In the body, nanoparticles can be systemically distributed and then may affect secondary target organs, such as the central nervous system (CNS). Putative adverse effects on the CNS are rarely investigated to date. Here, we used a mixed primary cell model consisting mainly of neurons and astrocytes and a minor proportion of oligodendrocytes to analyze the effects of well-characterized 20 and 40 nm silver nanoparticles (SNP). Similar gold nanoparticles served as control and proved inert for all endpoints tested. SNP induced a strong size-dependent cytotoxicity. Additionally, in the low concentration range (up to 10 μg/ml of SNP), the further differentiated cultures were more sensitive to SNP treatment. For detailed studies, we used low/medium dose concentrations (up to 20 μg/ml) and found strong oxidative stress responses. Reactive oxygen species (ROS) were detected along with the formation of protein carbonyls and the induction of heme oxygenase-1. We observed an acute calcium response, which clearly preceded oxidative stress responses. ROS formation was reduced by antioxidants, whereas the calcium response could not be alleviated by antioxidants. Finally, we looked into the responses of neurons and astrocytes separately. Astrocytes were much more vulnerable to SNP treatment compared with neurons. Consistently, SNP were mainly taken up by astrocytes and not by neurons. Immunofluorescence studies of mixed cell cultures indicated stronger effects on astrocyte morphology. Altogether, we can demonstrate strong effects of SNP associated with calcium dysregulation and ROS formation in primary neural cells, which were detectable already at moderate dosages.

Background Recent new anatomical and histological features of craniocervical junction in dogs and cats were described providing evidence of differences between the carnivore species. No information on these structures in foxes exists.

Results Two parts of the alar ligaments were found. A longer one aroused from dens of axis to the internal (medial) surface of the occipital condyles and was called apical part. A shorter part originated from the entire length of the lateral edge of the dens of axis and terminated on the internal wall of the vertebral foramen of atlas and thus was called the lateral part. The transverse ligament of the atlas was widened in the mid region, above the dens of axis, and thickened at enthesis. Periosteal fibrocartilage was detected in the transverse ligament of the atlas at the enthesis, and sesamoid fibrocartilage was present on periphery in the middle of the ligament.

Conclusions The craniocervical junction in foxes differs in part from other carnivores such as dogs and cats but resembles that of mesaticephalic dogs. The sesamoid and periosteal fibrocartilage supports the transverse ligament of the atlas whereas the alar ligaments have no cartilage.

Arterial or venous thromboses are frequent clinical complications with the risk of fatal progression. Recent studies suggest the disruption of angiogenesis in the course of thrombus resolution as the underlying pathomechanism. Very similar to the situation in human patients, equine vessels have been described to be particularly susceptible to thrombosis. In contrast to humans, equine donors are readily available to obtain organs and tissues for isolation of endothelial cells. Objective of this study was to isolate equine endothelial cells and develop an angiogenesis assay from primary cultures. Macrovascular endothelial cells were obtained from jugular veins and carotid arteries of nine horses, one of which suffered from inflammatory processes. After enzymatic isolation, the cells were incubated in different selective primary media. Phenotypic identification of endothelial cells was accomplished by morphology and positive staining to von Willebrand factor. The reliable, inexpensive, and standardized combination of methods presented here resulted in pure endothelial cultures for angiogenesis assays that can be used in any cell culture laboratory. Inverted phase microscopy and life cell imaging was used to characterize the stages of the angiogenic cascade of the endothelial cells. Life cell imaging gave new insights into the in vitro formation of capillary like structures including exocytosis of microparticles from endothelial cells before integration into the three-dimensional structure. We hypothesize that a specific population of endothelial cells showing a highly active migration pattern in life cell imaging might play a role in the resolution of thrombosis.

In this study the macroscopic and microscopic structure of the liver of a fast growing, meat-type turkey line (British United turkeys BUT Big 6, n = 25) and a wild-type turkey line (Wild Canadian turkey, n = 48) were compared at the age of 4, 8, 12, 16, and 20 wk. Because the growth plates of long bones were still detectable in the 20-week-old wild-type turkeys, indicating immaturity, a group of 8 wild-type turkeys at the age of 24 wk was included in the original scope of the study. Over the term of the study, the body and liver weights of birds from the meat-type turkey line increased at a faster rate than those of the wild-type turkey line. However, the relative liver weight of the meat-type turkeys declined (from 2.7 to 0.9%) to a greater extent than that of the wild-type turkeys (from 2.8 to 1.9%), suggesting a mismatch in development between muscle weights and liver weights of the meat-type turkeys. Signs of high levels of fat storage in the liver were detected in both lines but were greater in the wild-type turkey line, suggesting a better feed conversion by the extreme-genotype birds i.e., meat-type birds. For the first time, this study presents morphologic data on the structure and arrangement of the lymphatic tissue within the healthy turkey liver, describing two different types of lymphatic aggregations within the liver parenchyma, i.e., aggregations with and without fibrous capsules. Despite differences during development, both adult meat-type and adult wild-type turkeys had similar numbers of lymphatic aggregations.

Background Quantification of nanoparticle (NP) uptake in cells or tissues is very important for safety assessment. Often, electron microscopy based approaches are used for this purpose, which allow imaging at very high resolution. However, precise quantification of NP numbers in cells and tissues remains challenging. The aim of this study was to present a novel approach, that combines precise quantification of NPs in individual cells together with high resolution imaging of their intracellular distribution based on focused ion beam/ scanning electron microscopy (FIB/SEM) slice and view approaches.

Results We quantified cellular uptake of 75 nm diameter citrate stabilized silver NPs (Ag 75 Cit) into an individual human macrophage derived from monocytic THP-1 cells using a FIB/SEM slice and view approach. Cells were treated with 10 μg/ml for 24 h. We investigated a single cell and found in total 3138 ± 722 silver NPs inside this cell. Most of the silver NPs were located in large agglomerates, only a few were found in clusters of fewer than five NPs. Furthermore, we cross-checked our results by using inductively coupled plasma mass spectrometry and could confirm the FIB/SEM results.

Conclusions Our approach based on FIB/SEM slice and view is currently the only one that allows the quantification of the absolute dose of silver NPs in individual cells and at the same time to assess their intracellular distribution at high resolution. We therefore propose to use FIB/SEM slice and view to systematically analyse the cellular uptake of various NPs as a function of size, concentration and incubation time.

The histological characterization of the intestinal mucus layer is important for many scientific experiments investigating the interaction between intestinal microbiota, mucosal immune response and intestinal mucus production. The aim of this study was to examine and compare different fixation protocols for displaying and quantifying the intestinal mucus layer in piglets and to test which histomorphological parameters may correlate with the determined mucus layer thickness. Jejunal and colonal tissue samples of weaned piglets (n=10) were either frozen in liquid nitrogen or chemically fixed using methacarn solution. The frozen tissue samples were cryosectioned and subsequently postfixed using three different postfixatives: paraformaldehyde vapor, neutrally buffered formalin solution and ethanol solution. After dehydration, methacarn fixed tissues were embedded in paraffin wax. Both sections of cryopreserved and methacarn fixed tissue samples were stained with Alcian blue (AB)-PAS followed by the microscopically determination of the mucus layer thickness. Different pH values of the Alcian Blue staining solution and two mucus layer thickness measuring methods were compared. In addition, various histomorphological parameters of methacarn fixed tissue samples were evaluated including the number of goblet cells and the mucin staining area. Cryopreservation in combination with chemical postfixation led to mucus preservation in the colon of piglets allowing mucus thickness measurements. Mucus could be only partly preserved in cryosections of the jejunum impeding any quantitative description of the mucus layer thickness. The application of different postfixations, varying pH values of the AB solution and different mucus layer measuring methods led to comparable results regarding the mucus layer thickness. Methacarn fixation proved to be unsuitable for mucus depiction as only mucus patches were found in the jejunum or a detachment of the mucus layer from the epithelium was observed in the colon. Correlation analyses revealed that the proportion of the mucin staining area per crypt area (relative mucin staining) measured in methacarn fixed tissue samples corresponded to the colonal mucus layer thickness determined in cryopreserved tissue samples. In conclusion, the results showed that cryopreservation using liquid nitrogen followed by chemical postfixation and AB-PAS staining led to a reliable mucus preservation allowing a mucus thickness determination in the colon of pigs. Moreover, the detected relative mucin staining area may serve as a suitable histomorphological parameter for the assessment of the intestinal mucus layer thickness. The findings obtained in this study can be used for the implementation of an improved standard for the histological description of the mucus layer in the colon of pigs.

The current study evaluated the effects of different inclusion levels of microbial muramidase (Muramidase 007, DSM Nutritional Products) on gastrointestinal functionality by determination of apparent ileal digestibility (AID) of nutrients, investigation of intestinal histomorphology, and quantification of resulting growth performance. Four maize-wheat-soybean experimental diets were produced without (C) and with different dosages of muramidase: low (L, 25,000 LSU(F)/kg), medium (M, 35,000 LSU(F)/kg), and high (H, 45,000 LSU(F)/kg); diets were fed to broilers for 35 d. At the end of the experiment, AID of ether extract (EE), crude protein (CP), Ca, and P were determined and samples of the mid-jejunum and -ileum were collected for histomorphological observations. Data were subjected to ANOVA analysis using the GLM procedure. Orthogonal polynomial contrasts were used to assess linear and quadratic effects of different levels of the muramidase. At the end of the trial, Muramidase 007 supplementation linearly increased body weight gain and decreased feed conversion ratio (FCR) (P ≤ 0.05). Adding the muramidase to broiler diets also linearly increased the European poultry efficiency factor (P ≤ 0.05). Inclusion of the muramidase in broiler diets linearly increased AID of CP, EE, and P (P ≤ 0.05), and the H group had a higher AID of EE and CP compared to C group (P ≤ 0.05). Microbial muramidase supplementation linearly increased ileal villus length to crypt depth ratio and decreased the number of ileal CD45 cells (P ≤ 0.05). Broilers fed M and H diets had fewer number of CD45 cells in the ileum compared to those in C group (P ≤ 0.05). In conclusion, the results of the present study demonstrated that inclusion of the microbial muramidase in broiler diets could increase AID of key nutrients and improve growth performance in broilers. Adding microbial muramidase to broiler diets can therefore be considered as an interesting prospect to improve gastrointestinal functionality. Biological mechanisms causing these improvements need to be studied further.

Rearing dual-purpose chickens is a practicable approach to avoid culling one-day-old male layer chicks. The present study examined the impact of a conventional fattening diet on the liver of a novel dual-purpose chicken line (Lohmann Dual, LD) in comparison to a broiler (Ross 308) chicken line. Age-related changes of structure and lipid content of the liver were assessed. One hundred twenty and newly hatched chicks (LD = 66, Ross = 54) were kept under the same husbandry conditions and fed a commercial diet for 5 weeks for Ross and 9 weeks for LD. Six birds of each line were examined weekly. Their body weight (BW) and liver mass were recorded. Microscopic structure and ultrastructure of the liver were investigated and the liver lipid content was measured using a pre-validated method. During the study period, liver mass increased with age, while normalized liver mass decreased. Furthermore, liver mass of Ross birds was greater than that of LD birds of the same BW. Overall, no significant differences were observed in the hepatic structure or ultrastructure between the two chicken lines. The hepatic lymphatic aggregations were without fibrous capsules and their number and area increased throughout the first week, then the values began to fluctuate with age in both chicken lines. The changes in the liver lipid content of the two chicken lines were within the normal physiological range over the term of the study. The liver lipid content correlated negatively with age and body weight in both lines. It was the highest on the first day then decreased until day 7 and thereafter did not change in both chicken lines. However, given the same body weight, the Ross chickens had a 9% greater liver lipid content than LD chickens. It is concluded that there is no apparent adverse effect of a high-energy diet on the liver of LD chickens.

The self-consistent Born approximation quantitatively fails to capture disorder effects in semimetals. We present an alternative, simple-to-use nonperturbative approach to calculate the disorder-induced self-energy. It requires a sufficient broadening of the quasiparticle pole and the solution of a differential equation on the imaginary frequency axis. We demonstrate the performance of our method for various paradigmatic semimetal Hamiltonians and compare our results to exact numerical reference data. For intermediate and strong disorder, our approach yields quantitatively correct momentum-resolved results. It is thus complementary to existing renormalization group treatments of weak disorder in semimetals.

We consider the Kondo effect, arising from a hydrogen impurity in graphene. As a first approximation, the strong covalent bond to a carbon atom removes that carbon atom without breaking the C3 rotation symmetry, and we retain only the Hubbard interaction on the three nearest neighbors of the removed carbon atom which then behave as magnetic impurities. These three impurity spins are coupled to three conduction channels with definite helicity, two of which support a diverging local density of states (LDOS) ∝1/[|ω|ln2(Λ/|ω|)] near the Dirac point ω→0 even though the bulk density of states vanishes linearly. We study the resulting three-impurity multichannel Kondo model using the numerical renormalization group method. For weak potential scattering, the ground state of the Kondo model is a particle-hole symmetric spin-1/2 doublet, with ferromagnetic coupling between the three impurity spins; for moderate potential scattering, the ground state becomes a particle-hole asymmetric spin singlet, with antiferromagnetic coupling between the three impurity spins. This behavior is inherited by the Anderson model containing the hydrogen impurity and all four carbon atoms in its vicinity.

We study the robustness of 3D intrinsic topogical order under external perturbations by investigating the paradigmatic microscopic model, the 3D toric code in an external magnetic field. Exact dualities as well as variational calculations reveal a ground-state phase diagram with first and second-order quantum phase transitions. The variational approach can be applied without further approximations only for certain field directions. In the general field case, an approximative scheme based on an expansion of the variational energy in orders of the variational parameters is developed. For the breakdown of the 3D intrinsic topological order, it is found that the (im-)mobility of the quasiparticle excitations is crucial in contrast to their fractional statistics.

We investigate a well‐preserved paleo subduction channel that preserves a coherent part of the European continental margin exposed in the central Tauern Window (Eastern Alps), with the aim of testing models of sheath fold nappe formation and exhumation. The subduction zone was active during Paleogene convergence of the European and Adriatic plates, after closure of the Alpine Tethyan ocean. New cross sections and structural data together with new petrological data document a recumbent, tens of kilometers‐scale sheath fold in the center of the Tauern Window that formed during pervasive top‐foreland shear while subducted at high‐pressure (HP) conditions (~2.0 GPa, 500 °C) close to maximum burial depth. The fold comprises an isoclinally folded thrust that transported relicts of the former Alpine Tethys onto a distal part of the former European continental margin. The passive margin stratigraphy is still well preserved in the fold and highlights the special character of this segment of the European continental margin. We argue that this segment formed a promontory to the margin, which was inherited from Mesozoic rifting. In accordance with classical sheath fold theory, this promontory may have acted as an initial structural perturbation to nucleate a fold that was passively amplified to a sheath fold during top‐foreland shear in the subduction zone. The fold was at least partly exhumed and juxtaposed with the surrounding lower pressure units by opposing top‐hinterland and top‐foreland shear zones above and below, respectively, that is, in the sense of a nappe fold formed during channel‐extrusion exhumation.

Creatinine and proteinuria are used to monitor kidney transplant patients. However, renal biopsies are needed to diagnose renal graft rejection. Here, we assessed whether the quantification of different urinary cells would allow non-invasive detection of rejection. Urinary cell numbers of CD4+ and CD8+ T cells, monocytes/macrophages, tubular epithelial cells (TEC), and podocalyxin(PDX)-positive cells were determined using flow cytometry and were compared to biopsy results. Urine samples of 63 renal transplant patients were analyzed. Patients with transplant rejection had higher amounts of urinary T cells than controls; however, patients who showed worsening graft function without rejection had similar numbers of T cells. T cells correlated with histological findings (interstitial inflammation p = 0.0005, r = 0.70; tubulitis p = 0.006, r = 0.58). Combining the amount of urinary T cells and TEC, or T cells and PDX+ cells, yielded a significant segregation of patients with rejection from patients without rejection (all p < 0.01, area under the curve 0.89-0.91). Urinary cell populations analyzed by flow cytometry have the potential to introduce new monitoring methods for kidney transplant patients. The combination of urinary T cells, TEC, and PDX-positive cells may allow non-invasive detection of transplant rejection.

CD33 is a myeloid-associated marker and belongs to the sialic acid-binding immunoglobulin (Ig)-like lectin (Siglec) family. Such types of receptors are highly expressed in acute myeloid leukemia, which could be used in its treatment. CD33 shows high variability in its expression levels with still unknown reasons. Here, we investigated the CD33 expression of monocytes in human blood samples processed at different temperatures and in dependence on their phagocytic activity against opsonized Escherichia coli. The samples were stained by fluorescently labelled anti-human CD14 to specify the monocyte population, anti-human CD33 antibodies to evaluate CD33 expression and analyzed by flow cytometry and confocal laser scanning microscopy. In blood samples kept at 37°C or first pre-chilled at 0°C with subsequent warming up to 37°C, the percentage of CD33-positive monocytes as well as their relative fluorescence intensity was up-regulated compared to samples kept constantly at 0°C. After exposure to E. coli the CD33 relative fluorescence intensity of the monocytes activated at 37°C was 3 to 4 times higher than that of those cells kept inactive at 0°C. Microscopic analysis showed internalisation of CD33 due to its enhanced expression on the surface followed by engulfment of E. coli.

Short‐duration, high‐impact precipitation events in the extratropics are invariably convective in nature, typically occur during the summer, and are projected to intensify under climate change. The occurrence of convective precipitation is strongly regulated by the diurnal convective cycle, peaking in the late afternoon. Here we perform very high resolution (convection‐permitting) regional climate model simulations to study the scaling of extreme precipitation under climate change across the diurnal cycle. We show that the future intensification of extreme precipitation has a strong diurnal signal and that intraday scaling far in excess of overall scaling, and indeed thermodynamic expectations, is possible. We additionally show that, under a strong climate change scenario, the probability maximum for the occurrence of heavy to extreme precipitation may shift from late afternoon to the overnight/morning period. We further identify the thermodynamic and dynamic mechanisms which modify future extreme environments, explaining both the future scaling's diurnal signal and departure from thermodynamic expectations.

Identification of countermeasures able to prevent disuse-induced muscle wasting is crucial to increase performance of crew members during space flight as well as ameliorate patient's clinical outcome after long immobilization periods. We report on the outcome of short but high-impact reactive jumps (JUMP) as countermeasure during 60 days of 6° head-down tilt (HDT) bed rest on myofiber size, type composition, capillarization, and oxidative capacity in tissue biopsies (pre/post/recovery) from the knee extensor vastus lateralis (VL) and deep calf soleus (SOL) muscle of 22 healthy male participants (Reactive jumps in a sledge, RSL-study 2015-2016, DLR:envihab, Cologne). Bed rest induced a slow-to-fast myofiber shift (type I ->II) with an increased prevalence of hybrid fibers in SOL after bed rest without jumps (control, CTRL, p = 0.016). In SOL, JUMP countermeasure in bed rest prevented both fast and slow myofiber cross-sectional area (CSA) decrements (p = 0.005) in CTRL group. In VL, bed rest only induced capillary rarefaction, as reflected by the decrease in local capillary-to-fiber ratio (LCFR) for both type II (pre vs. post/R + 10, p = 0.028/0.028) and type I myofibers (pre vs. R + 10, p = 0.012), which was not seen in the JUMP group. VO2 max Fiber (pL × mm-1 × min-1) calculated from succinate dehydrogenase (SDH)-stained cryosections (OD660 nm) showed no significant differences between groups. High-impact jump training in bed rest did not prevent disuse-induced myofiber atrophy in VL, mitigated phenotype transition (type I - >II) in SOL, and attenuated capillary rarefaction in the prime knee extensor VL however with little impact on oxidative capacity changes.