Pyrrolizidine alkaloids (PAs) are a class of secondary metabolites found in various unrelated angiosperm lineages including cool-season grasses (Poaceae, subfamily Pooideae). Thesinine conjugates, saturated forms of PA that are regarded as non-toxic, have been described to occur in the two grass species Lolium perenne and Festuca arundinacea (Poaceae, subfamily Pooideae). In a wider screen, we tested various species of the Pooideae lineage, grown under controlled conditions, for their ability to produce thesinine conjugates or related structures. Using an LC-MS based targeted metabolomics approach we were able to show that PA biosynthesis in grasses is limited to a group of very closely related Pooideae species that produce a limited diversity of PA structures. High variability in PA levels was observed even between individuals of the same species. These individual accumulation patterns are discussed with respect to a possible function and evolution of this type of alkaloid.

Neurons in layer VI of visual cortex represent one of the largest sources of nonretinal input to the dorsal lateral geniculate nucleus (dLGN) and play a major role in modulating the gain of thalamic signal transmission. However, little is known about how and when these descending projections arrive and make functional connections with dLGN cells. Here we used a transgenic mouse to visualize corticogeniculate projections to examine the timing of cortical innervation in dLGN. Corticogeniculate innervation occurred at postnatal ages and was delayed compared with the arrival of retinal afferents. Cortical fibers began to enter dLGN at postnatal day 3 (P3) to P4, a time when retinogeniculate innervation is complete. However, cortical projections did not fully innervate dLGN until eye opening (P12), well after the time when retinal inputs from the two eyes segregate to form nonoverlapping eye-specific domains. In vitro thalamic slice recordings revealed that newly arriving cortical axons form functional connections with dLGN cells. However, adult-like responses that exhibited paired pulse facilitation did not fully emerge until 2 weeks of age. Finally, surgical or genetic elimination of retinal input greatly accelerated the rate of corticogeniculate innervation, with axons invading between P2 and P3 and fully innervating dLGN by P8 to P10. However, recordings in genetically deafferented mice showed that corticogeniculate synapses continued to mature at the same rate as controls. These studies suggest that retinal and cortical innervation of dLGN is highly coordinated and that input from retina plays an important role in regulating the rate of corticogeniculate innervation.

In many species, neurons are unevenly distributed across the retina, leading to nonuniform analysis of specific visual features at certain locations in visual space. In recent years, the mouse has emerged as a premiere model for probing visual system function, development, and disease. Thus, achieving a detailed understanding of mouse visual circuit architecture is of paramount importance. The general belief is that mice possess a relatively even topographic distribution of retinal ganglion cells (RGCs)-the output neurons of the eye. However, mouse RGCs include ∼30 subtypes; each responds best to a specific feature in the visual scene and conveys that information to central targets. Given the crucial role of RGCs and the prominence of the mouse as a model, we asked how different RGC subtypes are distributed across the retina. We targeted and filled individual fluorescently tagged RGC subtypes from across the retinal surface and evaluated the dendritic arbor extent and soma size of each cell according to its specific retinotopic position. Three prominent RGC subtypes: On-Off direction selective RGCs, object-motion-sensitive RGCs, and a specialized subclass of nonimage-forming RGCs each had marked topographic variations in their dendritic arbor sizes. Moreover, the pattern of variation was distinct for each RGC subtype. Thus, there is increasing evidence that the mouse retina encodes visual space in a region-specific manner. As a consequence, some visual features are sampled far more densely at certain retinal locations than others. These findings have implications for central visual processing, perception, and behavior in this prominent model species.

Yield losses caused by fungal pathogens represent a major threat to global food production. One of the most devastating fungal wheat pathogens is Zymoseptoria tritici. Despite the importance of this fungus, the underlying mechanisms of plant-pathogen interactions are poorly understood. Here we present a conceptual framework based on coinfection assays, comparative metabolomics, and microbiome profiling to study the interaction of Z. tritici in susceptible and resistant wheat. We demonstrate that Z. tritici suppresses the production of immune-related metabolites in a susceptible cultivar. Remarkably, this fungus-induced immune suppression spreads within the leaf and even to other leaves, a phenomenon that we term "systemic induced susceptibility". Using a comparative metabolomics approach, we identify defense-related biosynthetic pathways that are suppressed and induced in susceptible and resistant cultivars, respectively. We show that these fungus-induced changes correlate with changes in the wheat leaf microbiome. Our findings suggest that immune suppression by this hemibiotrophic pathogen impacts specialized plant metabolism, alters its associated microbial communities, and renders wheat vulnerable to further infections.

Alpha-synuclein (aSyn) is a cytosolic, aggregation-prone protein that is associated with neurodegenerative disorders like Parkinson's disease. Interestingly, the protein can appear in different conformations, including monomeric and oligomeric forms as well as amyloid fibrils. Its individual structural constituents seem to be dependent on various factors and the composition of the respective cellular surroundings. Although under physiological conditions, most aSyn is found in the cytosol and synapses of neurons, aSyn can also be found in lysosomal compartments, where it gets degraded. We here compare the assembly speed, morphology, folding state, and spreading of aSyn at cytosolic pH (pH 7.4) and lysosomal pH (pH 5) using Thioflavin T, transmission electron microscopy, circular dichroism, and Fourier transform infrared spectroscopy. Interestingly, we found substantial differences between aSyn aggregation under neutral and acidic pH conditions, like those present in cytosolic and lysosomal cellular compartments. Also, lysosomal aSyn enriched from an aSyn-overexpressing cell line was able to seed aggregation in a concentration-dependent manner. Moreover, we observed that aSyn aggregates formed under in vitro lysosomal pH (pH 5) conditions were not stable at neutral pH and collapsed into partly soluble aggregates with changed structural characteristics. Our findings have meaningful implications in intracellular toxicity events as well as in lysis procedures for molecular and structural characterization of intracellular aSyn conformers.

Glaucoma is a neurodegenerative disease that features the death of retinal ganglion cells (RGCs) in the retina, often as a result of prolonged increases in intraocular pressure. We show that preventing the formation of neuroinflammatory reactive astrocytes prevents the death of RGCs normally seen in a mouse model of glaucoma. Furthermore, we show that these spared RGCs are electrophysiologically functional and thus still have potential value for the function and regeneration of the retina. Finally, we demonstrate that the death of RGCs depends on a combination of both an injury to the neurons and the presence of reactive astrocytes, suggesting a model that may explain why reactive astrocytes are toxic only in some circumstances. Altogether, these findings highlight reactive astrocytes as drivers of RGC death in a chronic neurodegenerative disease of the eye.

Ribbon synapses transmit information in sensory systems, but their development is not well understood. To test the hypothesis that ribbon assembly stabilizes nascent synapses, we performed simultaneous time-lapse imaging of fluorescently-tagged ribbons in retinal cone bipolar cells (BCs) and postsynaptic densities (PSD95-FP) of retinal ganglion cells (RGCs). Ribbons and PSD95-FP clusters were more stable when these components colocalized at synapses. However, synapse density on ON-alpha RGCs was unchanged in mice lacking ribbons (ribeye knockout). Wildtype BCs make both ribbon-containing and ribbon-free synapses with these GCs even at maturity. Ribbon assembly and cone BC-RGC synapse maintenance are thus regulated independently. Despite the absence of synaptic ribbons, RGCs continued to respond robustly to light stimuli, although quantitative examination of the responses revealed reduced frequency and contrast sensitivity.

Recent rodent microbiome experiments suggest that besides Akkermansia, Parasutterella sp. are important in type 2 diabetes and obesity development. In the present translational human study, we aimed to characterize Parasutterella in our European cross-sectional FoCus cohort (n = 1,544) followed by validation of the major results in an independent Canadian cohort (n = 438). In addition, we examined Parasutterella abundance in response to a weight loss intervention (n = 55). Parasutterella was positively associated with BMI and type 2 diabetes independently of the reduced microbiome α/β diversity and low-grade inflammation commonly found in obesity. Nutritional analysis revealed a positive association with the dietary intake of carbohydrates but not with fat or protein consumption. Out of 126 serum metabolites differentially detectable by untargeted HPLC-based MS-metabolomics, L-cysteine showed the strongest reduction in subjects with high Parasutterella abundance. This is of interest, since Parasutterella is a known high L-cysteine consumer and L-cysteine is known to improve blood glucose levels in rodents. Furthermore, metabolic network enrichment analysis identified an association of high Parasutterella abundance with the activation of the human fatty acid biosynthesis pathway suggesting a mechanism for body weight gain. This is supported by a significant reduction of the Parasutterella abundance during our weight loss intervention. Together, these data indicate a role for Parasutterella in human type 2 diabetes and obesity, whereby the link to L-cysteine might be relevant in type 2 diabetes development and the link to the fatty acid biosynthesis pathway for body weight gain in response to a carbohydrate-rich diet in obesity development.

The dorsal lateral geniculate nucleus (dLGN) of the mouse has been an important experimental model for understanding thalamic circuit development. The developmental remodeling of retinal projections has been the primary focus, however much less is known about the maturation of their synaptic targets, the relay cells of the dLGN. Here we examined the growth and maturation of relay cells during the first few weeks of life and addressed whether early retinal innervation affects their development. To accomplish this we utilized the math5 null (math5 (-/-) ) mouse, a mutant lacking retinal ganglion cells and central projections.

Sensory processing can be tuned by a neuron's integration area, the types of inputs, and the proportion and number of connections with those inputs. Integration areas often vary topographically to sample space differentially across regions. Here, we highlight two visual circuits in which topographic changes in the postsynaptic retinal ganglion cell (RGC) dendritic territories and their presynaptic bipolar cell (BC) axonal territories are either matched or unmatched. Despite this difference, in both circuits, the proportion of inputs from each BC type, i.e., synaptic convergence between specific BCs and RGCs, remained constant across varying dendritic territory sizes. Furthermore, synapse density between BCs and RGCs was invariant across topography. Our results demonstrate a wiring design, likely engaging homotypic axonal tiling of BCs, that ensures consistency in synaptic convergence between specific BC types onto their target RGCs while enabling independent regulation of pre- and postsynaptic territory sizes and synapse number between cell pairs.

Correlating light microscopic immunolabelling results with electron microscopic data is of great interest in many fields of biomedical research but typically requires very specialized, expensive equipment and complex procedures which are not available in most labs. In this technical study, we describe an easy and "low-tech"-equipment-requiring pre-embedding immunolabelling approach that allows correlation of light microscopical immunolabelling results with electron microscopic (EM) data as demonstrated by the example of immunolabelled synaptic ribbons from retinal rod photoreceptor synapses. This pre-embedding approach does not require specialized embedding devices but only commonly available equipment. The cryostat section-based procedure allows optimization of the pre-embedding immunolabelling conditions at the less laborious and time-consuming light microscopic (LM) level before the ultrastructural analyses of the immunolabelled structures can be performed on the same sample after ultrathin sectioning without further modification. The same photoreceptor synapse that has been first studied at the light microscopic level can be subsequently analyzed with this approach at the electron microscopic level at individual ultrathin sections or serial ultrathin sections from individual, identical synapses. Higher resolution EM analyses of the immunolabelled synapses can be performed with only minor modifications of the combined LM/EM procedure. The detergent-free procedure is applicable even for weakly fixed cryostat sections which is a relevant aspect for many antibodies that do not work with more strongly fixed biological samples.

Optic neuritis is one of the first manifestations of multiple sclerosis. Its pathogenesis is incompletely understood, but considered to be initiated by an auto-immune response directed against myelin sheaths of the optic nerve. Here, we demonstrate in two frequently used and well-validated mouse models of optic neuritis that ribbon synapses in the myelin-free retina are targeted by an auto-reactive immune system even before alterations in the optic nerve have developed. The auto-immune response is directed against two adhesion proteins (CASPR1/CNTN1) that are present both in the paranodal region of myelinated nerves as well as at retinal ribbon synapses. This occurs in parallel with altered synaptic vesicle cycling in retinal ribbon synapses and altered visual behavior before the onset of optic nerve demyelination. These findings indicate that early synaptic dysfunctions in the retina contribute to the pathology of optic neuritis in multiple sclerosis.

Retinal ganglion cell (RGC) loss is a hallmark of glaucoma and the second leading cause of blindness worldwide. The type and timing of cellular changes leading to RGC loss in glaucoma remain incompletely understood, including whether specific RGC subtypes are preferentially impacted at early stages of this disease. Here we applied the microbead occlusion model of glaucoma to different transgenic mouse lines, each expressing green fluorescent protein in 1-2 specific RGC subtypes. Targeted filling, reconstruction, and subsequent comparison of the genetically identified RGCs in control and bead-injected eyes revealed that some subtypes undergo significant dendritic rearrangements as early as 7 d following induction of elevated intraocular pressure (IOP). By comparing specific On-type, On-Off-type and Off-type RGCs, we found that RGCs that target the majority of their dendritic arbors to the scleral half or "Off" sublamina of the inner plexiform layer (IPL) undergo the greatest changes, whereas RGCs with the majority of their dendrites in the On sublamina did not alter their structure at this time point. Moreover, M1 intrinsically photosensitive RGCs, which functionally are On RGCs but structurally stratify their dendrites in the Off sublamina of the IPL, also underwent significant changes in dendritic structure 1 week after elevated IOP. Thus, our findings reveal that certain RGC subtypes manifest significant changes in dendritic structure after very brief exposure to elevated IOP. The observation that RGCs stratifying most of their dendrites in the Off sublamina are first to alter their structure may inform the development of new strategies to detect, monitor, and treat glaucoma in humans.

The Unc119 protein mediates transport of myristoylated proteins to the photoreceptor outer segment, a specialized primary cilium. This transport activity is regulated by the GTPase Arl3 as well as by Arl13b and Rp2 that control Arl3 activation/inactivation. Interestingly, Unc119 is also enriched in photoreceptor synapses and can bind to RIBEYE, the main component of synaptic ribbons. In the present study, we analyzed whether the known regulatory proteins, that control the Unc119-dependent myristoylated protein transport at the primary cilium, are also present at the photoreceptor synaptic ribbon complex by using high-resolution immunofluorescence and immunogold electron microscopy. We found Arl3 and Arl13b to be enriched at the synaptic ribbon whereas Rp2 was predominantly found on vesicles distributed within the entire terminal. These findings indicate that the synaptic ribbon could be involved in the discharge of Unc119-bound lipid-modified proteins. In agreement with this hypothesis, we found Nphp3 (Nephrocystin-3), a myristoylated, Unc119-dependent cargo protein enriched at the basal portion of the ribbon in close vicinity to the active zone. Mutations in Nphp3 are known to be associated with Senior-Løken Syndrome 3 (SLS3). Visual impairment and blindness in SLS3 might thus not only result from ciliary dysfunctions but also from malfunctions of the photoreceptor synapse.

In the present study, we generated and characterized a splice site-specific monoclonal antibody that selectively detects the calcineurin-binding dynamin1 splice variant dynamin1xb. Calcineurin is a Ca2+-regulated phosphatase that enhances dynamin1 activity and is an important Ca2+-sensing mediator of homeostatic synaptic plasticity in neurons. Using this dynamin1xb-specific antibody, we found dynamin1xb highly enriched in synapses of all analyzed brain regions. In photoreceptor ribbon synapses, dynamin1xb was enriched in close vicinity to the synaptic ribbon in a manner indicative of a peri-active zone immunolabeling. Interestingly, in dark-adapted mice we observed an enhanced and selective enrichment of dynamin1xb in both synaptic layers of the retina in comparison to light-adapted mice. This could be due to an illumination-dependent recruitment of dynamin1xb to retinal synapses and/or due to a darkness-induced increase of dynamin1xb biosynthesis. These latter findings indicate that dynamin1xb is part of a versatile and highly adjustable, activity-regulated endocytic synaptic machinery.

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most fatal malignancies and its development is highly associated with inflammatory processes such as chronic pancreatitis (CP). Since the succinate dehydrogenase subunit B (SDHB) is regarded as tumor suppressor that is lost during cancer development, this study investigated the impact of M1-macrophages as part of the inflammatory microenvironment on the expression as well as function of SDHB in benign and premalignant pancreatic ductal epithelial cells (PDECs). Immunohistochemical analyses on pancreatic tissue sections from CP patients and control individuals revealed a stronger SDHB expression in ducts of CP tissues being associated with a greater abundance of macrophages compared to ducts in control tissues. Accordingly, indirect co-culture with M1-macrophages led to clearly elevated SDHB expression and SDH activity in benign H6c7-pBp and premalignant H6c7-kras PDECs. While siRNA-mediated SDHB knockdown in these cells did not affect glucose and lactate uptake after co-culture, SDHB knockdown significantly promoted PDEC growth which was associated with increased proliferation and decreased effector caspase activity particularly in co-cultured PDECs. Overall, these data indicate that SDHB expression and SDH activity are increased in PDECs when exposed to pro-inflammatory macrophages as a counterregulatory mechanism to prevent excessive PDEC growth triggered by the inflammatory environment.

The Food Chain Plus (FoCus) cohort was launched in 2011 for population-based research related to metabolic inflammation. To characterize this novel pathology in a comprehensive manner, data collection included multiple omics layers such as phenomics, microbiomics, metabolomics, genomics, and metagenomics as well as nutrition profiling, taste perception phenotyping and social network analysis. The cohort was set-up to represent a Northern German population of the Kiel region. Two-step recruitment included the randomised enrolment of participants via residents' registration offices and via the Obesity Outpatient Centre of the University Medical Center Schleswig-Holstein (UKSH). Hence, both a population- and metabolic inflammation- based cohort was created. In total, 1795 individuals were analysed at baseline. Baseline data collection took place between 2011 and 2014, including 63% females and 37% males with an age range of 18-83 years. The median age of all participants was 52.0 years [IQR: 42.5; 63.0 years] and the median baseline BMI in the study population was 27.7 kg/m2 [IQR: 23.7; 35.9 kg/m2]. In the baseline cohort, 14.1% of participants had type 2 diabetes mellitus, which was more prevalent in the subjects of the metabolic inflammation group (MIG; 31.8%). Follow-up for the assessment of disease progression, as well as the onset of new diseases with changes in subject's phenotype, diet or lifestyle factors is planned every 5 years. The first follow-up period was finished in 2020 and included 820 subjects.

The regeneration of nerves of the peripheral nervous system after injuries is a complex process. This study presents a novel in vitro neurite regeneration concept to investigate the regeneration of neurons and their processes with different concentrations of neurotrophic factors. The core part of the concept is a transparent microfluidic neurite isolation (NI) device affixed on top of a microelectrode array (MEA), providing a fast and easy way to assess both the growth and the electrical activity of neurites. The NI-MEA isolates neurites from the culture with microchannels that serve as guidance tubes, equipped with microelectrodes. Thus, the NI-MEA allows neurite growth, as observed by microscopy, to be correlated with neurite electrical activity, as measured by electrophysiological recordings. To demonstrate proof of concept of neurite regeneration, we cultured cells from the superior cervical ganglion of postnatal mice under different concentrations of nerve growth factor (NGF). During the regeneration process, we observed an increase in the number of neurites entering the microchannels along with an increase in spike activity recorded by the microelectrodes in the microchannels. We also observed a concentration-dependent effect of neurotrophic factor on the excitability of the growing neurites, with neurites bathed in 20 ng/ml NGF exhibiting enhanced early growth. Thus, our neurite regeneration concept with the NI-MEA device allows further study of neurotrophic factors and reduces the requirement for in vivo experiments on the regeneration of peripheral nerves after injury.

The production and consumption of mare's milk in Europe has gained importance, mainly based on positive health effects and a lower allergenic potential as compared to cows' milk. The allergenicity of milk is to a certain extent affected by different genetic variants. In classical dairy species, much research has been conducted into the genetic variability of milk proteins, but the knowledge in horses is scarce. Here, we characterize two major forms of equine αS2-casein arising from genomic 1.3 kb in-frame deletion involving two coding exons, one of which represents an equid specific duplication. Findings at the DNA-level have been verified by cDNA sequencing from horse milk of mares with different genotypes. At the protein-level, we were able to show by SDS-page and in-gel digestion with subsequent LC-MS analysis that both proteins are actually expressed. The comparison with published sequences of other equids revealed that the deletion has probably occurred before the ancestor of present-day asses and zebras diverged from the horse lineage.

In this study we addressed the questions whether an Atlantic brown algae extract (BAE) affects diet induced obesity in mice and which would be the primary targets and underlying key mechanisms. Male C57 BL/6 mice were fed a hypercaloric diet, referred to as high fat diet (HFD), supplemented with a freeze-dried aqueous BAE from Saccorhiza polyschides (5 %) for 8 months. Compared to the control group, dietary BAE supplementation significantly attenuated increase in body weight and fat mass. We observed apparent metabolic improvement including normalization of blood glucose, reduced plasma leptin, reduced fecal bile salt hydrolase activity with lower microbial production of toxic bile acid metabolites in the gut and increased systemic bile acid circulation in BAE-fed mice counteracting adverse effects of long term HFD feeding. Survival of mice receiving dietary BAE supplementation appeared slightly enhanced; however, median and maximal life spans as well as hepatic mTOR activation were not significantly different between BAE and control mice. We suggest that the beneficial metabolic effects of our BAE are at least partly mediated by alterations in gut microbiota associated with fermentation of indigestible polysaccharides that are major components of brown algae such as alginates and fucoidans. We moreover propose a multi-factorial mechanism that involves profound alterations in bile acid homeostasis, changes in intestinal and systemic glucose metabolism likely including increased intestinal gluconeogenesis, increased activity of the intestinally derived hormone GLP-1 contributing to promote systemic insulin sensitivity, and inhibition of α-amylase activity, which expectably limits dietary carbohydrate digestion and glucose release.