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Accurate information on diet composition is central to understanding and conserving carnivore populations. Quantitative fatty acid signature analysis (QFASA) has emerged as a powerful tool for estimating the diets of predators, but ambiguities remain about the timeframe of QFASA estimates and the need to account for species-specific patterns of metabolism. We conducted a series of feeding experiments with four juvenile male brown bears (Ursus arctos) to (1) track the timing of changes in adipose tissue composition and QFASA diet estimates in response to a change in diet and (2) quantify the relationship between consumer and diet FA composition (i.e., determine "calibration coefficients"). Bears were fed three compositionally distinct diets for 90-120 days each. Two marine-based diets were intended to approximate the lipid content and composition of the wild diet of polar bears (U. maritimus). Bear adipose tissue composition changed quickly in the direction of the diet and showed evidence of stabilization after 60 days. During hibernation, FA profiles were initially stable but diet estimates after 10 weeks were sensitive to calibration coefficients. Calibration coefficients derived from the marine-based diets were broadly similar to each other and to published values from marine-fed mink (Mustela vison), which have been used as a model for free-ranging polar bears. For growing bears on a high-fat diet, the temporal window for QFASA estimates was 30-90 days. Although our results reinforce the importance of accurate calibration, the similarities across taxa and diets suggest it may be feasible to develop a generalized QFASA approach for mammalian carnivores.

The traditional and current use of Calotropis procera and C. gigantea, two soft-wooded, xerophytic shrubs of the family Apocynaceae, are reviewed against the background of the plants' chemical constituents and their biological properties. The focus is on the usage of the plants for building materials, natural pesticides, animal feed and bioremediative purposes.

Most biology undergraduates learn about mutations in multiple classrooms throughout their college career. Understanding personalised genome test results, genome editing controversies, and the appearance of new variants of viruses or antibiotic resistant bacteria all require foundational knowledge about mutations. However, the abstract nature of molecular processes surrounding mutations makes them one of the more difficult topics for students to understand and apply. Instructors need valid assessment tools to document student understanding and tailor their instructional methods to address student knowledge gaps. We describe here the development and validation of the Mutations Criterion Referenced Assessment (MuCRA). This formative assessment was developed through an iterative process involving expert feedback and student responses to both open-ended and multiple-choice questions. The final MuCRA is composed of 10 multiple-choice questions aligned with three learning objectives. The item difficulty for each question was between 0.32-0.65, while the discrimination index ranged from 0.31-0.75 and the reliability (KR20) for the MuCRA was 0.69. The congruence analyses demonstrated distractors are capturing student misconceptions in 9/10 questions. These data indicate that the MuCRA can be used to reliably assess student learning and common misconceptions about mutations.

Fundamental knowledge on free-ranging animals has been obtained through capture-based studies; however, these may be logistically intensive, financially expensive, and potentially inconsistent with local cultural values. Genetic mark–recapture using remote tissue sampling has emerged as a less invasive alternative to capture-based population surveys but provides fewer opportunities to collect samples and measurements for broader ecological studies. We compared lipid content, fatty acid (FA) composition, and diet estimates from adipose tissue of polar bears (Ursus maritimus) obtained from two collection methods: remote biopsies (n = 138) sampled from helicopters and hunter-collected tissue (n = 499) from bears harvested in Davis Strait and Gulf of Boothia, Nunavut, 2010 – 2018. Lipid content of adipose tissue was lower in remote biopsies than harvest samples likely because remote biopsies removed only the outermost layer of subcutaneous tissue, rather than the more metabolically dynamic innermost tissue obtained from harvest samples. In contrast, FA composition was similar between the two collection methods with relatively small proportional differences in individual FAs. For diet estimates in Davis Strait, collection method was not a predictor of prey contribution to diet. In Gulf of Boothia, collection method was a predictor for some prey types, but the differences were relatively minor; the rank order of prey types was similar (e.g., ringed seal; Pusa hispida was consistently the primary prey in diets) and prey proportions differed by < 6% between the collection methods. Results from both methods showed that diets varied by geographic area, season, year, age class, and sex. Our study demonstrates that adipose tissue from remote biopsy provides reliable estimates of polar bear diet based on FA analysis and can be used to monitor underlying ecological changes in Arctic marine food webs.

Supplemental Digital Content is available in the text., Background: Knowledge gaps remain in the epidemiology and clinical implications of myocardial injury in coronavirus disease 2019 (COVID-19). We aimed to determine the prevalence and outcomes of myocardial injury in severe COVID-19 compared with acute respiratory distress syndrome (ARDS) unrelated to COVID-19. Methods: We included intubated patients with COVID-19 from 5 hospitals between March 15 and June 11, 2020, with troponin levels assessed. We compared them with patients from a cohort study of myocardial injury in ARDS and performed survival analysis with primary outcome of in-hospital death associated with myocardial injury. In addition, we performed linear regression to identify clinical factors associated with myocardial injury in COVID-19. Results: Of 243 intubated patients with COVID-19, 51% had troponin levels above the upper limit of normal. Chronic kidney disease, lactate, ferritin, and fibrinogen were associated with myocardial injury. Mortality was 22.7% among patients with COVID-19 with troponin under the upper limit of normal and 61.5% for those with troponin levels >10 times the upper limit of normal (P

Organoid models of early tissue development have been produced for the intestine, brain, kidney and other organs, but similar approaches for the heart have been lacking. Here we generate complex, highly structured, three-dimensional heart-forming organoids (HFOs) by embedding human pluripotent stem cell aggregates in Matrigel followed by directed cardiac differentiation via biphasic WNT pathway modulation with small molecules. HFOs are composed of a myocardial layer lined by endocardial-like cells and surrounded by septum-transversum-like anlagen; they further contain spatially and molecularly distinct anterior versus posterior foregut endoderm tissues and a vascular network. The architecture of HFOs closely resembles aspects of early native heart anlagen before heart tube formation, which is known to require an interplay with foregut endoderm development. We apply HFOs to study genetic defects in vitro by demonstrating that NKX2.5-knockout HFOs show a phenotype reminiscent of cardiac malformations previously observed in transgenic mice., Heart-forming organoids model early cardiac development.

Titanium dioxide nanoparticles (TiO2-NPs) are widely manufactured and can be released into the environment, where they have the potential to severely affect fish and human health. This study was conducted to evaluate the effect of TiO2-NPs on the growth of fingerlings of Labeo rohita. TiO2-NPs were used in different concentrations (1 mg/kg, 10 mg/kg, 100 mg/kg and 1000 mg/kg) mixed in fish basal feed for a period of 12 weeks. The mortality rate was recorded once the fish were acclimatized, after TiO2-NPs exposure. The growth in length (cm) and weight (g) of the fish was measured to evaluate the time and concentration dependent growth of the fingerlings. A high mortality rate was observed for the fish exposed to high concentrations of TiO2-NPs from week 10 onwards. A gradual increase in the body weight and length of the fish was observed for all treatments until the 8th week, which thereafter changed differently for different treatments. It could be shown that TiO2-NPs exert concentration dependent bi-modal effects on fish growth with a longer exposure to high concentrations of TiO2-NPs having an adverse effect on the growth. Therefore, TiO2-NPs in natural water bodies may affect the aquaculture industry as well as inland fisheries.

BACKGROUND: B-cell affinity maturation in germinal center relies on regulated actin dynamics for cell migration and cell-to-cell communication. Activating mutations in the cytoskeletal regulator Wiskott-Aldrich syndrome protein (WASp) cause X-linked neutropenia (XLN) with reduced serum level of IgA. OBJECTIVE: We investigated the role of B cells in XLN pathogenesis. METHODS: We examined B cells from 6 XLN patients, 2 of whom had novel R268W and S271F mutations in WASp. By using immunized XLN mouse models that carry the corresponding patient mutations, WASp L272P or WASp I296T, we examined the B-cell response. RESULTS: XLN patients had normal naive B cells and plasmablasts, but reduced IgA+ B cells and memory B cells, and poor B-cell proliferation. On immunization, XLN mice had a 2-fold reduction in germinal center B cells in spleen, but with increased generation of plasmablasts and plasma cells. In vitro, XLN B cells showed reduced immunoglobulin class switching and aberrant cell division as well as increased production of immunoglobulin-switched plasma cells. CONCLUSIONS: Overactive WASp predisposes B cells for premature differentiation into plasma cells at the expense of cell proliferation and immunoglobulin class switching. ispartof: JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY vol:149 issue:3 pages:1069-1084 ispartof: location:United States status: published

Ecological theory suggests that demographic responses by populations to environmental change vary depending on whether individuals inhabit central or peripheral regions within the species’ geographic range. Here, we tested this prediction by comparing a population of ringed seals Pusa hispida located at high latitudes as part of their core range (core) with a population located at the southern extremity of their range (peripheral). First, we compared the two regions’ environmental trends in timing of sea-ice breakup and freeze-up, open-water duration and the North Atlantic Oscillation (NAO). We found that the core region shifted to progressively warmer conditions in the early 1990s; whereas, in the peripheral region, the warming trend shifted in 1999 to one with no warming trend but high inter-annual variability. Next, we examined how body condition, inferred from blubber depth, responded to temporal changes in sea-ice and climatic variables – variables that have been shown to influence ringed seal demography. Core seals displayed minimal seasonal changes in body condition; whereas peripheral seals displayed a 20–60% amplitude seasonal change in body condition with a phase shift to earlier initiation of fat accumulation and loss. Finally, we tested for interannual differences and found that both core and peripheral seals responded similarly with decreased body condition following more positive NAO. Environmental variables influenced body condition in opposite directions between the two regions with core seals declining in body condition with later spring breakup and shorter open-water duration, whereas peripheral seals showed opposite relationships. Seals living at the core likely benefit from an evolved match between adaptation and environmental variation resulting in dampened seasonal and interannual fluctuations in body condition. Knowledge of how different populations respond to environmental change depending on geographic location within a species range can assist in anticipating population specific responses to climate warming.

Summary Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) represent an attractive model to investigate CM function and disease mechanisms. One characteristic marker of ventricular specificity of human CMs is expression of the ventricular, slow β-myosin heavy chain (MyHC), as opposed to the atrial, fast α-MyHC. The main aim of this study was to investigate at the single-cell level whether contraction kinetics and electrical activity of hESC-CMs are influenced by the relative expression of α-MyHC versus β-MyHC. For effective assignment of functional parameters to the expression of both MyHC isoforms at protein and mRNA levels in the very same hESC-CMs, we developed a single-cell mapping technique. Surprisingly, α- versus β-MyHC was not related to specific contractile or electrophysiological properties of the same cells. The multiparametric cell-by-cell analysis suggests that in hESC-CMs the expression of genes associated with electrical activity, contraction, calcium handling, and MyHCs is independently regulated., Highlights • Single-cell mapping technique allows multiparametric characterization of hESC-CMs • Twitch and action potential kinetics are independent of MyHC isoform in hESC-CMs • Myosin-mRNA levels are only weakly correlated with respective protein levels, In this article, Natalie Weber and colleagues developed a single-cell mapping technique for multiparametric analysis of hESC cardiomyocytes (hESC-CMs). Characterization of function (twitch and action potential) in direct relation to the expressed myosin heavy chain isoform by multiparametric analysis suggests that in hESC-CMs the expression of genes associated with electrical activity, contraction, calcium handling, and myosin heavy chains is independently regulated.

Polar bears (Ursus maritimus) and ringed seals (Pusa hispida) have a strong predator-prey relationship and are facing climate-associated Arctic habitat loss and harmful dietary exposure to total mercury (THg) and other pollutants. However, little is known about whether both species inhabiting the same area exhibit similar temporal patterns in Hg concentration, niche dynamics, and body fat indices. We used THg, δ13C, and δ15N values of western Hudson Bay polar bear hair (2004-2016) and ringed seal muscle samples (2003-2015) to investigate temporal trends of these variables and multidimensional niche metrics, as well as body fat indices for both species. We found a decline in THg concentration (by 3.8% per year) and δ13C (by 1.5‰) in ringed seals suggesting a change in feeding habits and carbon source use over time, whereas no significant changes occurred in polar bears. In contrast, the polar bear 3-dimensional niche size decreased by nearly half with no change in ringed seal niche size. The δ13C spacing between both species increased by approximately 1.5× suggesting different responses to annual changes in sympagic-pelagic carbon source production. Ringed seal body fat index was higher in years of earlier sea ice breakup with no change occurring in polar bears. These findings indicate that both species are responding differently to a changing environment suggesting a possible weakening of their predator-prey relationship in western Hudson Bay.

Climate warming and associated physical and biological changes will likely force widespread species redistribution, particularly in polar environments. However, tracking such distributional shifts is difficult. The dietary habits of apex predators, like polar bears (Ursus maritimus), may provide early signals of distributional change in prey populations. We used harvest-based sampling to investigate the spatial feeding patterns of polar bears across Nunavut from 2010 to 2018 (n = 1570) and identify spatiotemporal clusters of different prey based on predator diet estimates. Quantitative fatty acid signature analysis and the Getis-Ord Gi* statistic identified spatial clusters of high or low dietary proportions (i.e., “hot spots” and “cold spots”) reflecting seasonal and spatial availability of prey. Ringed seal (Pusa hispida) was the primary prey of bears throughout Nunavut followed by bearded seal (Erignathus barbatus), although proportional consumption varied spatially. A consistent ringed seal consumption hot spot was found in Gulf of Boothia indicating the importance of year-round availability of ringed seals. Spatial clusters of bearded seal and Atlantic walrus (Odobenus rosmarus rosmarus) throughout Foxe Basin suggested overlapping seasonal distributions and high regional abundance. Bears had consistently high dietary levels of harbour seal (Phoca vitulina) around Southampton Island and along the western coast of Hudson Bay suggesting a possible year-round concentration of this prey. Hot spots of harp seal (Pagophilus groenlandicus) consumption were evident throughout Davis Strait and a spring-summer hot spot around Jones Sound was consistent with harp seal migratory patterns. Year-round beluga whale (Delphinapterus leucas) hot spots were found along eastern Baffin Island and southern Viscount Melville Sound providing new knowledge of local conditions that promote polar bear predation or scavenging. Narwhal (Monodon monoceros) were less susceptible to predation with only one spatial cluster of high consumption appearing during spring-summer in Barrow Strait. Bowhead whale (Balaena mysticetus) hot spots occurred around south-western Foxe Basin and seasonally in southern Viscount Melville Sound suggesting carcasses are locally accessible to bears and may act as a supplemental food source in particular areas and seasons. The congruence of polar bear feeding habits and known prey distribution suggests polar bears serve as ecological indicators and ongoing monitoring of their diets may reveal regional and broad-scale changes in prey population distributions and Arctic ecosystem functioning.

The salivary gland can be permanently impaired by radiation treatment for head and neck cancers. Efforts at tissue regeneration have focused on saliva-producing acinar cells. However, myoepithelial cells are also critical to gland function, but mechanisms that regulate their differentiation are poorly defined. To study myoepithelial differentiation, we employed mSG-PAC1 murine salivary gland epithelial cells. We demonstrate that mSG-PAC1 spheroids exhibit phenotypic plasticity between pro-acinar and myoepithelial cell fates. Increased expression of pro-acinar/acinar or myoepithelial RNAs was identified from spheroids cultured under different media conditions by microarray followed by gene-set enrichment analysis. Spheroids cultured with different medium components expressed proteins typical of either acinar or myoepithelial cells, as detected by immunocytochemistry. We demonstrate that the pattern of TAZ expression in the epithelial compartment of the differentiating murine salivary gland correlates with the expression of the myoepithelial marker alpha-SMA, as is the case for TAZ expression in mSG-PAC1 spheroids. Our analysis also indicates that YAP/TAZ target genes are upregulated together with myoepithelial markers. Importantly, siRNA targeting of TAZ expression in mSG-PAC1 spheroids diminished the expression of myoepithelial markers. Our results in thisin vitrocell model implicate TAZ signaling in myoepithelial differentiation.

Polar bears (Ursus maritimus) rely on sea ice for hunting marine mammal prey. Declining sea ice conditions associated with climate warming have negatively affected polar bears, especially in the southern portion of their range. At higher latitudes, the transition from multi-year ice to thinner annual ice has been hypothesized to increase biological productivity and potentially improve polar bear foraging conditions. To investigate this possibility, we used quantitative fatty acid signature analysis to characterize the diet composition of 148 polar bears in two high-latitude subpopulations from 2012 to 2014: (1) Viscount Melville Sound, where little is known about marine mammal ecology, and (2) Northern Beaufort Sea, a subpopulation considered stable with comparatively more ecological data. We used adipose tissue lipid content as an index of body condition. To characterize long-term habitat conditions, we examined trends in sea ice metrics from 1979 to 2014 in both regions. Although the diets of bears in both subpopulations were dominated by ringed seal (Pusa hispida, mean biomass consumption = 45%), bears in Viscount Melville Sound showed higher proportional consumption of beluga whale (Delphinapterus leucas; mean biomass consumption = 37%) than any other polar bear subpopulation studied to date. Although the three-year duration of our study precludes long-term insights, relatively lighter sea ice conditions in Viscount Melville Sound were associated with reduced consumption of preferred prey (i.e., ringed seal), especially among female polar bears. Further, polar bears in Viscount Melville sound were in poorer body condition than those in the Northern Beaufort Sea. Our results do not indicate that declining sea ice has had any positive effect on polar bear foraging at high-latitudes.

Mammals adapted to unpredictable and low-energy environments often evolve a “bet-hedging” life history strategy characterized by less costly reproductive outputs over a longer and slower-growing life. In contrast, species adapted to more predictable (i.e., low variation) and higher energy environments may evolve greater fecundity over a shorter and faster-growing life. We tested whether this known interspecific pattern also occurs within a species. We compared life history traits of the ringed seal (Pusa hispida) in the Canadian High Arctic to those closer to the southern limit of the species' circumpolar distribution. We found that northern seals grew slower than southern seals (Brody growth coefficient), achieved a greater asymptotic body weight (82 and 69 kg vs. 74 and 54 kg female and male, respectively), reached sexual maturity later (6.1 years vs. 4.5 years), had lower fecundity (1.8 years vs. 1.3 years interbirth interval), longer average lifespan (5 years vs. 3 years median age), and greater movements (1,269 vs. 681 km). Mating systems also likely differed with northern seals showing morphological evidence of a promiscuous mating system with potential sperm competition as indicated by greater relative testes size. The northern region was also characterized by more unpredictable environmental timing of seasonal events, such as spring sea ice breakup. Life history variation between the intraspecific groups of seals appears to agree with interspecific patterns and provides a better understanding of how species' life history parameters shift in concert with environmental conditions.

Targeting of tumor immune escape mechanisms holds enormous therapeutic potential. Still, most patients progress under immune checkpoint blockade and some even become hyperprogressors. To investigate how cancer cells respond to activated but ineffective T cells, we challenged peptide-loaded MCF-7 breast cancer cells with antigen-specific CD8+ T cells in which lytic granules had been destroyed by pretreatment with Concanamycin A. Gene expression analysis after coculture revealed simultaneous induction of PD-L1, IDO1, CEACAM1, and further immunoregulatory checkpoints in breast cancer cells. Strikingly, we further observed gene signatures characteristic for dedifferentiation and acquisition of pluripotency markers including Yamanaka factors. Cognate interaction with nonlytic CD8+ T cells also increased the proportion of stem cell–like cancer cells in a cell-to-cell contact- or (at least) proximity-dependent manner in various cell lines and in primary breast cancer cell cultures; this induction of stem cell–like properties was confirmed by enhanced tumor-forming capacity in immunodeficient mice. Resulting tumors were characterized by enhanced cell density, higher proliferation rates, and increased propensity for lymphoid metastasis. These findings describe a widely underappreciated pathway for immune escape, namely immune-mediated dedifferentiation of breast cancer cells, which is associated with profound changes in gene expression and cellular behavior. As the enhanced malignant potential of cancer cells after nonlytic cognate interactions with CD8+ T cells enables increased tumor growth and metastasis in BALB/cnu/nu mice, the described mechanism may provide a possible explanation for the clinical phenomenon of hyperprogression in response to unsuccessful immunotherapy. Significance: This study shows that ineffective immune responses not only fail to clear a malignancy, but can also activate pathways in cancer cells that promote stemness and tumor-seeding capacity.

Extracellular matrix (ECM) degrading enzymes produced by Clostridium perfringens may play an important role during the initial phases of avian necrotic enteritis by facilitating toxin entry in the intestinal mucosa and destruction of the tissue. C. perfringens is known to produce several ECM-degrading proteases, such as kappa toxin, an extracellular collagenase that is encoded by the colA gene. In this study, the colA gene sequence of a collection of 48 C. perfringens strains, including pathogenic (i.e. toxinotype G) and commensal (i.e. toxinotype A) chicken derived strains and strains originating from other host species, was analyzed. Although the colA gene showed a high level of conservation (>96% nucleotide sequence identity), several gene variants carrying different nonsense mutations in the colA gene were identified, leading to the definition of four truncated collagenase variant types (I-IV). Collagenase variant types I, III and IV have a (nearly) complete collagenase unit but lack parts of the C-terminal recruitment domains, whereas collagenase variant types II misses the N-terminal part of collagenase unit. Gene fragments encoding a truncated collagenase were mainly linked with necrotic enteritis associated C. perfringens type G strains with collagenase variant types I and II being the most prevalent types. Gelatin zymography revealed that both recombinant full-length and variant type I collagenase have active auto-cleavage products. Moreover, both recombinant fragments were capable of degrading type I as well as type IV collagen, although variant type I collagenase showed a higher relative activity against collagen type IV as compared to full-length collagenase. Consequently, these smaller truncated collagenases might be able to break down collagen type IV in the epithelial basement membrane of the intestinal villi and so contribute to the initiation of the pathological process leading to necrotic enteritis.

Ecological flexibility of a species reflects its ability to cope with environmental change. Although polar bears (Ursus maritimus) are experiencing changes in foraging opportunities due to sea ice loss, regional prey availability and environmental conditions will influence the rate and severity of these effects. We examined changes in polar bear diet and the influence of sea ice characteristics in Foxe Basin over an 18-year period. We combined previous fatty acid data from bears harvested from 1999 to 2003 (n = 82) with additional data from 2010 to 2018 (n = 397). Polar bear diets were diverse; however, ringed seal (Pusa hispida) was the primary prey throughout the sample period. Prey contribution varied temporally and spatially, and by intrinsic factors, while the frequency of prey in diets varied over time suggesting that diet estimates reflect the variability in available prey. Bowhead whale (Balaena mysticetus), although still a minor dietary component, has more than doubled in frequency of occurrence in diets in recent years in association with increased scavenging opportunities. Higher dietary levels of beluga whale (Delphinapterus leucas) and harbour seal (Phoca vitulina) were linked to later breakup date suggesting heavier ice conditions may promote access to both prey species. The flexible foraging strategies of bears in Foxe Basin may help mitigate their vulnerability to changes in prey distribution and habitat conditions. Our results provide insights into the importance of alternative and supplemental food sources for polar bears during phenological changes in ice conditions that will likely have consequences to Arctic community structure as warming continues.

Publisher Summary The polar bear is, on average, the largest of the eight species of bears found worldwide and is completely white. Ecologically, it is the only marine bear. Typically, the body is stocky, lacks a shoulder hump, and has a longer neck in relation to the rest of the body than other ursids. Polar bears are thought to have originated from brown bears, 1.3 million years ago. Polar bears and brown bears are capable of interbreeding in zoos and the young are fertile. Although in the wild, there is little overlap in the habitats during the breeding season, there has been one confirmed. Polar bears are distributed in ice-covered waters throughout the circumpolar Arctic. Although small numbers of animals may be found in the permanent multiyear pack ice of the central arctic basin, their preferred habitat is the annual ice over continental shelves of the continents and islands around the coastline of the Arctic Ocean where overall biological productivity and densities of ringed seals, their primary prey, are greatest. Regional concentrations and seasonal movement patterns of polar bears are influenced primarily by the type and distribution of sea ice and by the density and distribution of seals. Polar bears that have continuous access to sea ice continue to hunt throughout the year. Their hunting methods and rates of success change with the seasons and vary in different areas. The most common method of hunting is to lie beside a breathing hole waiting for a seal to surface and breathe. Polar bears have a remarkable ability to store large amounts of fat during periods when prey, mainly seals, are available and then fast for protracted periods when food is not available.

Cryptococcus neoformans: (H99W) was serially passaged in the invertebrate wax moth Galleria mellonella fifteen times to study how fungal virulence evolves under selection and whether those adaptations affect virulence. The G. mellonella passaged strain (P15) and the pre-passage H99W strains were used to infect three different host models of C. neoformans: C. elegans, G. mellonella, and Balb/c mice. While there was no difference in survival in the invertebrate models, P15 killed mice faster than H99W through both intratracheal and intravenous routes of infection and mice infected intravenously with P15 showed higher fungal burden in the brain. Characterization of the major virulence factors of C. neoformans found that P15 had increased capsule size, GXM release, and melanization. Whole genome sequencing of P15 and H99W revealed two mutations in P15, an insertion in the promoter region of NADH dehydrogenase (CNAG_09000) and an insertion in the LMP1 gene (CNAG_06765). Both ATP production and metabolic rate were higher in P15 compared to H99W. Quantitative RT-PCR suggested that the increased ATP was due to increased RNA levels of NADH dehydrogenase. Thus, adaptation to growth in hemocytes resulted in increased production of ATP, increased metabolic rate, and increased virulence in mice. This was likely due to differential expression of virulence factors, which skewed the host immune response to a less efficient Th2 response, with higher levels of IL-4, IL-10, and TNF-α in the brain. Overall, serial passage experiments have increased our understanding of how this yeast evolves under innate immune selection pressure.

SARS-CoV-2 infection induces severe disease in a subpopulation of patients, but the underlying mechanisms remain unclear. We demonstrate robust IgM autoantibodies that recognize angiotensin converting enzyme-2 (ACE2) in 18/66 (27%) patients with severe COVID-19, which are rare (2/52; 3.8%) in hospitalized patients who are not ventilated. The antibodies do not undergo class-switching to IgG, suggesting a T-independent antibody response. Purified IgM from anti-ACE2 patients activates complement. Pathological analysis of lung obtained at autopsy shows endothelial cell staining for IgM in blood vessels in some patients. We propose that vascular endothelial ACE2 expression focuses the pathogenic effects of these autoantibodies on blood vessels, and contributes to the angiocentric pathology observed in some severe COVID-19 patients. These findings may have predictive and therapeutic implications.One-sentence summaryACE2 autoantibodies in severe COVID-19 have features of a T-independent immune response, and may mediate vascular damage.

CD81 plays a role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus. Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81-cholesterol association, but had disparate effects on HCV, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified an allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol unbound) or closed (cholesterol bound) conformation. The open mutant of CD81 exhibited reduced receptor activity whereas the closed mutant was enhanced. These data are consistent with cholesterol switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81-partner networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in health and disease.

To gain insight into how pathogens adapt to new hosts, Cryptococcus neoformans (H99W) was serially passaged in Galleria mellonella. The phenotypic characteristics of the passaged strain (P15) and H99W were evaluated. P15 grew faster in hemolymph than H99W, in vitro and in vivo, suggesting that adaptation had occurred. However, P15 was more susceptible to hydrogen peroxide in vitro, killed fewer mouse macrophages, and had less fungal burden in human ex vivo macrophages than H99W. Analysis of gene expression changes during Galleria infection showed only a few different genes involved in the reactive oxygen species response. As P15 sheds more GXM than H99W, P15 may have adapted by downregulating hemocyte hydrogen peroxide production, possibly through increased capsular glucuronoxylomannan (GXM) shedding. Hemocytes infected with P15 produced less hydrogen peroxide, and hydrogen peroxide production in response to GXM-shedding mutants was correlated with shed GXM. Histopathological examination of infected larvae showed increased numbers and sizes of immune nodules for P15 compared to H99W, suggesting an enhanced, but functionally defective, response to P15. These results could explain why this infection model does not always correlate with murine models. Overall, C. neoformans&rsquo, serial passage in G. mellonella resulted in a better understanding of how this yeast evolves under selection.

Macrophages serve as a first line of defense against infection with the facultative intracellular pathogen, Cryptococcus neoformans (Cn). However, the ability of these innate phagocytic cells to destroy ingested Cn is strongly influenced by polarization state with classically (M1) activated macrophages better able to control cryptococcal infections than alternatively (M2) activated cells. While earlier studies have demonstrated that intracellular Cn minimally affects the expression of M1 and M2 markers, the impact on the broader transcriptome associated with these states remains unclear. To investigate this, an in vitro cell culture model of intracellular infection together with RNA sequencing-based transcriptome profiling was used to measure the impact of Cn infection on gene expression in both polarization states. The gene expression profile of both M1 and M2 cells was extensively altered to become more like naive (M0) macrophages. Gene ontology analysis suggested that this involved changes in the activity of the Janus kinase-signal transducers and activators of transcription (JAK-STAT), p53, and nuclear factor-κB (NF-κB) pathways. Analyses of the principle polarization markers at the protein-level also revealed discrepancies between the RNA- and protein-level responses. In contrast to earlier studies, intracellular Cn was found to increase protein levels of the M1 marker iNos. In addition, common gene expression changes were identified that occurred post-Cn infection, independent of polarization state. This included upregulation of the transcriptional co-regulator Cited1, which was also apparent at the protein level in M1-polarized macrophages. These changes constitute a transcriptional signature of macrophage Cn infection and provide new insights into how Cn impacts gene expression and the phenotype of host phagocytes.

Animal structural body size and condition are often measured to evaluate individual health, identify responses to environmental change and food availability, and relate food availability to effects on reproduction and survival. A variety of condition metrics have been developed but relationships between these metrics and vital rates are rarely validated. Identifying an optimal approach to estimate the body condition of polar bears is needed to improve monitoring of their response to decline in sea ice habitat. Therefore, we examined relationships between several commonly used condition indices (CI), body mass, and size with female reproductive success and cub survival among polar bears (Ursus maritimus) measured in two subpopulations over three decades. To improve measurement and application of morphometrics and CIs, we also examined whether CIs are independent of age and structural size-an important assumption for monitoring temporal trends-and factors affecting measurement precision and accuracy. Maternal CIs and mass measured the fall prior to denning were related to cub production. Similarly, maternal CIs, mass, and length were related to the mass of cubs or yearlings that accompanied her. However, maternal body mass, but not CIs, measured in the spring was related to cub production and only maternal mass and length were related to the probability of cub survival. These results suggest that CIs may not be better indicators of fitness than body mass in part because CIs remove variation associated with body size that is important in affecting fitness. Further, CIs exhibited variable relationships with age for growing bears and were lower for longer bears despite body length being related to cub survival and female reproductive success. These results are consistent with findings from other species indicating that body mass is a useful metric to link environmental conditions and population dynamics.

Selenium is an essential trace element which, at adequate levels, presents different beneficial biological effects, such as cancer regression, tissue development and protection against oxidative damage. The positive effects of this element are related to the expression of selenoproteins and their ability to modulate the immune system and the oxidative stress response. In Chagas disease and sleeping sickness, selenium supplementation has shown blood parasitism reduction and the alleviation of specific aspects of the diseases, such as diminishing anemia in sleeping sickness or minimization of myocardial and right ventricular chamber damage in Chagasdisease. Although the influence of selenium in trypanosomiasis has been investigated, the direct effects of sodium selenite supplementation on trypanosome cells are poorly understood. Treatment of Trypanosoma cruzi cultures with low selenium doses demonstrated different results, according to the parasite evolutive form analyzed. Epimastigote cultures supplemented with 100 nM of sodium selenite presented cell growth increment, which varies from 10 to 40% according to the parasite strain assayed. Selenium concentration around 600nM leads to a 30% increase in the amastigote form number, whereas, at the same dose, the mammal host cell presented no cellular growth alteration. For the bloodstream form, the results agree with theliterature, and all sodium selenite concentrations tested, demonstrated a reduction in parasite viability. The data suggest that selenium supplementation, under specific conditions, could increase T. cruzi viability, demonstrating that a strategy for using selenium as an adjuvant in Chagas disease treatment requires additional experimentation. KEY WORDS: Selenium; trypanosomiasis; Chagas disease; Trypanosoma cruzi.

BackgroundRisk factors for poor outcomes from COVID-19 are emerging among US cohorts, but patient trajectories during hospitalization ranging from mild-moderate, severe, and death and the factors associated with these outcomes have been underexplored.MethodsWe performed a cohort analysis of consecutive COVID-19 hospital admissions at 5 Johns Hopkins hospitals in the Baltimore/DC area between March 4 and April 24, 2020. Disease severity and outcomes were classified using the WHO COVID-19 disease severity ordinal scale. Cox proportional-hazards regressions were performed to assess relationships between demographics, clinical features and progression to severe disease or death.Results832 COVID-19 patients were hospitalized; 633 (76.1%) were discharged, 113 (13.6%) died, and 85 (10.2%) remained hospitalized. Among those discharged, 518 (82%) had mild/moderate and 116 (18%) had severe illness. Mortality was statistically significantly associated with increasing age per 10 years (adjusted hazard ratio (aHR) 1.54; 95%CI 1.28-1.84), nursing home residence (aHR 2.13, 95%CI 1.41-3.23), Charlson comorbidity index (1.13; 95% CI 1.02-1.26), respiratory rate (aHR 1.13; 95%CI 1.09-1.17), D-dimer greater than 1mg/dL (aHR 2.79; 95% 1.53-5.09), and detectable troponin (aHR 2.79; 95%CI 1.53-5.09). In patients under 60, only male sex (aHR 1.7;95%CI 1.11-2.58), increasing body mass index (BMI) (aHR1.25 1.14-1.37), Charlson score (aHR 1.27; 1.1-1.46) and respiratory rate (aHR 1.16; 95%CI 1.13-1.2) were associated with severe illness or death.ConclusionsA combination of demographic and clinical features on admission is strongly associated with progression to severe disease or death in a US cohort of COVID-19 patients. Younger patients have distinct risk factors for poor outcomes.

Macrophages serve as a first line of defense against infection with the facultative intracellular pathogen, Cryptococcus neoformans (Cn). However, the ability of these innate phagocytic cells to destroy ingested Cn is strongly influenced by polarization state with classically (M1) activated macrophages better able to control cryptococcal infections than alternatively (M2) activated cells. While earlier studies have demonstrated that intracellular Cn minimally affects the expression of M1 and M2 markers, the impact on the broader transcriptome associated with these states remains unclear. To investigate this, we used an in vitro cell culture model of intracellular infection together with RNA sequencing-based transcriptome profiling to measure the impact of Cn infection on gene expression in both polarization states. The gene expression profile of both M1 and M2 cells was extensively altered to become more like naive (M0) macrophages. Gene ontology analysis suggested that this involved changes in the activity of the Janus kinase-signal transducers and activators of transcription (JAK-STAT), p53, and nuclear factor-κB (NF-κB) pathways. Analyses of the principle polarization markers at the protein-level also revealed discrepancies between the RNA- and protein-level responses. In contrast to earlier studies, intracellular Cn was found to increase protein levels of the M1 marker iNos. In addition, we identified common gene expression changes that occurred post-Cn infection, independent of polarization state. This included upregulation of the transcriptional co-regulator Cited1, which was also apparent at the protein level. These changes constitute a transcriptional signature of macrophage Cn infection and provide new insights into how Cn impacts gene expression and the phenotype of host phagocytes.

The genus Naegleria, of the free-living amoeba (FLA) group, has been investigated mainly due to its human health impact, resulting in deadly infections and their worldwide distribution on freshwater systems. Naegleria fowleri, colloquially known as the &ldquo, brain-eating amoeba,&rdquo, is the most studied Naegleria species because it causes primary amoebic meningoencephalitis (PAM) of high lethality. The assessment of FLA biodiversity is fundamental to evaluate the presence of pathogenic species and the possibility of human contamination. However, the knowledge of FLA distribution in Brazil is unknown, and to rectify this situation, we present research on identifying Naegleria spp. in the Monjolinho River as a model study. The river is a public Brazilian freshwater source that crosses the city of S&atilde, o Carlos, in S&atilde, o Paulo state, Brazil. Five distinct sampling sites were examined through limnological features, trophozoites culturing, and PCR against internal transcribed spacer (ITS) regions and 5.8S rRNA sequences. The results identified N. philippinensis, N. canariensisi, N. australiensis, N. gruberi, N. dobsoni sequences, as well as a Hartmannella sequence. The methodology delineated here represents the first Brazilian Naegleria spp. study on a freshwater system. Our results stress the urgency of a large scale evaluation of the presence of free-living amoebas in Brazil.

Prevotella spp. form one of the most dominant bacterial genera in the human gut. Multiple Prevotella spp. can co-exist in some but not all individuals consuming plant-based diets. To functionally investigate the relationship between Prevotella competition and diet, we isolated several novel Prevotella spp. from the mouse gut, performed genome and metatranscriptome analysis as well as a series of competition experiments under different dietary conditions. These experiments revealed that diverse dominant Prevotella species compete for similar metabolic niches in vivo, which is linked to the upregulation of specific Polysaccharide Utilization Loci (PULs). Complex plant-derived polysaccharides are required, and particularly arabinoxylan is sufficient, for the expansion of Prevotella spp. The most dominant Prevotella species encodes a specific tandem-repeat trsusC/D PUL, which is conserved in highly abundant Prevotella copri strains further expanded in humans consuming a vegan diet. Our data suggest efficient (arabino)xylan-utilization as a factor contributing to Prevotella dominance.

CD40 ligand (TNFSF5/CD154/CD40L), a member of the tumor necrosis factor (TNF) superfamily is a key regulator of the immune system. The cognate receptor CD40 (TNFRSF5) is expressed broadly on antigen-presenting cells and many tumor types, and has emerged as an attractive target for immunologic cancer treatment. Most of the CD40 targeting drugs in clinical development are antibodies which display some disadvantages: their activity typically depends on Fcγ receptor-mediated crosslinking, and depletion of CD40-expressing immune cells by antibody-dependent cellular cytotoxicity compromises an efficient antitumor response. To overcome the inadequacies of antibodies, we have developed the hexavalent receptor agonist (HERA) Technology. HERA compounds are fusion proteins composed of 3 receptor binding domains in a single chain arrangement, linked to an Fc-silenced human IgG1 thereby generating a hexavalent molecule. HERA-CD40L provides efficient receptor agonism on CD40-expressing cells and, importantly, does not require FcγR-mediated crosslinking. Strong activation of NFκB signaling was observed upon treatment of B cells with HERA-CD40L. Monocyte treatment with HERA-CD40L promoted differentiation towards the M1 spectrum and repolarization of M2 spectrum macrophages towards the M1 spectrum phenotype. Treatment of in vitro co-cultures of T and B cells with HERA-CD40L-triggered robust antitumor activation of T cells, which depended upon direct interaction with B cells. In contrast, bivalent anti-CD40 antibodies and trivalent soluble CD40L displayed weak activity which critically depended on crosslinking. In vivo, a murine surrogate of HERA-CD40L-stimulated clonal expansion of OT-I-specific murine CD8 T cells and showed single agent antitumor activity in the CD40 syngeneic MC38-CEA mouse model of colorectal cancer, suggesting an involvement of the immune system in controlling tumor growth. We conclude that HERA-CD40L is able to establish robust antitumor immune responses both in vitro and in vivo.

The genus Naegleria, of free-living amoeba (FLA) group, has been investigated mainly due to its human health impact resulting in deadly infections and their worldwide distribution on freshwater systems. Naegleria fowleri, colloquially known as the “brain-eating amoeba”, is the most studied Naegleria species because it causes Primary Amoebic Meningoencephalitis (PAM) of high lethality. The assessment of FLA biodiversity is fundamental to evaluate the presence of pathogenic species and the possibility of human contamination. However, the knowledge of FLA distribution in Brazil is unknown, and to rectify this situation we present a research on identifying Naegleria spp. in the Monjolinho River, as a model study. The river is a public Brazilian freshwater source that crosses the city of São Carlos. Five distinct sampling sites were examined through limnological features, trophozoites culturing and PCR against internal transcribed spacers (ITS) regions and 5.8S rRNA sequence. The results identified N. philippinensis, N. canariensisi, N. australiensis, N.gruberi, N. dobsoni sequences, as well as a Vahlkampfia sequence. The methodology delineated here represents the first Brazilian Naegleria spp. study on a freshwater system. Our result stresses the urgency of a large scale evaluation of the presence of free-living amoebas in Brazil.

International audience; AbstractIntestinal health is critically important for the welfare and performance of poultry. Enteric diseases that cause gut barrier failure result in high economic losses. Up till now there is no reliable faecal marker to measure gut barrier failure under field conditions. Therefore, the aim of the present study was to identify a faecal protein marker for diminished intestinal barrier function due to enteric diseases in broilers. To assess this, experimental necrotic enteritis and coccidiosis in broilers were used as models for gut barrier failure. Ovotransferrin was identified as a marker for gut barrier failure using a proteomics approach on samples from chickens with necrotic enteritis. These results were confirmed via ELISA on samples derived from both necrotic enteritis and coccidiosis trials, where faecal ovotransferrin levels were significantly correlated with the severity of gut barrier failure caused by either coccidiosis or necrotic enteritis. This indicates that faecal ovotransferrin quantification may represent a valuable tool to measure gut barrier failure caused by enteric pathogens.

The ability to predict the agronomic performance of single-crosses with high precision is essential for selecting superior candidates for hybrid breeding. With recent technological advances, thousands of new parent lines, and, consequently, millions of new hybrid combinations are possible in each breeding cycle, yet only a few hundred can be produced and phenotyped in multi-environment yield trials. Well established prediction approaches such as best linear unbiased prediction (BLUP) using pedigree data and whole-genome prediction using genomic data are limited in capturing epistasis and interactions occurring within and among downstream biological strata such as transcriptome and metabolome. Because mRNA and small RNA (sRNA) sequences are involved in transcriptional, translational and post-translational processes, we expect them to provide information influencing several biological strata. However, using sRNA data of parent lines to predict hybrid performance has not yet been addressed. Here, we gathered genomic, transcriptomic (mRNA and sRNA) and metabolomic data of parent lines to evaluate the ability of the data to predict the performance of untested hybrids for important agronomic traits in grain maize. We found a considerable interaction for predictive ability between predictor and trait, with mRNA data being a superior predictor for grain yield and genomic data for grain dry matter content, while sRNA performed relatively poorly for both traits. Combining mRNA and genomic data as predictors resulted in high predictive abilities across both traits and combining other predictors improved prediction over that of the individual predictors alone. We conclude that downstream “omics” can complement genomics for hybrid prediction, and, thereby, contribute to more efficient selection of hybrid candidates.

Naegleria fowleri is a pathogenic amoeboflagellate most prominently known for its role as the etiological agent of the Primary Amoebic Meningoencephalitis (PAM), a disease that afflicts the central nervous system and is fatal in more than 95% of the reported cases. Although being fatal and with potential risks for an increase in the occurrence of the pathogen in populated areas, the organism receives little public health attention. A great underestimation in the number of PAM cases reported is assumed, taking into account the difficulty in obtaining an accurate diagnosis. In this review, we summarize different techniques and methods used in the identification of the protozoan in clinical and environmental samples. Since it remains unclear whether the protozoan infection can be successfully treated with the currently available drugs, we proceed to discuss the current PAM therapeutic strategies and its effectiveness. Finally, novel compounds for potential treatments are discussed as well as research on vaccine development against PAM.

Zusammenfassung: Grunberg, H., Cezanne, R., Eckstein, J., Eichler, M., Kempf, H., Meinunger, L., Preussing, M., Putzmann, F., Scholz, P., Thiel, H., Thiemann, R. & Hentschel, J. 2017. Neue und bemerkenswerte Flechtenfunde in Thuringen. – Herzogia 30: 463–482. Es werden sowohl Erstnachweise als auch Funde von extrem seltenen oder vom Aussterben bedrohten Flechtenarten in Thuringen mitgeteilt. Neufunde sind Absconditella lignicola, Acrocordia cavata, Agonimia allobata, A. flabelliformis, A. vouauxii, Alyxoria ochrocheila, Bacidia arceutina, B. trachona, B. viridifarinosa, Bacidina neosquamulosa, Caloplaca luteoalba, Candelariella plumbea, Cyphelium karelicum, Gregorella humida, Lecanactis dilleniana, Lecania sylvestris, Lempholemma isidiodes, Strigula jamesii, Thelenella muscorum, Umbilicaria subglabra und Verruculopsis lecideoides. Bacidia rosella, Caloplaca cerina, Heppia lutosa und Parmelina quercina waren in der Roten Liste Thuringens bereits als ausgestorben verzeichnet. Sie konnten nach langer Zeit wi...

CD40 ligand is a member of the TNF superfamily (TNF-SF) and a key regulator of the immune system. Its cognate receptor CD40 is expressed on antigen-presenting cells and on many tumor types, and has emerged as an attractive target for immunological cancer treatment. We developed HERA-CD40L, a construct composed of a trivalent single-chain CD40L-receptor-binding-domain (scCD40L-RBD) linked to a silenced human IgG1-Fc-domain thereby generating a hexavalent molecule. We showed previously that HERA-CD40L mimics the natural ligand, thereby inducing potent agonistic activity which is independent of FcγR mediated crosslinking and superior to anti-CD40 benchmark antibodies. For combinatorial cancer immunotherapy we have created bispecific molecules by combining the HERA-CD40L scaffold with antibody derived domains targeting different classes of tumor associated antigens. These bispecific fusion proteins combine the potent co-stimulatory CD40-agonist with either direct tumor-cell targeting and/or additional immunomodulatory activities in the tumor microenvironment. To evaluate the different fusion protein formats, the tumor associated antigens CEA, PD-L1 and CD95L were chosen as model-targets. In addition to the hexavalent targeted HERA-CD40L, trivalent targeted fusion proteins, employing the scCD40L-RBD as building block, were created as well. All engineering prototypes were produced in CHO-S cells and purified, resulting in highly pure non-aggregating protein lots as demonstrated by SDS-PAGE and HPLC-SEC. Functional binding to their respective targets was shown by ELISA and to proof biological in vitro activity luciferase reporter gene assays were employed. The basic underlying immunological processes have been investigated in vitro. The trivalent CD40L, the trivalent CD40L-bispecifics anti-CD95L-scCD40L-RBD and anti-CEA-scCD40L-RBD as well as anti-CD40, anti-PD-L1 or anti-CD95L antibodies did not activate human monocytes, even if the antibodies were co-administered with trivalent CD40L. In contrast, the hexavalent HERA-CD40L, the hexavalent bifunctional CD40L-constructs and the trivalent anti-PD-L1-scCD40L-RBD-construct induced strong activation/maturation of the monocytes as indicated by CD83, CD86, HLA-DR upregulation which was accompanied by increased chemokine receptor (CD54, CCR7) and PD-L1 expression. When co-cultured with CD3 positive T cells, these pre-activated monocytes led to subsequent activation of CD4 as well as CD8 positive T cells, indicated by increased expression of CD25, CD69 and CD54. This clearly shows that the close proximity of anti-PD-L1 and trivalent CD40L within one molecule makes a huge difference for biological activity. Hence, these novel bispecific constructs are a promising therapeutic approach to promote anti-tumor immune responses. Citation Format: Matthias Schroeder, Katharina Billian-Frey, Jaromir Sykora, Mauricio Redondo-Mueller, Karl Heinonen, Jamie Frankish, Christian Gieffers, Meinolf Thiemann, Oliver Hill. Generation and characterization of novel bispecific molecules combining single-chain-CD40L with anti-CEA, anti-CD95L or anti-PD-L1 targeting moieties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1587.

Global climate changes are magnified in the Arctic and are having an especially dramatic effect on the spatial and temporal distribution and the thickness traits of sea ice. Decline of Arctic sea ice may lead to qualitative and/or quantitative changes in diet and reduced body condition (i.e. adipose tissue stores) of ice-associated apex predators such as polar bears (Ursus maritimus). This may further affect their tissue concentrations of lipophilic pollutants. We determined how variations in adipose tissue stores associated to both breeding status and spatial changes in sea ice conditions and diet influence concentrations and biotransformation of lipophilic persistent organic pollutants (POPs). We collected 112 blood and fat samples from female polar bears (Ursus maritimus) of different breeding status (alone, with cubs of the year, or with yearlings) during two seasons (April and September) in 2012 and 2013 at three locations of Svalbard, Norway, with contrasted sea ice conditions. We inferred diet from nitrogen and carbon stable isotope ratios in red blood cells and fatty acid composition in adipose tissue. Relative to diet, body condition, which was negatively related to sea ice extent at both temporal and spatial scales, was the most important predictor for concentrations of POPs in plasma and fat, whereas diet showed a minor influence. Additionally, fatter females were more efficient at biotransforming PCBs than were leaner ones. Breeding status influenced the concentrations of less lipophilic compounds such as β-hexachlorocyclohexane, which were lower in females with yearlings, probably due to excretion into milk and subsequent offloading to young. In conclusion, our results indicate that declining sea ice indirectly leads to increased concentrations of lipophilic pollutants in polar bears mediated through reduced feeding opportunities and declining body condition rather than changes in diet composition.

Tri-axial accelerometers have been used to remotely identify the behaviors of a wide range of taxa. Assigning behaviors to accelerometer data often involves the use of captive animals or surrogate species, as their accelerometer signatures are generally assumed to be similar to those of their wild counterparts. However, this has rarely been tested. Validated accelerometer data are needed for polar bears Ursus maritimus to understand how habitat conditions may influence behavior and energy demands. We used accelerometer and water conductivity data to remotely distinguish 10 polar bear behaviors. We calibrated accelerometer and conductivity data collected from collars with behaviors observed from video-recorded captive polar bears and brown bears U. arctos, and with video from camera collars deployed on free-ranging polar bears on sea ice and on land. We used random forest models to predict behaviors and found strong ability to discriminate the most common wild polar bear behaviors using a combination of accelerometer and conductivity sensor data from captive or wild polar bears. In contrast, models using data from captive brown bears failed to reliably distinguish most active behaviors in wild polar bears. Our ability to discriminate behavior was greatest when species- and habitat-specific data from wild individuals were used to train models. Data from captive individuals may be suitable for calibrating accelerometers, but may provide reduced ability to discriminate some behaviors. The accelerometer calibrations developed here provide a method to quantify polar bear behaviors to evaluate the impacts of declines in Arctic sea ice.

The integrin-mediated interaction of cells with components of the extracellular matrix (ECM) regulates many cellular processes including cell division. Cytokinesis is the last step of cell division and is critical for normal development and tissue homeostasis as it ensures the proper segregation of genetic and cytoplasmic material between daughter cells. Cytokinesis failure leads to defects in development and tissue differentiation, as well as tumorigenesis. Abscission of intercellular bridge that connects presumptive daughter cells is the last step of cell division. The mitotic kinesin-like protein 1 (MKLP1) plays a central role in positioning the abscission machinery. Here, we show that α6 integrins promote successful cytokinesis in salivary gland epithelial cells by regulating the expression of MKLP1. RNAi-mediated depletion of α6 integrins inhibits cytokinesis and the expression of MKLP1 and p90 ribosomal-S6-kinase 2 (RSK2). Depletion of RSK2 results in similar defects in cytokinesis and also inhibits the expression of MKLP1, suggesting that the expression of RSK2 is required downstream of integrins to promote MKLP1 expression and successful cytokinesis. RNAi-mediated depletion of RSK2 in embryonic salivary glands in organ culture also results in the inhibition of cytokinesis and MKLP1 expression, indicating the physiological significance of this pathway.

The composition of the intestinal microbiota influences the outcome of enteric infections in human and mice. However, the role of specific members and their metabolites contributing to disease severity is largely unknown. Using isogenic mouse lines harboring distinct microbiota communities, we observed highly variable disease kinetics of enteric Citrobacter rodentium colonization after infection. Transfer of communities from susceptible and resistant mice into germ-free mice verified that the varying susceptibilities are determined by microbiota composition. The strongest differences in colonization were observed in the cecum and could be maintained in vitro by coculturing cecal bacteria with C. rodentium. Cohousing of animals as well as the transfer of cultivable bacteria from resistant to susceptible mice led to variable outcomes in the recipient mice. Microbiome analysis revealed that a higher abundance of butyrate-producing bacteria was associated with the resistant phenotype. Quantification of short-chain fatty acid (SCFA) levels before and after infection revealed increased concentrations of acetate, butyrate and propionate in mice with delayed colonization. Addition of physiological concentrations of butyrate, but not of acetate and/or propionate strongly impaired growth of C. rodentium in vitro. In vivo supplementation of susceptible, antibiotic-treated and germ-free mice with butyrate led to the same level of protection, notably only when cecal butyrate concentration reached a concentration higher than 50 nmol/mg indicating a critical threshold for protection. In the recent years, commensal-derived primary and secondary bacterial metabolites emerged as potent modulators of hosts susceptibility to infection. Our results provide evidence that variations in SCFA production in mice fed fibre-rich chow-based diets modulate susceptibility to colonization with Enterobacteriaceae not only in antibiotic-disturbed ecosystems but even in undisturbed microbial communities. These findings emphasise the need for microbiota normalization across laboratory mouse lines for infection experiments with the model-pathogen C. rodentium independent of investigations of diet and antibiotic usage., Author summary The distinct composition of the gut microbiota in each individual results in variable metabolic activity and output of these communities, which influences the host, including resistance to enteric pathogens. Lack of reproducibility in biomedical research is nowadays frequently attributed to the microbiota, but little is known about which specific members and metabolites contribute to disease severity. Here, we use genetically identical mouse lines with variable microbiota compositions on a standardized diet and observed highly variable colonization with the enteric pathogen Citrobacter rodentium without antibiotics intervention. We found the same differences in formerly germ-free animals harbouring the respective donors microbiota and also in vitro by coculturing cecal bacteria from resistant and susceptible animals with C. rodentium showing that the phenotype is fully dependent on differences in the microbiota. We analysed the microbiome composition and found a higher abundance of butyrate-producing bacteria as well as increased levels of butyrate in resistant mice. By supplementation of susceptible and germ-free animals with butyrate, we could significantly lower the levels of colonization highlighting that commensal-derived primary and secondary bacterial metabolites are highly variable between laboratory animals from different vendors and are potent modulators of hosts susceptibility to infection with C. rodentium.

Loss-of-function mutations of the SCN5A gene encoding for the sodium channel α-subunit NaV1.5 result in the autosomal dominant hereditary disease Brugada Syndrome (BrS) with a high risk of sudden cardiac death in the adult. We here engineered human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the CRISPR/Cas9 introduced BrS-mutation p.A735V-NaV1.5 (g.2204C > T in exon 14 of SCN5A) as a novel model independent of patient´s genetic background. Recent studies raised concern regarding the use of hiPSC-CMs for studying adult-onset hereditary diseases due to cells’ immature phenotype. To tackle this concern, long-term cultivation of hiPSC-CMs on a stiff matrix (27–42 days) was applied to promote maturation. Patch clamp recordings of A735V mutated hiPSC-CMs revealed a substantially reduced upstroke velocity and sodium current density, a prominent rightward shift of the steady state activation curve and decelerated recovery from inactivation as compared to isogenic hiPSC-CMs controls. These observations were substantiated by a comparative study on mutant A735V-NaV1.5 channels heterologously expressed in HEK293T cells. In contrast to mutated hiPSC-CMs, a leftward shift of sodium channel inactivation was not observed in HEK293T, emphasizing the importance of investigating mechanisms of BrS in independent systems. Overall, our approach supports hiPSC-CMs’ relevance for investigating channelopathies in a dish.

Radiation therapy for head and neck cancers results in permanent damage to the saliva producing acinar compartment of the salivary gland. To date, a pure pro-acinar cell line to study underlying mechanisms of acinar cell differentiation in culture has not been described. Here, we report the establishment of a pro-acinar (mSG-PAC1) and ductal (mSG-DUC1) cell line, from the murine submandibular salivary gland (SMG), which recapitulate developmental milestones in differentiation. mSG-DUC1 cells express the ductal markers, keratin-7 and keratin-19, and form lumenized spheroids. mSG-PAC1 cells express the pro-acinar markers SOX10 and aquaporin-5. Using the mSG-PAC1 cell line, we demonstrate that FGF2 regulates specific steps during acinar cell maturation. FGF2 up-regulates aquaporin-5 and the expression of the α3 and α6 subunits of the α3β1 and α6β1 integrins that are known to promote SMG morphogenesis and differentiation. mSG-DUC1 and mSG-PAC1 cells were derived from genetically modified mice, homozygous for floxed alleles of the integrin α3 subunit. Similar to SMGs from α3-null mice, deletion of α3 alleles in mSG-PAC1 cells results in the up-regulation of E-cadherin and the down-regulation of CDC42. Our data indicate that mSG-DUC1 and mSG-PAC1 cells will serve as important tools to gain mechanistic insight into salivary gland morphogenesis and differentiation.

Background Glucocorticoid-induced TNFR-related protein (TNFRSF18, GITR, CD357), expressed by T cells, and its ligand (TNFSF18, GITRL), expressed by myeloid populations, provide co-stimulatory signals that boost T cell activity. Due to the important role that GITR plays in regulating immune functions, agonistic stimulation of GITR is a promising therapeutic concept. Multiple strategies to induce GITR signaling have been investigated. The limited clinical efficacy of antibody-based GITR agonists results from structural and functional characteristics of antibodies that are unsuitable for stimulating the well-defined trimeric members of the TNFRSF. Methods To overcome limitations of antibody-based TNFRSF agonists, we have developed HERA-GITRL, a fully human hexavalent TNF receptor agonist (HERA) targeting GITR and mimicking the natural signaling concept. HERA-GITRL is composed of a trivalent but single-chain GITRL-receptor-binding-domain (scGITRL-RBD) unit fused to an IgG1 derived silenced Fc-domain serving as dimerization scaffold. A specific mouse surrogate, mmHERA-GITRL, was also generated to examine in vivo activity in respective mouse tumor models. Results For functional characterization of HERA-GITRL in vitro, human immune cells were isolated from healthy-donor blood and stimulated with anti-CD3 antibody in the presence of HERA-GITRL. Consistently, HERA-GITRL increased the activity of T cells, including proliferation and differentiation, even in the presence of regulatory T cells. In line with these findings, mmHERA-GITRL enhanced antigen-specific clonal expansion of both CD4+ (OT-II) and CD8+ (OT-I) T cells in vivo while having no effect on non-specific T cells. In addition, mmHERA-GITRL showed single-agent anti-tumor activity in two subcutaneous syngeneic colon cancer models (CT26wt and MC38-CEA). Importantly, this activity is independent of its FcγR-binding functionality, as both mmHERA-GITRL with a functional Fc- and a silenced Fc-domain showed similar tumor growth inhibition. Finally, in a direct in vitro comparison to a bivalent clinical benchmark anti-GITR antibody and a trivalent GITRL, only the hexavalent HERA-GITRL showed full biological activity independent of additional crosslinking. Conclusion In this manuscript, we describe the development of HERA-GITRL, a true GITR agonist with a clearly defined mechanism of action. By clustering six receptor chains in a spatially well-defined manner, HERA-GITRL induces potent agonistic activity without being dependent on additional FcγR-mediated crosslinking. Electronic supplementary material The online version of this article (10.1186/s40425-019-0671-4) contains supplementary material, which is available to authorized users.