Shed light on photosynthetic organisms: a physical perspective to correct light measurements.

The requirements for novel and innovative production systems expedite research on light emitting diode-based illumination in a life science context. In course of these rapid developments, the scientific community is in need of a consensus regarding to the characterization and presentation of the applied lighting conditions. This publication aims to establish a basic understanding of photon physics and propose guidelines for the conclusive usage of light related quantities. To illustrate the challenges in data handling, six different light sources were measured and characterized. Furthermore, a stepwise conversion within and in-between physical systems is presented, and an opportunity to extract information from weak data sets is demonstrated. The proposed calculations indicated flexibility in data handling, but revealed partial inaccuracy for colored light emitting diodes with spectral power distribution maxima far-off 550 nm compared to spectrometer-based measurements and conversions. Furthermore, it could be shown, that when comparing light properties, the determination of photometric quantities is incorrect to describe lighting systems for photosynthetic organism and the usage of luxmeter or similar photometric sensors should be avoided. The presented guidelines shall support scientists in applying a consistent and precise characterization of their illumination regimes, tailored to their requirements to avoid ambiguous communication and the generation of incorrect and thus incomparable data based on wrong quantities and units, such as lumen or lux, in future research.

Lossy Image Compression in a Preclinical Multimodal Imaging Study.

The growing use of multimodal high-resolution volumetric data in pre-clinical studies leads to challenges related to the management and handling of the large amount of these datasets. Contrarily to the clinical context, currently there are no standard guidelines to regulate the use of image compression in pre-clinical contexts as a potential alleviation of this problem. In this work, the authors study the application of lossy image coding to compress high-resolution volumetric biomedical data. The impact of compression on the metrics and interpretation of volumetric data was quantified for a correlated multimodal imaging study to characterize murine tumor vasculature, using volumetric high-resolution episcopic microscopy (HREM), micro-computed tomography (µCT), and micro-magnetic resonance imaging (µMRI). The effects of compression were assessed by measuring task-specific performances of several biomedical experts who interpreted and labeled multiple data volumes compressed at different degrees. We defined trade-offs between data volume reduction and preservation of visual information, which ensured the preservation of relevant vasculature morphology at maximum compression efficiency across scales. Using the Jaccard Index (JI) and the average Hausdorff Distance (HD) after vasculature segmentation, we could demonstrate that, in this study, compression that yields to a 256-fold reduction of the data size allowed to keep the error induced by compression below the inter-observer variability, with minimal impact on the assessment of the tumor vasculature across scales.

Regulation of Oxygenic Photosynthesis during Trophic Transitions in the Green Alga Chromochloris zofingiensis.

Light and nutrients are critical regulators of photosynthesis and metabolism in plants and algae. Many algae have the metabolic flexibility to grow photoautotrophically, heterotrophically, or mixotrophically. Here, we describe reversible Glc-dependent repression/activation of oxygenic photosynthesis in the unicellular green alga Chromochloris zofingiensis. We observed rapid and reversible changes in photosynthesis, in the photosynthetic apparatus, in thylakoid ultrastructure, and in energy stores including lipids and starch. Following Glc addition in the light, C. zofingiensis shuts off photosynthesis within days and accumulates large amounts of commercially relevant bioproducts, including triacylglycerols and the high-value nutraceutical ketocarotenoid astaxanthin, while increasing culture biomass. RNA sequencing reveals reversible changes in the transcriptome that form the basis of this metabolic regulation. Functional enrichment analyses show that Glc represses photosynthetic pathways while ketocarotenoid biosynthesis and heterotrophic carbon metabolism are upregulated. Because sugars play fundamental regulatory roles in gene expression, physiology, metabolism, and growth in both plants and animals, we have developed a simple algal model system to investigate conserved eukaryotic sugar responses as well as mechanisms of thylakoid breakdown and biogenesis in chloroplasts. Understanding regulation of photosynthesis and metabolism in algae could enable bioengineering to reroute metabolism toward beneficial bioproducts for energy, food, pharmaceuticals, and human health.

Biodegradation of different PET variants from food containers by Ideonella sakaiensis.

The accumulation of macro-, micro- and nano-plastic wastes in the environment is a major global concern, as these materials are resilient to degradation processes. However, microorganisms have evolved their own biological means to metabolize these petroleum-derived polymers, e.g., Ideonella sakaiensis has recently been found to be capable of utilizing polyethylene terephthalate (PET) as its sole carbon source. This study aims to prove its potential capacity to biodegrade two commercial PET materials, obtained from food packaging containers. Plastic pieces of different crystallinity were simultaneously introduced to Ideonella sakaiensis during a seven-week lasting investigation. Loss in weight, appearance of plastics, as well as growth of Ideonella sakaiensis-through quantitative real-time PCR-were determined. Both plastics were found enzymatically attacked in a two-stage degradation process, reaching biodegradation capacities of up to 96%. Interestingly, the transparent, high crystallinity PET was almost fully degraded first, followed by the colored low-crystallinity PET. Results of quantitative real-time PCR-based gene copy numbers were found in line with experimental results, thus underlining its potential of this method to be applied in future studies with Ideonella sakaiensis.

An observational study of wounds treated with hydro-responsive wound dressings.

OBJECTIVE: Acute and hard-to-heal wounds are a significant burden to both a patient's quality of life and resources in healthcare systems. Here, we evaluate the outcomes of a non-comparative case series study in which Ringer's solution-preactivated polyacrylate dressings were used to treat acute and hard-to-heal wounds (the presence of Ringer's solution provides a wound dressing that allows, upon application, the immediate hydration of the underlying wound tissue). METHOD: Patients with acute and hard-to-heal wounds were enrolled into an open-labelled, non-comparative observational study. Patients were treated with Ringer's solution-preactivated polyacrylate dressings to enable wound debridement and wound cleansing for up to 12 weeks. RESULTS: A total of 303 patients were enrolled in the study and 278 were included in the analysis. Wound size decreased, from a median of 3.6cm2 (interquartile range (IQR): 1.2-9.3] at baseline to a median of 2.6cm2 (IQR: 1.1-7.8] at 84 days. Relative wound area reduction (WAR) was 43.1% at 84 days and estimated probability of achievement of a WAR of ≥40% and ≥60% was 68.7% and 53.4%, respectively. Median time to achieve a WAR of ≥40% and ≥60% was 54 days and 75 days, respectively. The median percentage of wound area covered by fibrin had decreased from 50.0% to 10% and granulation tissue had increased from 25% to 50% after 84 days. In addition, periwound skin condition, local signs of infection and pain all showed improvement. The majority of the wounds were assessed as 'healed' or 'better' at the conclusion of the evaluation period. CONCLUSION: Based on the findings of this study, the use of Ringer's solution-preactivated polyacrylate dressings in daily practice has the potential to improve clinical outcomes, including healing, in patients with acute and hard-to-heal wounds.

Cross-modality imaging of bisphosphonate-treated murine jawbones.

In this proof-of-principle study, we established and implemented a cross-modality imaging (CMI) pipeline to characterize and compare bisphosphonate (BIS)-treated jawbones of Sprague-Dawley rats after tooth extraction after physical therapies (photobiomodulation and extracorporeal shockwave therapy (PBMT and ESWT)). We showcase the feasibility of such a CMI approach and its compatibility across imaging modalities to probe the same region of interest (ROI) of the same jawbone. Jawbones were imaged in toto in 3D using micro-Computed Tomography to identify ROIs for subsequent sequential 2D analysis using well-established technologies such as Atomic Force Microscopy and Scanning Electron Microscopy, and recent imaging approaches in biomedical settings, such as micro-X-Ray Fluorescence Spectroscopy. By combining these four modalities, multiscale information on the morphology, topography, mechanical stiffness (Young's modulus), and calcium, zinc and phosphorus concentrations of the bone was collected. Based on the CMI pipeline, we characterized and compared the jawbones of a previously published clinically relevant rat model of BIS-related osteonecrosis of the jawbone (BRONJ) before and after treatment with BISs, PBMT and ESWT. While we did not find that physical therapies altered the mechanical and elemental jawbone parameters with significance (probably due to the small sample size of only up to 5 samples per group), both ESWT and PBMT reduced pore thicknesses and bone-to-enamel distances significantly compared to the controls. Although focused on BIS-treated jawbones, the established CMI platform can be beneficial in the study of bone-related diseases in general (such as osteoarthritis or -porosis) to acquire complementary hallmarks and better characterize disease status and alleviation potentials.

FRET in Dyads with Orthogonal Chromophores and Minimal Spectral Overlap.

A dyad of orthogonally arranged chromophores of benzoperylenehexacarboxylictrisimide (energy donor, D) and terrylenetetracarboxylic bisimide (energy acceptor, A) was synthesized where a quantitative energy transfer (FRET) from D to A was found, although not only are both transition moments orthogonally arranged but the spectral overlap between the fluorescence of the energy donor and the absorption of the acceptor is also small. Even an appreciable further diminishing of the overlap in a dyad with phenylbenzoxazole as the donor could not inhibit the energy transfer. Thus, the application of the FRET theory to real systems is estimated to be limited where molecular vibrations seem to play a key role in the energy transfer.

Chromosome-level genome assembly and transcriptome of the green alga Chromochloris zofingiensis illuminates astaxanthin production.

Microalgae have potential to help meet energy and food demands without exacerbating environmental problems. There is interest in the unicellular green alga Chromochloris zofingiensis, because it produces lipids for biofuels and a highly valuable carotenoid nutraceutical, astaxanthin. To advance understanding of its biology and facilitate commercial development, we present a C. zofingiensis chromosome-level nuclear genome, organelle genomes, and transcriptome from diverse growth conditions. The assembly, derived from a combination of short- and long-read sequencing in conjunction with optical mapping, revealed a compact genome of ∼58 Mbp distributed over 19 chromosomes containing 15,274 predicted protein-coding genes. The genome has uniform gene density over chromosomes, low repetitive sequence content (∼6%), and a high fraction of protein-coding sequence (∼39%) with relatively long coding exons and few coding introns. Functional annotation of gene models identified orthologous families for the majority (∼73%) of genes. Synteny analysis uncovered localized but scrambled blocks of genes in putative orthologous relationships with other green algae. Two genes encoding beta-ketolase (BKT), the key enzyme synthesizing astaxanthin, were found in the genome, and both were up-regulated by high light. Isolation and molecular analysis of astaxanthin-deficient mutants showed that BKT1 is required for the production of astaxanthin. Moreover, the transcriptome under high light exposure revealed candidate genes that could be involved in critical yet missing steps of astaxanthin biosynthesis, including ABC transporters, cytochrome P450 enzymes, and an acyltransferase. The high-quality genome and transcriptome provide insight into the green algal lineage and carotenoid production.

Age-dependent dissociation of ATP synthase dimers and loss of inner-membrane cristae in mitochondria.

Aging is one of the most fundamental, yet least understood biological processes that affect all forms of eukaryotic life. Mitochondria are intimately involved in aging, but the underlying molecular mechanisms are largely unknown. Electron cryotomography of whole mitochondria from the aging model organism Podospora anserina revealed profound age-dependent changes in membrane architecture. With increasing age, the typical cristae disappear and the inner membrane vesiculates. The ATP synthase dimers that form rows at the cristae tips dissociate into monomers in inner-membrane vesicles, and the membrane curvature at the ATP synthase inverts. Dissociation of the ATP synthase dimer may involve the peptidyl prolyl isomerase cyclophilin D. Finally, the outer membrane ruptures near large contact-site complexes, releasing apoptogens into the cytoplasm. Inner-membrane vesiculation and dissociation of ATP synthase dimers would impair the ability of mitochondria to supply the cell with sufficient ATP to maintain essential cellular functions.

Selective Retina Therapy in Acute and Chronic-Recurrent Central Serous Chorioretinopathy.

PURPOSE: Selective retina therapy (SRT), the confined laser heating and destruction of retinal pigment epithelial cells, has been shown to treat acute types of central serous chorioretinopathy (CSC) successfully without damaging the photoreceptors and thus avoiding laser-induced scotoma. However, a benefit of laser treatment for chronic forms of CSC is questionable. In this study, the efficacy of SRT by means of the previously used 1.7-µs and shorter 300-ns pulse duration was evaluated for both types of CSC, also considering re-treatment for nonresponders. MATERIAL AND METHODS: In a two-center trial, 26 patients were treated with SRT for acute (n = 10) and chronic-recurrent CSC (n = 16). All patients presented with subretinal fluid (SRF) in OCT and leakage in fluorescein angiography (FA). SRT was performed using a prototype SRT laser system (frequency-doubled Q-switched Nd:YLF-laser, wavelength 527 nm) with adjustable pulse duration. The following irradiation settings were used: a train of 30 laser pulses with a repetition rate of 100 Hz and pulse durations of 300 ns and 1.7 µs, pulse energy 120-200 µJ, retinal spot size 200 µm. Because SRT lesions are invisible, FA was always performed 1 h after treatment to demonstrate laser outcome (5-8 single spots in the area of leakage). In cases where energy was too low, as indicated by missing FA leakage, energy was adjusted and the patient re-treated immediately. Observation intervals were after 4 weeks and 3 months. In case of nonimprovement of the disease after 3 months, re-treatment was considered. RESULTS: Of 10 patients with active CSC that presents focal leakage in FA, 5 had completely resolved fluid after 4 weeks and all 10 after 3 months. Mean visual acuity increased from 76.6 ETDRS letters to 85.0 ETDRS letters 3 months after SRT. Chronic-recurrent CSC was characterized by less severe SRF at baseline in OCT and weaker leakage in FA than in acute types. Visual acuity changed from baseline 71.6 to 72.8 ETDRS letters after 3 months. At this time, SRF was absent in 3 out of 16 patients (19%), FA leakage had come to a complete stop in 6 out of 16 patients (38%). In 6 of the remaining chronic CSC patients, repeated SRT with higher pulse energy was considered because of persistent leakage activity. After the re-treatment, SRF resolved completely in 5 patients (83.3%) after only 25 days. CONCLUSION: SRT showed promising results in treating acute CSC, but was less effective in chronic cases. Interestingly, re-treatment resulted in enhanced fluid resolution and dry conditions after a considerably shorter time in most patients. Therefore, SRT including re-treatment if necessary might be a valuable CSC treatment alternative even in chronic-recurrent cases.

Automated segmentation of cell organelles in volume electron microscopy using deep learning.

Recent advances in computing power triggered the use of artificial intelligence in image analysis in life sciences. To train these algorithms, a large enough set of certified labeled data is required. The trained neural network is then capable of producing accurate instance segmentation results that will then need to be re-assembled into the original dataset: the entire process requires substantial expertise and time to achieve quantifiable results. To speed-up the process, from cell organelle detection to quantification across electron microscopy modalities, we propose a deep-learning based approach for fast automatic outline segmentation (FAMOUS), that involves organelle detection combined with image morphology, and 3D meshing to automatically segment, visualize and quantify cell organelles within volume electron microscopy datasets. From start to finish, FAMOUS provides full segmentation results within a week on previously unseen datasets. FAMOUS was showcased on a HeLa cell dataset acquired using a focused ion beam scanning electron microscope, and on yeast cells acquired by transmission electron tomography. RESEARCH HIGHLIGHTS: Introducing a rapid, multimodal machine-learning workflow for the automatic segmentation of 3D cell organelles. Successfully applied to a variety of volume electron microscopy datasets and cell lines. Outperforming manual segmentation methods in time and accuracy. Enabling high-throughput quantitative cell biology.

Crowded chromatin is not sufficient for heterochromatin formation and not required for its maintenance.

In contrast to cytoplasmic organelles, which are usually separated from the rest of the cell by phospholipid membranes, nuclear compartments are readily accessible to diffusing proteins and must rely on different mechanisms to maintain their integrity. Specific interactions between scaffolding proteins are known to have important roles for the formation and maintenance of nuclear structures. General physical mechanisms such as molecular crowding, phase separation or colloidal behavior have also been suggested, but their physiological significance remains uncertain. For macromolecular crowding, a role in the maintenance of nucleoli and promyelocytic leukemia (PML) nuclear bodies has been shown. Here, we tested whether a modulation of the compaction state of chromatin, which directly influences the local crowding state, has an impact on the formation and maintenance of densely packed heterochromatin. By osmotic perturbations, we could modify the packing state of chromatin in a controlled manner and show that chromatin compaction, which is associated with increased crowding conditions, is not, per se, sufficient to initiate the formation of new bona fide heterochromatin structures nor is it necessary to maintain already established heterochromatin domains. In consequence, if an increase in crowding induced by chromatin compaction maybe an early step in heterochromatin formation, specific protein-protein interactions are nevertheless required to make heterochromatin long lasting and independent of the crowding state.

Changes in dialysis treatment modalities during institution of flat rate reimbursement and quality assurance programs.

Dialysis procedure rates in Germany were changed in 2002 from per-session to weekly flat rate payments, and quality assurance was introduced in 2009 with defined treatment targets for spKt/V, dialysis frequency, treatment time, and hemoglobin. In order to understand trends in treatment parameters before and after the introduction of these changes, we analyzed data from 407 to 618 prevalent patients each year (hemodialysis over 90 days) in 14-21 centers in cross-sections of the Dialysis Outcomes and Practice Patterns Study (phases 1-4, 1998-2011). Descriptive statistics were used to report differences over time in the four quality assurance parameters along with erythropoietin-stimulating agent (ESA) and intravenous iron doses. Time trends were analyzed using linear mixed models adjusted for patient demographics and comorbidities. The proportion of patients with short treatment times (less than 4 h) and low spKt/V (below 1.2) improved throughout the study and was lowest after implementation of quality assurance. Hemoglobin levels have increased since 1998 and remained consistent since 2005, with only 8-10% of patients below 10 g/dl. About 90% of patients were prescribed ESAs, with the dose declining since peaking in 2006. Intravenous iron use was highest in 2011. Hence, trends to improve quality metrics for hemodialysis have been established in Germany even after introduction of flat rate reimbursement. Thus, analysis of facility practice patterns is needed to maintain quality of care in a cost-containment environment.

U-Net based vessel segmentation for murine brains with small micro-magnetic resonance imaging reference datasets.

Identification and quantitative segmentation of individual blood vessels in mice visualized with preclinical imaging techniques is a tedious, manual or semiautomated task that can require weeks of reviewing hundreds of levels of individual data sets. Preclinical imaging, such as micro-magnetic resonance imaging (µMRI) can produce tomographic datasets of murine vasculature across length scales and organs, which is of outmost importance to study tumor progression, angiogenesis, or vascular risk factors for diseases such as Alzheimer's. Training a neural network capable of accurate segmentation results requires a sufficiently large amount of labelled data, which takes a long time to compile. Recently, several reasonably automated approaches have emerged in the preclinical context but still require significant manual input and are less accurate than the deep learning approach presented in this paper-quantified by the Dice score. In this work, the implementation of a shallow, three-dimensional U-Net architecture for the segmentation of vessels in murine brains is presented, which is (1) open-source, (2) can be achieved with a small dataset (in this work only 8 µMRI imaging stacks of mouse brains were available), and (3) requires only a small subset of labelled training data. The presented model is evaluated together with two post-processing methodologies using a cross-validation, which results in an average Dice score of 61.34% in its best setup. The results show, that the methodology is able to detect blood vessels faster and more reliably compared to state-of-the-art vesselness filters with an average Dice score of 43.88% for the used dataset.

CryoEM reveals that ribosomes in microsporidian spores are locked in a dimeric hibernating state.

Translational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of electron cryo-tomography and single-particle electron cryo-microscopy. We show that microsporidian spores contain hibernating ribosomes that are locked in a dimeric (100S) state, which is formed by a unique dimerization mechanism involving the beak region. The ribosomes within the dimer are fully assembled, suggesting that they are ready to be activated once the host cell is invaded. This study provides structural evidence for dimerization acting as a mechanism for ribosomal hibernation in microsporidia, and therefore demonstrates that eukaryotes utilize this mechanism in translational control.

Gyrate atrophy: clinical and genetic findings in a female without arginine-restricted diet during her first 39 years of life and report of a new OAT gene mutation.

We report the clinical and genetic data obtained at a 17-year follow-up examination of a patient with gyrate atrophy, without an arginine-restricted diet. Patient examinations included visual acuity (VA), perimetry, biomicroscopy, funduscopy, fundus photography, fundus autofluorescence (FAF), spectral-domain optical coherence tomography (OCT), and standard full-field electroretinography (ERG). Blood samples were taken for measurement of serum ornithine level and molecular genetic analysis of the OAT gene. The female was 22 years of age when gyrate atrophy was diagnosed based on peripheral chorioretinal atrophy and an increased ornithine level. Reexamination after 17 years revealed a reduced VA (0.25 OU), dense cataract, extensive peripheral chorioretinal atrophy, a further increased ornithine level, but only slow progression of visual field constriction, and still detectable ERG amplitudes. FAF was absent in the atrophic periphery and almost homogeneous at the posterior pole except parafoveally. OCT showed interruption of the foveal inner/outer segment junction and parafoveal microcystoid spaces. After cataract surgery, VA increased to the same values as those found at the age of 22 years (0.5 OD, 0.6 OS). Molecular analysis revealed a new deletion c.532_536delTGGGG (p.Trp178X) and a known mutation c.897C>G (p.Tyr299X) in the OAT gene. Although the patient had refused to diet during her first 39 years of life, the gyrate atrophy showed a very slow progression. FAF allows evaluating the integrity of the retinal pigment epithelium and may help to delimit gyrate atrophy from choroideremia. Interruption of foveal inner/outer segment junction and cystoid macula edema appears in gyrate atrophy.

A versatile standard for bathochromic fluorescence based on intramolecular FRET.

A perylene and a terrylene tetracarboxylic bisimide dyad was prepared in which an efficient energy transfer from the former to the latter is observed. The absorption spectrum of this compound covers a broad range. Bathochromic fluorescence with a high quantum yield was obtained independent of excitation wavelengths (λ < 655 nm). The dyad can be recommended for the use of calibrating fluorescence spectrometers, as well as a fluorescence standard in the bathochromic region.

Correlative multimodal imaging: Building a community.

Few would have thought that when Porter and colleagues used light microscopy to target their cell of interest to be analyzed in the electron microscope in the 1940s, that Correlative Imaging would develop into the thriving field it is today. Even though the first use of Correlative Light Electron Microscopy (CLEM) was established in the 1940s, it is only since the year 2000 that there has been a real surge in the application of CLEM technology. The power of CLEM is recognized in the scientific community as evidenced by the growing number of publications and dedicated sessions at scientific meetings. The field is also broadening, incorporating a multitude of other techniques including preclinical research and diagnostics, and slowly but surely the overarching field of Correlative Multimodality Imaging (CMI) is taking its place as an established technique and a research area in its own right. In this chapter, we will look at the initiatives that are being developed within the scientific world to build a coherent CMI community, with a particular emphasis on the developments in Europe. To achieve this aim, the community will need to design mechanisms for the interdisciplinary exchange of knowledge and benefits, set up training schemes, and develop standards for CMI technology and its data.

High-Resolution Episcopic Microscopy (HREM) in Multimodal Imaging Approaches.

High-resolution episcopic microscopy (HREM) is a three-dimensional (3D) episcopic imaging modality based on the acquisition of two-dimensional (2D) images from the cut surface of a block of tissue embedded in resin. Such images, acquired serially through the entire length/depth of the tissue block, are aligned and stacked for 3D reconstruction. HREM has proven to be specifically advantageous when integrated in correlative multimodal imaging (CMI) pipelines. CMI creates a composite and zoomable view of exactly the same specimen and region of interest by (sequentially) correlating two or more modalities. CMI combines complementary modalities to gain holistic structural, functional, and chemical information of the entire sample and place molecular details into their overall spatiotemporal multiscale context. HREM has an advantage over in vivo 3D imaging techniques on account of better histomorphologic resolution while simultaneously providing volume data. HREM also has certain advantages over ex vivo light microscopy modalities. The latter can provide better cellular resolution but usually covers a limited area or volume of tissue, with limited 3D structural context. HREM has predominantly filled a niche in the phenotyping of embryos and characterisation of anatomic developmental abnormalities in various species. Under the umbrella of CMI, when combined with histopathology in a mutually complementary manner, HREM could find wider application in additional nonclinical and translational areas. HREM, being a modified histology technique, could also be incorporated into specialised preclinical pathology workflows. This review will highlight HREM as a versatile imaging platform in CMI approaches and present its benefits and limitations.