygen isotope analysis has proved to be a particularly powerful technique for investigating the formation and evolution of early-formed differentiated asteroids and has provided unique insights into the interrelationships between various groups of achondrites. A clear example of this is seenin samples that lie close to the terrestrial fractionation line (TFL). Based on the data from conventional oxygen isotope analysis, it was suggested that the main-group pallasites, the howardite eucrite diogenite suite (HEDs) and mesosiderites could all be derived from a single common parent body. However,high precision analysis demonstrates that main-group pallasites have a Δ17O composition that is fully resolvable from that of the HEDs and mesosiderites, indicating the involvement of at least two parent bodies. The range of Δ17O values exhibited by an achondrite group provides a useful means of assessing the extent to which their parent body underwent melting and isotopic homogenization. Oxygen isotope analysis can also highlight relationships between ungrouped achondrites and the more well-populated groups. A clear example of this is the proposed link between the evolved GRA 06128/9 meteorites and the brachinites. The evidence from oxygen isotopes, in conjunction with that from other techniques, indicates that we have samples from approximately 110 asteroidal parent bodies (∼60 irons, ∼35 achondrites and stony-iron, and ∼15 chondrites) in our global meteorite collection. However, compared to the likely size of the original protoplanetary asteroid population, this is an extremely low value. In addition, almost all of the differentiated samples (achondrites, stony-iron and irons) are derived from parent bodies that were highly disrupted early in their evolution. High-precision oxygen isotope analysis of achondrites provides some important insights into the origin of mass-independent variation in the early Solar System. In particular, the evidence from various primitive achondrite groups indicates that both the slope 1 (Y&R) and CCAM lines are of primordial significance. Δ17O differences between water ice and silicate-rich solids were probably the initial source of the slope 1 anomaly. These phases most likely acquired their isotopic composition as a result of UV photo-dissociation of CO that took place either in the early solar nebula or precursor giant molecular cloud. Such small-scale isotopic heterogeneities were propagated into larger-sized bodies, such as asteroids and planets, as a result of early Solar System processes, including dehydration, aqueous alteration,melting and collisional interactions

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Unravelling the interaction targets and metabolic fate of docosahexaenoyl ethanolamide

Poly-unsaturated fatty acids (PUFAs) and their metabolites have well-described immune regulating functions. In general, n-6 PUFAs and related metabolites possess pro-inflammatory functionality whereas n-3 PUFAs and related metabolites possess less pro-inflammatory or even anti-inflammatory properties. In this thesis the metabolism and anti-inflammatory properties of the endogenously produced n-3 PUFA derivative docosahexaenoyl ethanolamide (DHEA) are described. In chapter 2 a literature overview of the metabolic and immune regulating properties of PUFAs and their acyl derivatives is given. It is described that DHEA levels in murine and human plasma and tissues depend on the dietary intake of n-3 PUFAs. Herein DHEA is not the end-product but oxygenation of DHEA by enzymes like CYP450 and LOX-15 is described. This observation showed that studying the metabolism and immune regulatory roles of n-3 PUFAs and n-3 PUFA derivatives is complex. Recent studies revealed that the use of chemical probes, which are synthetic chemical PUFA or PUFA related compounds with bio-orthogonal handles, can provide an interesting alternative to conventional immunological tools to investigate the immune regulating effects of the PUFAs.In chapter 3, bi-functional synthetic chemical probes were used to map the interactions of the potent anti-inflammatory regulator DHEA and the n-6 PUFA ethanolamine derivative arachidonoyl ethanolamide (AEA) in 1.0 μg/mL LPS stimulated murine RAW264.7 macrophages. Diazirine and alkyne moieties were installed into DHEA and AEA related structures to allow for covalent coupling to proteins using 366 nm UV-light, and subsequent CuAAC click coupling to a fluorescent probe or biotin. Despite the small chemical alterations present in the probe when compared to native DHEA, the bi-functional chemical derived DHEA probes displayed similar anti-inflammatory properties compared to the natural DHEA, on IL-6 and prostaglandin E2 (PGE2) reduction. Uptake of the PUFA derived probes was successful and resulted in cytosolic localisation in the suggested regions of the ER, Golgi-system, and showed vesicular compartmentalisation. Chemical proteomic interactome mapping showed that both AEA and DHEA interacted with the oxygenation enzyme cyclooxygenase 2 (COX-2), but also with peroxiredoxin I, peroxiredoxin IV, different proteins involved in the Rho GTPase signalling pathway, and Rac1 and its related interactome proteins. Immunolabelling studies showed co-localisation as prerequisite for molecular interaction between the PUFA derived amides with COX-2 and Rac1. Bio-informatic analysis of the proteome interactors suggested regulatory roles for DHEA in cytoskeletal remodelling and cell migration, and also in ROS scavenging. Future studies are required to interpret the immunological consequences of the chemical proteomic interactions obtained.To study the interaction between DHEA and COX-2, chapter 4 describes the development of a cell free COX-2 enzymatic assay. First, the enzymatic assay is verified using the known COX-2 substrates arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). After validation of the assay the N-acyl ethanolamine derived PUFAs AEA, eicosapentaenoyl ethanolamide (EPEA), and DHEA were added to the enzymatic assay. For AEA production of prostaglandin ethanolamide E2 PGE2-EA, PGD2-EA, 11-hydroxyeicosatetraenoyl ethanolamide (11-HETE-EA), and 15-HETE-EA were identified, verifying the validity of the methodology. For EPEA the novel products prostaglandin E3 ethanolamide (PGE3-EA), and 11-hydroxypentaenoyl ethanolamide 11-HEPE-EA (and the postulated products 14- and 18-HEPE-EA) were obtained, and DHEA conversion resulted in 13- and 16-hydroxydocosahexaenoyl ethanolamide (13- and 16-HDHEA). Metabolic conversion of EPEA and DHEA was limited resulting in synthetic yields of 0.7% for PGE3-EA, 1.3% for 16-HDHEA, and 1.4% for 13-HDHEA. Production of 13- and 16-HDHEA was also confirmed in 1.0 μg/mL LPS-stimulated RAW264.7 macrophages, in which it was shown that both DHEA addition and LPS-stimulation are required for the production of 13- and 16-HDHEA. Addition of the selective COX-2 inhibitor celecoxib resulted in blocking of 13- and 16-HDHEA synthesis, proving that 13- and 16-HDHEA synthesis in vitro was mediated by COX-2. Incubations with 10 μM DHEA in 1.0 μg/mL LPS-stimulated RAW264.7 macrophages resulted in production of 7.3 (± 2.3) pmol of 13-HDHEA and 8.6 (± 3.5) pmol of 16-HDHEA per 106 RAW macrophage cells.In chapter 5 the immune regulating effect of 13-HDHEA and 16-HDHEA were tested. It was shown that the novel compounds 13- and 16-HDHEA reduced the production of inflammatory cytokines TNFα and IL-1β in 1.0 μg/mL LPS-stimulated RAW264.7 macrophages, but did not affect levels of nitric oxide (NO), IL-6, and the prostaglandins PGE2 and PGD2. Transcriptomic analysis revealed a shift in upregulation of several anti-inflammatory related genes and reduction of pro-inflammatory related genes, especially downstream of toll-like receptor 4 (TLR4) and its regulators. Furthermore, 13- and 16-HDHEA showed distinct anti-inflammatory gene regulation compared to DHEA, but the anti-inflammatory regulation of DHEA was more potent than its COX-2 metabolites in 1.0 μg/mL LPS-stimulated RAW264.7 macrophages. It was therefore proposed that COX-2 metabolism of DHEA could also acts as a regulatory mechanism to limit the anti-inflammatory properties of DHEA.In chapter 6 the effects of intraperitoneally injected (i.p.) DHEA were tested in a DSS induced colitis model in C57Bl/6 mice. The mice received 2% DSS in drinking water (ad libitum) for five days and were injected with a vehicle, 10 mg/kg DHEA, or 15 mg/kg DHEA each day, starting one day before DSS administration and ending one day after the final DSS administration. It was shown that DHEA reduced phenotypic colitis markers like body weight and rectal bleeding, and improved stool consistency. Pathophysiological markers in the colon such as colon length, cellular infiltration, and neutrophil activity were not significantly affected by the DHEA treatment. DHEA metabolism in the liver of these mice was investigated showing that i.p. DHEA treatment resulted in increased DHEA levels in the liver, but DHEA metabolites of COX-2 were not identified. AEA levels and oxylipin levels were not affected in the livers of those mice, which is most likely explained by the absence of COX-2 mRNA gene expression. In conclusion, i.p. DHEA treatment reduced phenotypic colitis markers, but did not affect classical markers of colon damage. Apart from hepatic DHEA levels, no significant metabolic effects were observed in the livers.The thesis is concluded by a general discussion and summary regarding the findings

Immunomodulating effects of 13- and 16-hydroxylated docosahexaenoyl ethanolamide in LPS stimulated RAW264.7 macrophages

Docosahexaenoyl ethanolamide (DHEA), the ethanolamine conjugate of the n-3 long chain polyunsaturated fatty acid docosahexaenoic acid, is endogenously present in the human circulation and in tissues. Its immunomodulating properties have been (partly) attributed to an interaction with the cyclooxygenase-2 (COX-2) enzyme. Recently, we discovered that COX-2 converts DHEA into two oxygenated metabolites, 13- and 16-hydroxylated-DHEA (13- and 16-HDHEA, respectively). It remained unclear whether these oxygenated metabolites also display immunomodulating properties like their parent DHEA. In the current study we investigated the immunomodulating properties of 13- and 16-HDHEA in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The compounds reduced production of tumor necrosis factor alpha (TNFα), interleukin (IL)-1β and IL-1Ra, but did not affect nitric oxide (NO) and IL-6 release. Transcriptome analysis showed that the compounds inhibited the LPS-mediated induction of pro-inflammatory genes (InhbA, Ifit1) and suggested potential inhibition of regulators such as toll-like receptor 4 (TLR4), MyD88, and interferon regulatory factor 3 (IRF3), whereas anti-inflammatory genes (SerpinB2) and potential regulators IL-10, sirtuin 1 (Sirt-1), fluticasone propionate were induced. Additionally, transcriptome analysis of 13-HDHEA suggests a potential anti-angiogenic role. In contrast to the known oxylipin-lowering effects of DHEA, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses revealed that 13- and 16-HDHEA did not affect oxylipin formation. Overall, the anti-inflammatory effects of 13-HDHEA and 16-HDHEA are less pronounced compared to their parent molecule DHEA. Together, this suggests that the COX-2 metabolism of DHEA may act as a regulatory mechanism to limit the anti-inflammatory properties of DHEA

Novel COX-2 products of n-3 polyunsaturated fatty acid-ethanolamine-conjugates identified in RAW264.7 macrophages

Cyclooxygenase 2 (COX-2) plays a key role in the regulation of inflammation by catalyzing the oxygenation of PUFAs to prostaglandins (PGs) and hydroperoxides. Next to this, COX-2 can metabolize neutral lipids, including endocannabinoid-like esters and amides. We developed an LC-HRMS-based human recombinant (h)COX-2 screening assay to examine its ability to also convert n-3 PUFA-derived N-acylethanolamines. Our assay yields known hCOX-2-derived products from established PUFAs and anandamide. Subsequently, we proved that eicosapentaenoylethanolamide (EPEA), the N-acylethanolamine derivative of EPA, is converted into PGE3-ethanolamide (PGE3-EA), and into 11-, 14-, and 18-hydroxyeicosapentaenoyl-EA (11-, 14-, and 18-HEPE-EA, respectively). Interestingly, we demonstrated that docosahexaenoylethanolamide (DHEA) is converted by hCOX-2 into the previously unknown metabolites, 13- and 16-hydroxy-DHEA (13- and 16-HDHEA, respectively). These products were also produced by lipopolysaccharide-stimulated RAW267.4 macrophages incubated with DHEA. No oxygenated DHEA metabolites were detected when the selective COX-2 inhibitor, celecoxib, was added to the cells, further underlining the role of COX-2 in the formation of the novel hydroxylated products. This work demonstrates for the first time that DHEA and EPEA are converted by COX-2 into previously unknown hydroxylated metabolites and invites future studies toward the biological effects of these metabolites.</p

The role of n-3 PUFA-derived fatty acid derivatives and their oxygenated metabolites in the modulation of inflammation

Notwithstanding the ongoing debate on their full potential in health and disease, there is general consensus that n-3 PUFAs play important physiological roles. Increasing dietary n-3 PUFA intake results in increased DHA and EPA content in cell membranes as well as an increase in n-3 derived oxylipin and -endocannabinoid concentrations, like fatty acid amides and glycerol-esters. These shifts are believed to (partly) explain the pharmacological and anti-inflammatory effects of n-3 PUFAs. Recent studies discovered that n-3 PUFA-derived endocannabinoids can be further metabolized by the oxidative enzymes CYP-450, LOX and COX, similar to the n-6 derived endocannabinoids. Interestingly, these oxidized n-3 PUFA derived endocannabinoids of eicosapentaenoyl ethanolamide (EPEA) and docosahexaenoyl ethanolamide (DHEA) have higher anti-inflammatory and anti-proliferative potential than their precursors. In this review, an overview of recently discovered n-3 PUFA derived endocannabinoids and their metabolites is provided. In addition, the use of chemical probes will be presented as a promising technique to study the n-3 PUFA and n-3 PUFA metabolism within the field of lipid biochemistry.</p

Analysis of Omega-3 Fatty Acid-Derived N-Acylethanolamines in Biological Matrices

The adequate quantification of endocannabinoids and related N-acylethanolamines can be complex due to their low endogenous levels, structural diversity, and metabolism. Therefore, advanced analytical approaches, involving LC–MS, are required to quantify these molecules in plasma, tissues, and other matrices. It has been shown that endocannabinoid congeners synthesized from n-3 poly-unsaturated fatty acids (n-3 PUFAs), such as docosahexaenoylethanolamide (DHEA) and eicosapentaenoylethanolamide (EPEA), have interesting immunomodulatory and tumor-inhibiting properties. Recent work has shown that DHEA and EPEA can be further enzymatically metabolized by cyclo-oxygenase 2 (COX-2), forming oxygenated metabolites. Here, an LC–MS-based method for the quantification of the n-3 PUFA-derived endocannabinoid congeners DHEA and EPEA is described, which is also suited to measure a wider spectrum of endocannabinoids. The chapter contains a step-by-step protocol for the analysis of (n-3) endocannabinoids in plasma, including sample collection and solid phase extraction, LC–MS analysis, and data processing. In addition, protocol modifications are provided to allow quantification of n-3 PUFA-derived endocannabinoids and their COX-2 metabolites in tissues and cell culture media. Finally, conditions that alter endocannabinoid concentrations are briefly discussed

Highly Specific Binding on Antifouling Zwitterionic Polymer-Coated Microbeads as Measured by Flow Cytometry

Micron- and nano-sized particles are extensively used in various biomedical applications. However, their performance is often drastically hampered by the nonspecific adsorption of biomolecules, a process called biofouling, which can cause false-positive and false-negative outcomes in diagnostic tests. Although antifouling coatings have been extensively studied on flat surfaces, their use on micro- and nanoparticles remains largely unexplored, despite the widespread experimental (specifically, clinical) uncertainties that arise because of biofouling. Here, we describe the preparation of magnetic micron-sized beads coated with zwitterionic sulfobetaine polymer brushes that display strong antifouling characteristics. These coated beads can then be equipped with recognition elements of choice, to enable the specific binding of target molecules. First, we present a proof of principle with biotin-functionalized beads that are able to specifically bind fluorescently labeled streptavidin from a complex mixture of serum proteins. Moreover, we show the versatility of the method by demonstrating that it is also possible to functionalize the beads with mannose moieties to specifically bind the carbohydrate-binding protein concanavalin A. Flow cytometry was used to show that thus-modified beads only bind specifically targeted proteins, with minimal/near-zero nonspecific protein adsorption from other proteins that are present. These antifouling zwitterionic polymer-coated beads, therefore, provide a significant advancement for the many bead-based diagnostic and other biosensing applications that require stringent antifouling conditions