Investigation of Hydrocolloid Plant Polysaccharides as Potential Candidates to Mimic the Functions of MUC5B in Saliva.

The successful substitution of complex physiological fluids, such as human saliva, remains a major challenge in drug development. Although there are a large number of saliva substitutes on the market, their efficacy is often inadequate due to short residence time in the mouth, unpleasant mouthfeel, or insufficient protection of the teeth. Therefore, systems need to be identified that mimic the functions of saliva, in particular the salivary mucin MUC5B and the unique physiological properties of saliva. To this end, plant extracts known to contain hydrocolloid polysaccharides and to have mucus-forming properties were studied to evaluate their suitability as saliva substitutes. The aqueous plant extracts of Calendula officinalis, Fucus sp. thalli, and lichenan from Lichen islandicus were examined for composition using a range of techniques, including GC-MS, NMR, SEC, assessment of pH, osmolality, buffering capacity, viscoelasticity, viscoelastic interactions with human saliva, hydrocolloid network formation, and in vitro cell adhesion. For this purpose, a physiologically adapted adhesive test was developed using human buccal epithelial cells. The results show that lichenan is the most promising candidate to mimic the properties of MUC5B. By adjusting the pH, osmolality, and buffering capacity with K2HPO4, it was shown that lichenan exhibited high cell adhesion, with a maximum detachment force that was comparable to that of unstimulated whole mouth saliva.

Effect of astaxanthin in type-2 diabetes -induced APPxhQC transgenic and NTG mice.

OBJECTIVES: Aggregation and misfolding of amyloid beta (Aß) and tau proteins, suggested to arise from post-translational modification processes, are thought to be the main cause of Alzheimer's disease (AD). Additionally, a plethora of evidence exists that links metabolic dysfunctions such as obesity, type 2 diabetes (T2D), and dyslipidemia to the pathogenesis of AD. We thus investigated the combinatory effect of T2D and human glutaminyl cyclase activity (pyroglutamylation), on the pathology of AD and whether astaxanthin (ASX) treatment ameliorates accompanying pathophysiological manifestations. METHODS: Male transgenic AD mice, APPxhQC, expressing human APP751 with the Swedish and the London mutation and human glutaminyl cyclase (hQC) enzyme and their non-transgenic (NTG) littermates were used. Both APPxhQC and NTG mice were allocated to 3 groups, control, T2D-control, and T2D-ASX. Mice were fed control or high fat diet ± ASX for 13 weeks starting at an age of 11-12 months. High fat diet fed mice were further treated with streptozocin for T2D induction. Effects of genotype, T2D induction, and ASX treatment were evaluated by analysing glycemic readouts, lipid concentration, Aß deposition, hippocampus-dependent cognitive function and nutrient sensing using immunosorbent assay, ELISA-based assays, western blotting, immunofluorescence staining, and behavioral testing via Morris water maze (MWM), respectively. RESULTS: APPxhQC mice presented a higher glucose sensitivity compared to NTG mice. T2D-induced brain dysfunction was more severe in NTG compared to the APPxhQC mice. T2D induction impaired memory functions while increasing hepatic LC3B, ABCA1, and p65 levels in NTG mice. T2D induction resulted in a progressive shift of Aß from the soluble to insoluble form in APPxhQC mice. ASX treatment reversed T2D-induced memory dysfunction in NTG mice and in parallel increased hepatic pAKT while decreasing p65 and increasing cerebral p-S6rp and p65 levels. ASX treatment reduced soluble Aß38 and Aß40 and insoluble Aß40 levels in T2D-induced APPxhQC mice. CONCLUSIONS: We demonstrate that T2D induction in APPxhQC mice poses additional risk for AD pathology as seen by increased Aß deposition. Although ASX treatment reduced Aß expression in T2D-induced APPxhQC mice and rescued T2D-induced memory impairment in NTG mice, ASX treatment alone may not be effective in cases of T2D comorbidity and AD.

Western diet-induced ultrastructural changes in mouse pancreatic acinar cells.

Mouse models of diet-induced type 2 diabetes mellitus provide powerful tools for studying the structural and physiological changes that are related to the disease progression. In this study, diabetic-like glucose dysregulation was induced in mice by feeding them a western diet, and light and transmission electron microscopy were used to study the ultrastructural changes in the pancreatic acinar cells. Acinar necrosis and vacuolization of the cytoplasm were the most prominent features. Furthermore, we observed intracellular and extracellular accumulation of lipid compounds in the form of lipid droplets, structural enlargement of the cisternae of the rough endoplasmic reticulum (RER), and altered mitochondrial morphology, with mitochondria lacking the typical organization of the inner membrane. Last, autophagic structures, i.e., autophagosomes, autolysosomes, and residual bodies, were abundant within the acinar cells of western diet-fed mice, and the autolysosomes contained lipids and material of varying electron density. While diets inducing obesity and type 2 diabetes are clearly associated with structural changes and dysfunction of the endocrine pancreas, we here demonstrate the strong effect of dietary intervention on the structure of acinar cells in the exocrine part of the organ before detectable changes in plasma amylase activity, which may help us better understand the development of non-alcoholic fatty pancreas disease and its association with endo- and exocrine dysfunction.

Targeting structural flexibility in low density lipoprotein by integrating cryo-electron microscopy and high-speed atomic force microscopy.

Low-density lipoprotein (LDL) plays a crucial role in cholesterol metabolism. Responsible for cholesterol transport from the liver to the organs, LDL accumulation in the arteries is a primary cause of cardiovascular diseases, such as atherosclerosis. This work focuses on the fundamental question of the LDL molecular structure, as well as the topology and molecular motions of apolipoprotein B-100 (apo B-100), which is addressed by single-particle cryo-electron microscopy (cryo-EM) and high-speed atomic force microscopy (HS-AFM). Our results suggest a revised model of the LDL core organization with respect to the cholesterol ester (CE) arrangement. In addition, a high-density region close to the flattened poles could be identified, likely enriched in free cholesterol. The most remarkable new details are two protrusions on the LDL surface, attributed to the protein apo B-100. HS-AFM adds the dimension of time and reveals for the first time a highly dynamic direct description of LDL, where we could follow large domain fluctuations of the protrusions in real time. To tackle the inherent flexibility and heterogeneity of LDL, the cryo-EM maps are further assessed by 3D variability analysis. Our study gives a detailed explanation how to approach the intrinsic flexibility of a complex system comprising lipids and protein.

Astaxanthin enhances autophagy, amyloid beta clearance and exerts anti-inflammatory effects in in vitro models of Alzheimer's disease-related blood brain barrier dysfunction and inflammation.

Defective degradation and clearance of amyloid-ß as well as inflammation per se are crucial players in the pathology of Alzheimer's disease (AD). A defective transport across the blood-brain barrier is causative for amyloid-ß (Aß) accumulation in the brain, provoking amyloid plaque formation. Using primary porcine brain capillary endothelial cells and murine organotypic hippocampal slice cultures as in vitro models of AD, we investigated the effects of the antioxidant astaxanthin (ASX) on Aß clearance and neuroinflammation. We report that ASX enhanced the clearance of misfolded proteins in primary porcine brain capillary endothelial cells by inducing autophagy and altered the Aß processing pathway. We observed a reduction in the expression levels of intracellular and secreted amyloid precursor protein/Aß accompanied by an increase in ABC transporters ABCA1, ABCG1 as well as low density lipoprotein receptor-related protein 1 mRNA levels. Furthermore, ASX treatment increased autophagic flux as evidenced by increased lipidation of LC3B-II as well as reduced protein expression of phosphorylated S6 ribosomal protein and mTOR. In LPS-stimulated brain slices, ASX exerted anti-inflammatory effects by reducing the secretion of inflammatory cytokines while shifting microglia polarization from M1 to M2 phenotype. Our data suggest ASX as potential therapeutic compound ameliorating AD-related blood brain barrier impairment and inflammation.

A multi-step machine learning approach for accelerating QbD-based process development of protein spray drying.

This study proposes a new material-efficient multi-step machine learning (ML) approach for the development of a design space (DS) for spray drying proteins. Typically, a DS is developed by performing a design of experiments (DoE) with the spray dryer and the protein of interest, followed by deriving the DoE models via multi-variate regression. This approach was followed as a benchmark to the ML approach. The more complex the process and required accuracy of the final model is, the more experiments are necessary. However, most biologics are expensive and thus experiments should be kept to a minimum. Therefore, the suitability of using a surrogate material and ML for the development of a DS was investigated. To this end, a DoE was performed with the surrogate and the data used for training the ML approach. The ML and DoE model predictions were compared to measurements of three protein-based validation runs. The suitability of using lactose as surrogate was investigated and advantages of the proposed approach were demonstrated. Limitations were identified at protein concentrations >35 mg/ml and particle sizes of x50>6 µm. Within the investigated DS protein secondary structure was preserved, and most process settings, resulted in yields >75% and residual moisture <10 wt%.

Investigation of the Influence of Wound-Treatment-Relevant Buffer Systems on the Colloidal and Optical Properties of Gold Nanoparticles.

Biocompatible gold nanoparticles (AuNPs) are used in wound healing due to their radical scavenging activity. They shorten wound healing time by, for example, improving re-epithelialization and promoting the formation of new connective tissue. Another approach that promotes wound healing through cell proliferation while inhibiting bacterial growth is an acidic microenvironment, which can be achieved with acid-forming buffers. Accordingly, a combination of these two approaches appears promising and is the focus of the present study. Here, 18 nm and 56 nm gold NP (Au) were prepared with Turkevich reduction synthesis using design-of-experiments methodology, and the influence of pH and ionic strength on their behaviour was investigated. The citrate buffer had a pronounced effect on the stability of AuNPs due to the more complex intermolecular interactions, which was also confirmed by the changes in optical properties. In contrast, AuNPs dispersed in lactate and phosphate buffer were stable at therapeutically relevant ionic strength, regardless of their size. Simulation of the local pH distribution near the particle surface also showed a steep pH gradient for particles smaller than 100 nm. This suggests that the healing potential is further enhanced by a more acidic environment at the particle surface, making this strategy a promising approach.

Autolysis Affects the Iron Cargo of Ferritins in Neurons and Glial Cells at Different Rates in the Human Brain.

Iron is known to accumulate in neurological disorders, so a careful balance of the iron concentration is essential for healthy brain functioning. An imbalance in iron homeostasis could arise due to the dysfunction of proteins involved in iron homeostasis. Here, we focus on ferritin-the primary iron storage protein of the brain. In this study, we aimed to improve a method to measure ferritin-bound iron in the human post-mortem brain, and to discern its distribution in particular cell types and brain regions. Though it is known that glial cells and neurons differ in their ferritin concentration, the change in the number and distribution of iron-filled ferritin cores between different cell types during autolysis has not been revealed yet. Here, we show the cellular and region-wide distribution of ferritin in the human brain using state-of-the-art analytical electron microscopy. We validated the concentration of iron-filled ferritin cores to the absolute iron concentration measured by quantitative MRI and inductively coupled plasma mass spectrometry. We show that ferritins lose iron from their cores with the progression of autolysis whereas the overall iron concentrations were unaffected. Although the highest concentration of ferritin was found in glial cells, as the total ferritin concentration increased in a patient, ferritin accumulated more in neurons than in glial cells. Summed up, our findings point out the unique behaviour of neurons in storing iron during autolysis and explain the differences between the absolute iron concentrations and iron-filled ferritin in a cell-type-dependent manner in the human brain. The rate of loss of the iron-filled ferritin cores during autolysis is higher in neurons than in glial cells.

Ultrastructural changes in esophageal tissue undergoing stretch tests with possible impact on tissue engineering and long gap esophageal repairs performed under tension.

Esophageal biomechanical studies are being performed to understand structural changes resulting from stretches during repair of esophageal atresias as well as to obtain biomechanical values for tissue-engineered esophagus. The present study offers insights into ultrastructural changes after stretching of the ovine esophagus using uniaxial stretch tests. In vitro uniaxial stretching was performed on esophagi (n = 16) obtained from the abattoir within 4-6 h of 1-month-old lambs. Esophagi were divided into 4 groups (4 esophagi/group): control, Group1 (G1), Group2 (G2), Group3 (G3) stretched to 20%, 30% and 40% of their original length respectively. Force and lengthening were measured with 5 cycles performed on every specimen. Transmission electron microscopic (TEM) studies were performed on the 4 groups. During observational TEM study of the control group there were no significant differences in muscle cell structure or extracellular matrix. In all stretched groups varying degrees of alterations were identified. The degree of damage correlated linearly with the increasing level of stretch. Distance between the cells showed significant difference between the groups (control (µ = 0.41 µm, SD = 0.26), G1 (µ = 1.36 µm, SD = 1.21), G2 (µ = 2.8 µm, SD = 1.83), and G3 (µ = 3.01 µm, SD = 2.06). The diameter of the cells (control µ = 19.87 µm, SD = 3.81; G1 µ = 20.38 µm, SD = 4.45; G2 µ = 21.7 µm, SD = 6.58; G3 µ = 24.48 µm, SD = 6.69) and the distance between myofibrils (control µ = 0.23 µm, SD = 0.08; G1 µ = 0.27 µm, SD = 0.08; G2 µ = 0.4 µm, SD = 0.15; G3 µ = 0.61 µm, SD = 0.2) were significantly different as well ( p < 0.05 was considered to be significant). Esophageal stretching > 30% alters the regular intracellular and extracellular structure of the esophageal muscle and leads to disruption of intra- and extracellular bonds. These findings could provide valuable insights into alterations in the microscopic structure of the esophagus in esophageal atresias repaired under tension as well as the basis for mechanical characterization for tissue engineering of the esophagus.

Needles to Spheres: Evaluation of inkjet printing as a particle shape enhancement tool.

Active pharmaceutical ingredients (APIs) often reveal shapes challenging to process, e.g. acicular structures, and exhibit reduced bioavailability induced by slow dissolution rate. Leveraging the API particles' surface and bulk properties offers an attractive pathway to circumvent these challenges. Inkjet printing is an attractive processing technique able to tackle these limitations already in initial stages when little material is available, while particle properties are maintained over the entire production scale. Additionally, it is applicable to a wide range of formulations and offers the possibility of co-processing with a variety of excipients to improve the API's bioavailability. This study addresses the optimization of particle shapes for processability enhancement and demonstrates the successful application of inkjet printing to engineer spherical lacosamide particles, which are usually highly acicular. By optimizing the ink formulation, adapting the substrate-liquid interface and tailoring the heat transfer to the particle, spherical particles in the vicinity of 100 µm, with improved flow properties compared to the bulk material, were produced. Furthermore, the particle size was tailored reproducibly by adjusting the deposited ink volume per cycle and the number of printing cycles. Therefore, the present study shows a novel, reliable, scalable and economical strategy to overcome challenging particle morphologies by co-processing an API with suitable excipients.

Development of a Workflow to Engineer Tailored Microparticles Via Inkjet Printing.

PURPOSE: New drug development and delivery approaches result in an ever-increasing demand for tailored microparticles with defined sizes and structures. Inkjet printing technologies could be promising new processes to engineer particles with defined characteristics, as they are created to precisely deliver liquid droplets with high uniformity. METHODS: D-mannitol was used as a model compound alone or co-processed with the pore former agent ammonium bicarbonate, and the polymer polyethylene glycol 200. Firstly, a drop shape analyzer was used to characterize and understand ink/substrate interactions, evaporation, and solidification kinetics. Consequently, the process was transferred to a laboratory-scale inkjet printer and the resulting particles collected, characterized and compared to others obtained via an industrial standard technique. RESULTS: The droplet shape analysis allowed to understand how 3D structures are formed and helped define the formulation and process parameters for inkjet printing. By adjusting the drop number and process waveform, spherical particles with a mean size of approximately 100 µm were obtained. The addition of pore former and polymer allowed to tailor the crystallization kinetics, resulting in particles with a different surface (i.e., spike-like surface) and bulk (e.g. porous and non-porous) structure. CONCLUSION: The workflow described enabled the production of 3D structures via inkjet printing, demonstrating that this technique can be a promising approach to engineer microparticles.

Functional remodelling of perinuclear mitochondria alters nucleoplasmic Ca2+ signalling in heart failure.

Mitochondrial dysfunction in cardiomyocytes is a hallmark of heart failure development. Although initial studies recognized the importance of different mitochondrial subpopulations, there is a striking lack of direct comparison of intrafibrillar (IF) versus perinuclear (PN) mitochondria during the development of HF. Here, we use multiple approaches to examine the morphology and functional properties of IF versus PN mitochondria in pressure overload-induced cardiac remodelling in mice, and in non-failing and failing human cardiomyocytes. We demonstrate that PN mitochondria from failing cardiomyocytes are more susceptible to depolarization of mitochondrial membrane potential, reactive oxygen species generation and impairment in Ca2+ uptake compared with IF mitochondria at baseline and under physiological stress protocol. We also demonstrate, for the first time to our knowledge, that under normal conditions PN mitochondrial Ca2+ uptake shapes nucleoplasmic Ca2+ transients (CaTs) and limits nucleoplasmic Ca2+ loading. The loss of PN mitochondrial Ca2+ buffering capacity translates into increased nucleoplasmic CaTs and may explain disproportionate rise in nucleoplasmic [Ca2+] in failing cardiomyocytes at increased stimulation frequencies. Therefore, a previously unidentified benefit of restoring the mitochondrial Ca2+ uptake may be normalization of nuclear Ca2+ signalling and alleviation of altered excitation-transcription, which could be an important therapeutic approach to prevent adverse cardiac remodelling. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

Deficiency of B vitamins leads to cholesterol-independent atherogenic transformation of the aorta.

Atherosclerosis, the leading cause of cardiovascular disease responsible for the majority of deaths worldwide, cannot be sufficiently explained by established risk factors, including hypercholesterolemia. Elevated plasma homocysteine is an independent risk factor for atherosclerosis and is strongly linked to cardiovascular mortality. However, the role of homocysteine in atherosclerosis is still insufficiently understood. Previous research in this area has been also hampered by the lack of reproducible in vivo models of atherosclerosis that resemble the human situation. Here, we have developed and applied an automated system for vessel wall injury that leads to more homogenous damage and more pronounced atherosclerotic plaque development, even at low balloon pressure. Our automated system helped to glean vital details of cholesterol-independent changes in the aortic wall of balloon-injured rabbits. We show that deficiency of B vitamins, which are required for homocysteine degradation, leads to atherogenic transformation of the aorta resulting in accumulation of macrophages and lipids, impairment of its biomechanical properties and disorganization of aortic collagen/elastin in the absence of hypercholesterolemia. A combination of B vitamin deficiency and hypercholesterolemia leads to thickening of the aorta, decreased aortic water diffusion, increased LDL-cholesterol and impaired vascular reactivity compared to any single condition. Our findings suggest that deficiency of B vitamins leads to atherogenic transformation of the aorta even in the absence of hypercholesterolemia and aggravates atherosclerosis development in its presence.

MICU1 controls spatial membrane potential gradients and guides Ca2+ fluxes within mitochondrial substructures.

Mitochondrial ultrastructure represents a pinnacle of form and function, with the inner mitochondrial membrane (IMM) forming isolated pockets of cristae membrane (CM), separated from the inner-boundary membrane (IBM) by cristae junctions (CJ). Applying structured illumination and electron microscopy, a novel and fundamental function of MICU1 in mediating Ca2+ control over spatial membrane potential gradients (SMPGs) between CM and IMS was identified. We unveiled alterations of SMPGs by transient CJ openings when Ca2+ binds to MICU1 resulting in spatial cristae depolarization. This Ca2+/MICU1-mediated plasticity of the CJ further provides the mechanistic bedrock of the biphasic mitochondrial Ca2+ uptake kinetics via the mitochondrial Ca2+ uniporter (MCU) during intracellular Ca2+ release: Initially, high Ca2+ opens CJ via Ca2+/MICU1 and allows instant Ca2+ uptake across the CM through constantly active MCU. Second, MCU disseminates into the IBM, thus establishing Ca2+ uptake across the IBM that circumvents the CM. Under the condition of MICU1 methylation by PRMT1 in aging or cancer, UCP2 that binds to methylated MICU1 destabilizes CJ, disrupts SMPGs, and facilitates fast Ca2+ uptake via the CM.

Three-dimensional SEM, TEM, and STEM for analysis of large-scale biological systems.

A major aim in structural cell biology is to analyze intact cells in three dimensions, visualize subcellular structures, and even localize proteins at the best possible resolution in three dimensions. Though recently developed electron microscopy tools such as electron tomography, or three-dimensional (3D) scanning electron microscopy, offer great resolution in three dimensions, the challenge is that, the better the resolution, usually the smaller the volume under investigation. Several different approaches to overcome this challenge were presented at the Microscopy Conference in Vienna in 2021. These tools include array tomography, batch tomography, or scanning transmission electron tomography, all of which can nowadays be extended toward correlative light and electron tomography, with greatly increased 3D information. Here, we review these tools, describe the underlying procedures, and discuss their advantages and limits.

Thiolated Chitosan Conjugated Liposomes for Oral Delivery of Selenium Nanoparticles.

This study aimed to design a hybrid oral liposomal delivery system for selenium nanoparticles (Lip-SeNPs) to improve the bioavailability of selenium. Thiolated chitosan, a multifunctional polymer with mucoadhesive properties, was used for surface functionalization of Lip-SeNPs. Selenium nanoparticle (SeNP)-loaded liposomes were manufactured by a single step microfluidics-assisted chemical reduction and assembling process. Subsequently, chitosan-N-acetylcysteine was covalently conjugated to the preformed Lip-SeNPs. The Lip-SeNPs were characterized in terms of composition, morphology, size, zeta potential, lipid organization, loading efficiency and radical scavenging activity. A co-culture system (Caco-2:HT29-MTX) that integrates mucus secreting and enterocyte-like cell types was used as a model of the human intestinal epithelium to determine adsorption, mucus penetration, release and transport properties of Lip-SeNPs in vitro. Thiolated Lip-SeNPs were positively charged with an average size of about 250 nm. Thiolated Lip-SeNPs tightly adhered to the mucus layer without penetrating the enterocytes. This finding was consistent with ex vivo adsorption studies using freshly excised porcine small intestinal tissues. Due to the improved mucoadhesion and retention in a simulated microenvironment of the small intestine, thiolated Lip-SeNPs might be a promising tool for oral selenium delivery.

Adipose triglyceride lipase-mediated lipid catabolism is essential for bronchiolar regeneration.

The lung airways are constantly exposed to inhaled toxic substances, resulting in cellular damage that is repaired by local expansion of resident bronchiolar epithelial club cells. Disturbed bronchiolar epithelial damage repair lies at the core of many prevalent lung diseases, including chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, and lung cancer. However, it is still not known how bronchiolar club cell energy metabolism contributes to this process. Here, we show that adipose triglyceride lipase (ATGL), the rate-limiting enzyme for intracellular lipolysis, is critical for normal club cell function in mice. Deletion of the gene encoding ATGL, Pnpla2 (also known as Atgl), induced substantial triglyceride accumulation, decreased mitochondrial numbers, and decreased mitochondrial respiration in club cells. This defect manifested as bronchiolar epithelial thickening and increased airway resistance under baseline conditions. After naphthalene­induced epithelial denudation, a regenerative defect was apparent. Mechanistically, dysfunctional PPARα lipid-signaling underlies this phenotype because (a) ATGL was needed for PPARα lipid-signaling in regenerating bronchioles and (b) administration of the specific PPARα agonist WY14643 restored normal bronchiolar club cell ultrastructure and regenerative potential. Our data emphasize the importance of the cellular energy metabolism for lung epithelial regeneration and highlight the significance of ATGL-mediated lipid catabolism for lung health.

An ultrastructural 3D reconstruction method for observing the arrangement of collagen fibrils and proteoglycans in the human aortic wall under mechanical load.

An insight into changes of soft biological tissue ultrastructures under loading conditions is essential to understand their response to mechanical stimuli. Therefore, this study offers an approach to investigate the arrangement of collagen fibrils and proteoglycans (PGs), which are located within the mechanically loaded aortic wall. The human aortic samples were either fixed directly with glutaraldehyde in the load-free state or subjected to a planar biaxial extension test prior to fixation. The aortic ultrastructure was recorded using electron tomography. Collagen fibrils and PGs were segmented using convolutional neural networks, particularly the ESPNet model. The 3D ultrastructural reconstructions revealed a complex organization of collagen fibrils and PGs. In particular, we observed that not all PGs are attached to the collagen fibrils, but some fill the spaces between the fibrils with a clear distance to the collagen. The complex organization cannot be fully captured or can be severely misinterpreted in 2D. The approach developed opens up practical possibilities, including the quantification of the spatial relationship between collagen fibrils and PGs as a function of the mechanical load. Such quantification can also be used to compare tissues under different conditions, e.g., healthy and diseased, to improve or develop new material models. STATEMENT OF SIGNIFICANCE: The developed approach enables the 3D reconstruction of collagen fibrils and proteoglycans as they are embedded in the loaded human aortic wall. This methodological pipeline comprises the knowledge of arterial mechanics, imaging with transmission electron microscopy and electron tomography, segmentation of 3D image data sets with convolutional neural networks and finally offers a unique insight into the ultrastructural changes in the aortic tissue caused by mechanical stimuli.

Ultrastructure of spherites in the midgut diverticula and Malpighian tubules of the harvestman Amilenus aurantiacus during the winter diapause.

Amilenus aurantiacus overwinter in diapause, a natural starvation period, in hypogean habitats. The structure of spherites in the midgut diverticula (MD) and Malpighian tubules (MT) has been studied comparatively by light microscopy and TEM to detect eventual differences in mineral consumption in the beginning and at the end of the starvation period in these organs (MD and MT) associated with digestive processes. The chemical composition of spherites was examined by combining energy-dispersive X-ray spectroscopy (EDXS), electron energy-loss spectroscopy (EELS) and energy-filtered TEM (EFTEM). The structure of the spherites changed during overwintering in both organs. At the beginning of overwintering, the spherites were composed of densely packed concentric layers of electron-dense and electron-lucent material. In the middle and at the end of overwintering, the electron-lucent layers between the layers of material indicated the loss of some material. The chemical composition of the spherites changed only in the MD; at the beginning of overwintering, these contained Si, O, C and Fe, while later there was no more Fe. In contrast, spherites in the MT were composed of Si, O, C and Ca throughout overwintering. A less intensive exploitation of the MD spherites was probably due to complete cessation of digestive and other cell activity in this organ during the winter diapause; activity of the MT slowed, but continued removing the cell metabolites.

The complex synaptic pathways onto a looming-detector neuron revealed using serial block-face scanning electron microscopy.

The ability of locusts to detect looming stimuli and avoid collisions or predators depends on a neuronal circuit in the locust's optic lobe. Although comprehensively studied for over three decades, there are still major questions about the computational steps of this circuit. We used fourth instar larvae of Locusta migratoria to describe the connection between the lobula giant movement detector 1 (LGMD1) neuron in the lobula complex and the upstream neuropil, the medulla. Serial block-face scanning electron microscopy (SBEM) was used to characterize the morphology of the connecting neurons termed trans-medullary afferent (TmA) neurons and their synaptic connectivity. This enabled us to trace neurons over several hundred micrometers between the medulla and the lobula complex while identifying their synapses. We traced two different TmA neurons, each from a different individual, from their synapses with the LGMD in the lobula complex up into the medulla and describe their synaptic relationships. There is not a simple downstream transmission of the signal from a lamina neuron onto these TmA neurons; there is also a feedback loop in place with TmA neurons making outputs as well as receiving inputs. More than one type of neuron shapes the signal of the TmA neurons in the medulla. We found both columnar and trans-columnar neurons connected with the traced TmA neurons in the medulla. These findings indicate that there are computational steps in the medulla that have not been included in models of the neuronal pathway for looming detection.