Revealing the Chemical Composition of Birch Pollen Grains by Raman Spectroscopic Imaging.

The investigation of the biochemical composition of pollen grains is of the utmost interest for several environmental aspects, such as their allergenic potential and their changes in growth conditions due to climatic factors. In order to fully understand the composition of pollen grains, not only is an in-depth analysis of their molecular components necessary but also spatial information of, e.g., the thickness of the outer shell, should be recorded. However, there is a lack of studies using molecular imaging methods for a spatially resolved biochemical composition on a single-grain level. In this study, Raman spectroscopy was implemented as an analytical tool to investigate birch pollen by imaging single pollen grains and analyzing their spectral profiles. The imaging modality allowed us to reveal the layered structure of pollen grains based on the biochemical information of the recorded Raman spectra. Seven different birch pollen species collected at two different locations in Germany were investigated and compared. Using chemometric algorithms such as hierarchical cluster analysis and multiple-curve resolution, several components of the grain wall, such as sporopollenin, as well as the inner core presenting high starch concentrations, were identified and quantified. Differences in the concentrations of, e.g., sporopollenin, lipids and proteins in the pollen species at the two different collection sites were found, and are discussed in connection with germination and other growth processes.

Identification of tissue sections from decellularized liver scaffolds for repopulation experiments.

BACKGROUND: Biological organ engineering is a novel experimental approach to generate functional liver grafts by decellularization and repopulation. Currently, healthy organs of small or large animals and human organs with preexisting liver diseases are used to optimize decellularization and repopulation.However, the effects of morphological changes on allo- and xenogeneic cell-scaffold interactions during repopulation procedure, e.g., using scaffold-sections, are unknown. We present a sequential morphological workflow to identify murine liver scaffold-sections with well-preserved microarchitecture. METHODS: Native livers (CONT, n = 9) and livers with experimentally induced pathologies (hepatics steatosis: STEA, n = 7; hepatic fibrosis induced by bile duct ligation: BDL, n = 9; nodular regenerative hyperplasia induced by 90% partial hepatectomy: PH, n = 8) were decellularized using SDS and Triton X-100 to generate cell-free scaffolds. Scaffold-sections were assessed using a sequential morphological workflow consisting of macroscopic, microscopic and morphological evaluation: (1) The scaffold was evaluated by a macroscopic decellularization score. (2) Regions without visible tissue remnants were localized for sampling and histological processing. Subsequent microscopical examination served to identify tissue samples without cell remnants. (3) Only cell-free tissue sections were subjected to detailed liver-specific morphological assessment using a histological and immunohistochemical decellularization score. RESULTS: Decellularization was feasible in 33 livers, which were subjected to the sequential morphological workflow. In 11 of 33 scaffolds we achieved a good macroscopic decellularization result (CONT: 3 scaffolds; STEA: 3 scaffolds; BDL: 3 scaffolds; PH: 2 scaffolds). The microscopic assessment resulted in the selection of 88 cell-free tissue sections (CONT: 15 sections; STEA: 38 sections; BDL: 30 sections; PH: 5 sections). In 27 of those sections we obtained a good histological decellularization result (CONT: 3 sections; STEA: 6 sections; BDL: 17 sections; PH: 1 section). All experimental groups contained sections with a good immunohistochemical decellularization result (CONT: 6 sections; STEA: 5 sections; BDL: 4 sections; PH: 1 section). DISCUSSION: Decellularization was possible in all experimental groups, irrespectively of the underlying morphological alteration. Furthermore, our proposed sequential morphological workflow was suitable to detect tissue sections with well-preserved hepatic microarchitecture, as needed for further repopulation experiments.

Semantic Segmentation of Intralobular and Extralobular Tissue from Liver Scaffold H&E Images.

Decellularized tissue is an important source for biological tissue engineering. Evaluation of the quality of decellularized tissue is performed using scanned images of hematoxylin-eosin stained (H&E) tissue sections and is usually dependent on the observer. The first step in creating a tool for the assessment of the quality of the liver scaffold without observer bias is the automatic segmentation of the whole slide image into three classes: the background, intralobular area, and extralobular area. Such segmentation enables to perform the texture analysis in the intralobular area of the liver scaffold, which is crucial part in the recellularization procedure. Existing semi-automatic methods for general segmentation (i.e., thresholding, watershed, etc.) do not meet the quality requirements. Moreover, there are no methods available to solve this task automatically. Given the low amount of training data, we proposed a two-stage method. The first stage is based on classification of simple hand-crafted descriptors of the pixels and their neighborhoods. This method is trained on partially annotated data. Its outputs are used for training of the second-stage approach, which is based on a convolutional neural network (CNN). Our architecture inspired by U-Net reaches very promising results, despite a very low amount of the training data. We provide qualitative and quantitative data for both stages. With the best training setup, we reach 90.70% recognition accuracy.

Novel morphological multi-scale evaluation system for quality assessment of decellularized liver scaffolds.

Decellularized scaffolds can serve as an excellent three-dimensional environment for cell repopulation. They maintain tissue-specific microarchitecture of extracellular matrix proteins with important spatial cues for cell adhesion, migration, growth, and differentiation. However, criteria for quality assessment of the three-dimensional structure of decellularized scaffolds are rather fragmented, usually study-specific, and mostly semi-quantitative. Thus, we aimed to develop a robust structural assessment system for decellularized porcine liver scaffolds. Five scaffolds of different quality were used to establish the new evaluation system. We combined conventional semi-quantitative scoring criteria with a quantitative scaffold evaluation based on automated image analysis. For the quantitation, we developed a specific open source software tool (ScaffAn) applying algorithms designed for texture analysis, segmentation, and skeletonization. ScaffAn calculates selected parameters characterizing structural features of porcine liver scaffolds such as the sinusoidal network. After evaluating individual scaffolds, the total scores predicted scaffold interaction with cells in terms of cell adhesion. Higher scores corresponded to higher numbers of cells attached to the scaffolds. Moreover, our analysis revealed that the conventional system could not identify fine differences between good quality scaffolds while the additional use of ScaffAn allowed discrimination. This led us to the conclusion that only using the combined score resulted in the best discrimination between different quality scaffolds. Overall, our newly defined evaluation system has the potential to select the liver scaffolds most suitable for recellularization, and can represent a step toward better success in liver tissue engineering.

Ramipril pretreatment worsened renal injury and survival despite a reduction in renal inflammation in experimentally induced sepsis in mice.

INTRODUCTION: The angiotensin converting enzyme inhibitor ramipril is a standard antihypertensive therapy for many patients. Because angiotensin II may promote inflammation, we were interested in whether basal pretreatment with ramipril may modify renal function and inflammation as well as systemic outcome in experimentally induced sepsis in mice. MATERIAL AND METHODS: Ramipril (10 mg/kg/day) pretreatment or placebo (NaCl) was given intraperitoneally for 5 days to C57BL6/J mice, followed by either sham operation or cecal ligation and puncture sepsis induction. Real-time polymerase chain reaction and immunological stains were used to evaluate renal gene and protein expression, respectively. Plasma creatinine, neutrophil-gelatinase associated lipocalin, and blood urea nitrogen were used as markers for renal function. A clinical severity score was determined. RESULTS: Administration of ramipril before cecal ligation and puncture surgery was associated with reduced renal inflammation but did not improved renal function and structure and even worsened the clinical status of septic mice. CONCLUSIONS: The data suggest that the effects of ramipril pretreatment are complex. Additional studies including monitoring of hemodynamic parameters are necessary to elucidate the exact mechanism(s) of this observation. In addition, the timing of the ramipril administration could be of importance.

[Video-based Analysis of Practical Skills - a Suitable Tool to Develop Surgical Training?] / Videoanalyse praktischer Fertigkeiten ­ ein geeignetes Tool zur Weiterentwicklung der chirurgischen Lehre?

BACKGROUND: Video-based analysis concepts are being is increasingly applied in medical education. These tools are mainly used to obtain information about the individual performance of a candidate and to provide feedback. The present study explores whether video-based analysis of practical skills can also be used for the development of surgical training. METHODS: First, the performance of students in semester 10 (n = 38) in a surgical suture exercise (duration: 3 min) was video-documented. The video material was then analysed using 10 specific criteria. The analysis then served as a basis for the development of error prevention exercises. In the following, the effects of the additional teaching units on the performance in the suture exercise were examined in a pilot study using a two-group comparison. RESULTS: The video sequences were reviewed independently by 2 experts. Typical errors could be observed in the handling of the surgical instruments, the handling of the suture material as well as in the motion sequence. Then, additional teaching units dealing with the identified error areas (handling of the instruments and the suture material) were developed. The results of the two-group comparison (before and after implementation of the new exercises) showed that completing the additional teaching units had a medium effect on the result quality of the suture exercise (Cohen's d = 0.73). CONCLUSION: Video analysis of practical skills seems to be a suitable basis for the development of surgical training. Typical errors can be identified in terms of type and frequency, and preventive exercises can be developed, which have a positive effect on the quality of the results of a practical task.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

Crystal structures of 4,4'-(disulfane-1,2-diyl)bis(5-methyl-2H-1,3-dithiol-2-one) and 4,4'-(diselanane-1,2-diyl)bis(5-methyl-2H-1,3-dithiol-2-one).

The two title compounds, C8H6O2S6 and C8H6O2S4Se2, are isotypic with very similar cell parameters. The complete mol-ecules constitute the asymmetric units, despite being chemically perfectly symmetric. The most prominant differences in the metrical parameters arise from the distinct sizes of sulfur and selenium in the dichalcogenide bridges, with C-S-S-C and C-Se-Se-C torsion angles of 70.70 (5) and 68.88 (3)°, respectively. The crystal packing is determined by weak non-classical hydrogen-bonding inter-actions. One carbonyl oxygen but not the other participates in C-H⋯O inter-actions zigzagging along the b axis, forming infinite chains. This is complemented by an intra-molecular C-H⋯S inter-action and further inter-molecular C-H⋯S (C-H⋯Se) inter-actions, resulting in a three-dimensional network. The inter-actions involving the bridging chalcogenides form chains protruding along the c axis.

MORG1+/- mice are protected from histological renal damage and inflammation in a murine model of endotoxemia.

BACKGROUND: The MAPK-organizer 1 (MORG1) play a scaffold function in the MAPK and/or the PHD3 signalling paths. Recently, we reported that MORG1+/- mice are protected from renal injury induced by systemic hypoxia and acute renal ischemia-reperfusion injury via increased hypoxia-inducible factors (HIFs). Here, we explore whether MORG1 heterozygosity could attenuate renal injury in a murine model of lipopolysaccharide (LPS) induced endotoxemia. METHODS: Endotoxemia was induced in mice by an intraperitoneal (i.p) application of 5 mg/kg BW LPS. The renal damage was estimated by periodic acid Schiff's staining; renal injury was evaluated by detection of urinary and plasma levels of neutrophil gelatinase-associated lipocalin and albumin/creatinine ratio via ELISAs. Renal mRNA expression was assessed by real-time PCR, whereas the protein expression was determined by immunohistochemistry or Western blotting. RESULTS: LPS administration increased tubular injury, microalbuminuria, IL-6 plasma levels and renal TNF-α expression in MORG1 +/+ mice. This was accompanied with enhanced infiltration of the inflammatory T-cells in renal tissue and activation of the NF-κB transcription factors. In contrast, endotoxemic MORG1 +/- showed significantly less tubular injury, reduced plasma IL-6 levels, significantly decreased renal TNF-α expression and T-cells infiltration. In support, the renal levels of activated caspase-3 were lower in endotoxemic MORG1 +/- mice compared with endotoxemic MORG1 +/+ mice. Interestingly, LPS application induced a significantly higher accumulation of renal HIF-2α in the kidneys of MORG1+/- mice than in wild-type mice, accompanied with a diminished phosphorylation of IκB-α and IKK α,ß and decreased iNOS mRNA in the renal tissues of the LPS-challenged MORG1+/- mice, indicating an inhibition of the NF-κB transcriptional activation. CONCLUSIONS: MORG1 heterozygosity protects against histological renal damage and shows anti-inflammatory effects in a murine endotoxemia model through modulation of HIF-2α stabilisation and/or simultaneous inhibition of the NF-κB signalling. Here, we show for the first time that MORG1 scaffold could represent the missing link between innate immunity and inflammation.

Recommendations to enhance constructivist-based learning in Interprofessional Education using video-based self-assessment.

INTRODUCTION: Interprofessional collaboration is crucial to the optimization of patient care. AIM: This paper aims to provide recommendations for implementing an innovative constructivist educational concept with the core element of video-based self-assessment. METHODOLOGY: A course for students in medicine, physiotherapy, and nursing was developed through interprofessional, cross-institutional collaboration. The course consisted of drawing on prior knowledge about the work done by each professional group in regard to a specific clinical scenario and an interprofessional treatment situation, filming a role play of this treatment situation, and a structured self-assessment of the role play. We evaluated the preparation and implementation of the three courses conducted thus far. Concrete recommendations for implementation were made based on evaluation sheets (students), open discussions (tutors, instructors, institutions) and recorded meeting minutes (project managers, project participants). RESULTS: Basic recommendations for implementation include: selecting appropriate criteria for self-assessment and a simulated situation that offers members of each professional group an equal opportunity to act in the role play. In terms of administrative implementation we recommend early coordination among the professions and educational institutions regarding the target groups, scheduling and attendance policy to ensure participant recruitment across all professions. Procedural planning should include developing teaching materials, such as the case vignette and treatment scenario, and providing technical equipment that can be operated intuitively in order to ensure efficient recording. CONCLUSION: These recommendations serve as an aid for implementing an innovative constructivist educational concept with video-based self-assessment at its core.

Preconditioned suppression of prolyl-hydroxylases attenuates renal injury but increases mortality in septic murine models.

BACKGROUND: Septic conditions contribute to tissue hypoxia, potentially leading to multiple organ failure, including acute kidney injury. The regulation of cellular adaptation to low oxygen levels is regulated by hypoxia-inducible transcription factors (HIFs). While the role of HIFs in ischaemia/reperfusion is more studied, their function in sepsis-induced renal injury is not well characterized. In this study, we investigated whether pharmacological activation of HIFs by suppression of prolyl-hydroxylases (PHDs) protects against septic acute kidney injury. METHODS: Two models of sepsis-caecal ligation and punction and peritoneal contamination and infection-were induced on 12-week-old C57BL6/J mice. Pharmacological inhibition of PHDs, leading to HIF activation, was achieved by intraperitoneal application of 3,4-dihydroxybenzoate (3,4-DHB) before sepsis. A quantitative real-time reverse transcription polymerase chain reaction, immunohistology and enzyme-linked immunosorbent assays were utilized to detect gene expression, renal protein levels and renal functional parameters, respectively. Tissue morphology was analysed by periodic acid-Schiff reaction. Early kidney injury was estimated by kidney injury molecule-1 analyses. Apoptosis was detected in situ by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling stain. The systemic effect of 3,4-DHB pretreatment in sepsis was analysed by 72-h survival studies. RESULTS: Pharmacological activation of HIFs before sepsis induction attenuated sepsis-related vacuolization and dilation of the proximal tubules, reduced tubular apoptosis and correlated to lower T-cell infiltration in renal tissue compared with the non-treated septic animals. PHD suppression elevated the basal renal HIF-1α expression and basal plasma concentrations of HIF targets erythropoietin and vascular endothelial growth factor. Whereas it preserved renal structure in both models, it improved renal function in a model-dependent manner. Moreover, inhibition of PHDs led to increased mortality in both models. Analysis of liver function showed increased organ destruction with massive glycogen loss and hepatocyte's apoptosis due to 3,4-DHB administration before sepsis induction. CONCLUSIONS: In summary, the pharmacological activation of HIFs by 3,4-DHB administration, although it showed renoprotective effects in sepsis-related kidney injury, induced more severe problems in other organs such as the liver during sepsis, leading to increased mortality.

Ultrasonic excitation affects friction interactions between food materials and cutting tools.

In the food industry, ultrasonic cutting is used to improve separation by a reduction of the cutting force. This reduction can be attributed to the modification of tool-workpiece interactions at the cutting edge and along the tool flanks because of the superposition of the cutting movement with ultrasonic vibration of the cutting tool. In this study, model experiments were used to analyze friction between the flanks of a cutting tool and the material to be cut. Friction force at a commercial cutting sonotrode was quantified using combined cutting-friction experiments, and sliding friction tests were carried out by adapting a standard draw-off assembly and using an ultrasonic welding sonotrode as sliding surface. The impact of material parameters, ultrasonic amplitude, and the texture of the contacting food surface on friction force was investigated. The results show that ultrasonic vibration significantly reduces the sliding friction force. While the amplitude showed no influence within the tested range, the texture of the contact surface of the food affects the intensity of ultrasonic transportation effects. These effects are a result of mechanical interactions and of changes in material properties of the contact layer, which are induced by the deformation of contact points, friction heating and absorption heating because of the dissipation of mechanical vibration energy.