Search

Cell culture experiments can support characterization of enzymatic activities in healthy and tumorous human tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) enables simultaneous measurement of several steroids from a single sample, facilitating analysis of molecular pathways involved in steroid biosynthesis. We developed a reliable but fast method for quantification of cortisol, cortisone and aldosterone in cell culture supernatant. Validation, including investigation of matrix-matched calibration, was performed for two different cell types. Utility of the method was demonstrated in the study of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity under conditions of glucocorticoid and mineralocorticoid excess in different cell types. Aldosterone, cortisol and cortisone were extracted by liquid-liquid extraction (LLE) with methyl tert-butyl ether from 1 mL of cell culture supernatant. Steroids were separated on a Kinetex biphenyl column (50 ×2.1 mm, 2.6 µm) with gradient elution of water and methanol containing 2 mM ammonium format and analysed in multiple reaction monitoring mode after positive electrospray ionization. Application of the method included cell culture experiments with two different primary cell types, human coronary artery smooth muscle cells (HCSMC) and human coronary artery endothelial cells (EC). Cells were treated with different concentrations of cortisol, aldosterone and mifepristone, a glucocorticoid receptor antagonist and quantitative PCR was performed. The method exhibits high precision (CV ≤ 6 %) and accuracy (deviation from nominal concentration ≤ 6 %) for concentrations above the limit of quantification (LoQ) which is 0.11, 0.56 and 0.69 nmol/L for aldosterone, cortisone and cortisol, respectively. Calibration curves did not differ when prepared in media or solvent. The method enabled us to confirm activity of HSD11B2 and concentration dependent conversion of cortisol to cortisone in HCSMC (median conversion ratio at 140 nM cortisol = 1.46 %). In contrast we did not observe any HSD11B2 activity in EC. Neither addition of high aldosterone, nor addition of 1 µM mifepristone had impact on glucocorticoid concentrations. Quantitative PCR revealed expression of HSD11B1 and HSD11B2 in HCSMC but not in EC. We present a fast and reliable method for quantification of cortisol, cortisone and aldosterone in cell culture supernatants. The method enabled us to study HSD11B2 activity in two different cell types and will support future experiments investigating mechanisms of target organ damage in conditions of glucocorticoid and mineralocorticoid excess., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Martin Bidlingmaier reports financial support was provided by German Research Foundation. Holger Schneider reports financial support was provided by German Research Foundation. Martin Reincke reports financial support was provided by German Research Foundation. Marily Theodoropoulou reports financial support was provided by German Research Foundation. Martin Reincke reports financial support was provided by European Research Council. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

As concerns about the environmental impacts of biowaste disposal increase, lactic acid bacterial fermentation is becoming increasingly popular. Current academic research is aimed at the process optimization by developing digital bioreactors. The primary focus is to develop a digital model mimicking the biochemical reactions. In the light of this, this paper intended to build a digital model of biochemical reactions during the fermentation process of both glucose and biowaste substrates, including white pasta and organic municipal waste. For this purpose, near-infrared (NIR) and mid-infrared (MIR) spectroscopy techniques were used to collect spectral information during the fermentation process. Next, the samples were analyzed by High Pressure Liquid Chromatography (HPLC) to measure their glucose, fructose, arabinose, xylose, disaccharide, lactic acid, and acetic acid contents. The results showed that learning algorithms trained on MIR spectra accurately estimated the biochemical reactions for both glucose and biowaste substrates. For the glucose substrate, the results showed R-squared of 0.97 and RMSE of 4.69 g/L for glucose, and R-squared of 0.98 and RMSE of 2.74 g/L for lactic acid. In the case of biowaste substrate, estimations included glucose (R-squared = 0.97, RMSE = 4.69 g/L), fructose (R-squared = 0.88, RMSE = 1.47 g/L), arabinose (R-squared = 0.98, RMSE = 0.55 g/L), xylose (R-squared = 0.93, RMSE = 1.11 g/L), disaccharide (R-squared = 0.90, RMSE = 0.55 g/L), total sugar (R-squared = 0.98, RMSE = 3.79 g/L), lactic acid (R-squared = 0.98, RMSE = 2.74 g/L), and acetic acid (R-squared = 0.97, RMSE = 0.36 g/L). Regarding NIR spectral data, the predictive models were accurate when the substrate was glucose, however, they failed to accurately estimate the chemical reactions in the case of biowaste substrate. The findings of this study can be used to fulfill the requirements for a continuous fermentation process with the objective of maximizing lactic acid production., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Living organisms control the formation of mineral skeletons and other structures through biomineralization. Major phylogenetic groups usually consistently follow a single biomineralization pathway. Foraminifera, which are very efficient marine calcifiers, making a substantial contribution to global carbonate production and global carbon sequestration, are regarded as an exception. This phylum has been commonly thought to follow two contrasting models of either in situ 'mineralization of extracellular matrix' attributed to hyaline rotaliid shells, or 'mineralization within intracellular vesicles' attributed to porcelaneous miliolid shells. Our previous results on rotaliids along with those on miliolids in this paper question such a wide divergence of biomineralization pathways within the same phylum of Foraminifera. We have found under a high-resolution scanning electron microscopy (SEM) that precipitation of high-Mg calcitic mesocrystals in porcelaneous shells takes place in situ and form a dense, chaotic meshwork of needle-like crystallites. We have not observed calcified needles that already precipitated in the transported vesicles, what challenges the previous model of miliolid mineralization. Hence, Foraminifera probably utilize less divergent calcification pathways, following the recently discovered biomineralization principles. Mesocrystalline chamber walls in both models are therefore most likely created by intravesicular accumulation of pre-formed liquid amorphous mineral phase deposited and crystallized within the extracellular organic matrix enclosed in a biologically controlled privileged space by active pseudopodial structures. Both calcification pathways evolved independently in the Paleozoic and are well conserved in two clades that represent different chamber formation modes., Competing Interests: ZD, JT, AP, MH, JB, MJ, NK, UB No competing interests declared, (© 2024, Dubicka et al.)

Production of commodity chemicals, such as benzene, toluene, ethylbenzene, and xylenes (BTEX), from renewable resources is key for a sustainable society. Biocatalysis enables one-pot multistep transformation of bioresources under mild conditions, yet it is often limited to biochemicals. Herein, we developed a non-natural three-enzyme cascade for one-pot conversion of biobased l-phenylalanine into ethylbenzene. The key rate-limiting photodecarboxylase was subjected to structure-guided semirational engineering, and a triple mutant CvFAP(Y466T/P460A/G462I) was obtained with a 6.3-fold higher productivity. With this improved photodecarboxylase, an optimized two-cell sequential process was developed to convert l-phenylalanine into ethylbenzene with 82 % conversion. The cascade reaction was integrated with fermentation to achieve the one-pot bioproduction of ethylbenzene from biobased glycerol, demonstrating the potential of cascade biocatalysis plus enzyme engineering for the production of biobased commodity chemicals., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the cholesterol-binding, lipid-raft associated protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here, we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore analysed a mouse line expressing the equivalent podocin isoform (podocin Δexon5 ) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocin Δexon5 and used targeted mass spectrometry and qPCR to compare protein and mRNA levels of podocin wildtype and podocin Δexon5 . After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes' foot process morphology.Mice homozygous for podocin Δexon5 were born heavily albuminuric and did not survive past the first 24 h after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocin Δexon5 , whereas mRNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes' slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocin Δexon5 were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels.The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex., (© 2023. The Author(s).)

Mammalian kidneys filter enormous volumes of water and small solutes, a filtration driven by the hydrostatic pressure in glomerular capillaries, which is considerably higher than in most other tissues. Interdigitating cellular processes of podocytes form the slits for fluid filtration connected by the membrane-like slit diaphragm cell junction containing a mechanosensitive ion channel complex and allow filtration while counteracting hydrostatic pressure. Several previous publications speculated that podocyte processes may display a preferable orientation on glomerular capillaries instead of a random distribution. However, for decades, the controversy over spatially oriented filtration slits could not be resolved due to technical limitations of imaging technologies. Here, we used advanced high-resolution, three-dimensional microscopy with high data throughput to assess spatial orientation of podocyte processes and filtration slits quantitatively. Filtration-slit-generating secondary processes preferentially align along the capillaries' longitudinal axis while primary processes are preferably perpendicular to the longitudinal direction. This preferential orientation required maturation in development of the mice but was lost in mice with kidney disease due to treatment with nephrotoxic serum or with underlying heterologous mutations in the podocyte foot process protein podocin. Thus, the observation that podocytes maintain a preferred spatial orientation of their processes on glomerular capillaries goes well in line with the role of podocyte foot processes as mechanical buttresses to counteract mechanical forces resulting from pressurized capillaries. Future studies are needed to establish how podocytes establish and maintain their orientation and why orientation is lost under pathological conditions., (Copyright © 2023 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

An enzyme cascade was established previously consisting of a recycling system with an l-amino acid oxidase (hcLAAO4) and a catalase (hCAT) for different α-keto acid co-substrates of (S)-selective amine transaminases (ATAs) in kinetic resolutions of racemic amines. Only 1 mol % of the co-substrate was required and l-amino acids instead of α-keto acids could be applied. However, soluble enzymes cannot be reused easily. Immobilization of hcLAAO4, hCAT and the (S)-selective ATA from Vibrio fluvialis (ATA-Vfl) was addressed here. Immobilization of the enzymes together rather than on separate beads showed higher reaction rates most likely due to fast co-substrate channeling between ATA-Vfl and hcLAAO4 due to their close proximity. Co-immobilization allowed further reduction of the co-substrate amount to 0.1 mol % most likely due to a more efficient H 2 O 2 -removal caused by the stabilized hCAT and its proximity to hcLAAO4. Finally, the co-immobilized enzyme cascade was reused in 3 cycles of preparative kinetic resolutions to produce (R)-1-PEA with high enantiomeric purity (97.3 %ee). Further recycling was inefficient due to the instability of ATA-Vfl, while hcLAAO4 and hCAT revealed high stability. An engineered ATA-Vfl-8M was used in the co-immobilized enzyme cascade to produce (R)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethanamine, an apremilast-intermediate, with a 1,000 fold lower input of the co-substrate., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel_I_48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved β-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel_I_48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources., (© 2023 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Amine transaminases (ATAs) are powerful biocatalysts for the stereoselective synthesis of chiral amines. Machine learning provides a promising approach for protein engineering, but activity prediction models for ATAs remain elusive due to the difficulty of obtaining high-quality training data. Thus, we first created variants of the ATA from Ruegeria sp. (3FCR) with improved catalytic activity (up to 2000-fold) as well as reversed stereoselectivity by a structure-dependent rational design and collected a high-quality dataset in this process. Subsequently, we designed a modified one-hot code to describe steric and electronic effects of substrates and residues within ATAs. Finally, we built a gradient boosting regression tree predictor for catalytic activity and stereoselectivity, and applied this for the data-driven design of optimized variants which then showed improved activity (up to 3-fold compared to the best variants previously identified). We also demonstrated that the model can predict the catalytic activity for ATA variants of another origin by retraining with a small set of additional data., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Morphological alterations at the kidney filtration barrier increase intrinsic capillary wall permeability resulting in albuminuria. However, automated, quantitative assessment of these morphological changes has not been possible with electron or light microscopy. Here we present a deep learning-based approach for segmentation and quantitative analysis of foot processes in images acquired with confocal and super-resolution fluorescence microscopy. Our method, Automatic Morphological Analysis of Podocytes (AMAP), accurately segments podocyte foot processes and quantifies their morphology. AMAP applied to a set of kidney diseases in patient biopsies and a mouse model of focal segmental glomerulosclerosis allowed for accurate and comprehensive quantification of various morphometric features. With the use of AMAP, detailed morphology of podocyte foot process effacement was found to differ between categories of kidney pathologies, showed detailed variability between diverse patients with the same clinical diagnosis, and correlated with levels of proteinuria. AMAP could potentially complement other readouts such as various omics, standard histologic/electron microscopy and blood/urine assays for future personalized diagnosis and treatment of kidney disease. Thus, our novel finding could have implications to afford an understanding of early phases of kidney disease progression and may provide supplemental information in precision diagnostics., (Copyright © 2023 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Significance Statement: AKI is a major clinical complication leading to high mortality, but intensive research over the past decades has not led to targeted preventive or therapeutic measures. In rodent models, caloric restriction (CR) and transient hypoxia significantly prevent AKI and a recent comparative transcriptome analysis of murine kidneys identified kynureninase (KYNU) as a shared downstream target. The present work shows that KYNU strongly contributes to CR-mediated protection as a key player in the de novo nicotinamide adenine dinucleotide biosynthesis pathway. Importantly, the link between CR and NAD+ biosynthesis could be recapitulated in a human cohort., Background: Clinical practice lacks strategies to treat AKI. Interestingly, preconditioning by hypoxia and caloric restriction (CR) is highly protective in rodent AKI models. However, the underlying molecular mechanisms of this process are unknown., Methods: Kynureninase (KYNU) knockout mice were generated by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and comparative transcriptome, proteome and metabolite analyses of murine kidneys pre- and post-ischemia-reperfusion injury in the context of CR or ad libitum diet were performed. In addition, acetyl-lysin enrichment and mass spectrometry were used to assess protein acetylation., Results: We identified KYNU as a downstream target of CR and show that KYNU strongly contributes to the protective effect of CR. The KYNU-dependent de novo nicotinamide adenine dinucleotide (NAD+) biosynthesis pathway is necessary for CR-associated maintenance of NAD+ levels. This finding is associated with reduced protein acetylation in CR-treated animals, specifically affecting enzymes in energy metabolism. Importantly, the effect of CR on de novo NAD+ biosynthesis pathway metabolites can be recapitulated in humans., Conclusions: CR induces the de novo NAD+ synthesis pathway in the context of IRI and is essential for its full nephroprotective potential. Differential protein acetylation may be the molecular mechanism underlying the relationship of NAD+, CR, and nephroprotection., (Copyright © 2023 by the American Society of Nephrology.)

Main Conclusion: The pool of carbon- and nitrogen-rich metabolites is quantitatively relevant in non-foliar photosynthetic organs during grain filling, which have a better response to water limitation than flag leaves. The response of durum wheat to contrasting water regimes has been extensively studied at leaf and agronomic level in previous studies, but the water stress effects on source-sink dynamics, particularly non-foliar photosynthetic organs, is more limited. Our study aims to investigate the response of different photosynthetic organs to water stress and to quantify the pool of carbon and nitrogen metabolites available for grain filling. Five durum wheat varieties were grown in field trials in the Spanish region of Castile and León under irrigated and rainfed conditions. Water stress led to a significant decrease in yield, biomass, and carbon and nitrogen assimilation, improved water use efficiency, and modified grain quality traits in the five varieties. The pool of carbon (glucose, glucose-6-phosphate, fructose, sucrose, starch, and malate) and nitrogen (glutamate, amino acids, proteins and chlorophylls) metabolites in leaf blades and sheaths, peduncles, awns, glumes and lemmas were also analysed. The results showed that the metabolism of the blades and peduncles was the most susceptible to water stress, while ear metabolism showed higher stability, particularly at mid-grain filling. Interestingly, the total metabolite content per organ highlighted that a large source of nutrients, which may be directly involved in grain filling, are found outside the blades, with the peduncles being quantitatively the most relevant. We conclude that yield improvements in our Mediterranean agro-ecosystem are highly linked to the success of shoots in producing ears and a higher number of grains, while grain filling is highly dependent on the capacity of non-foliar organs to fix CO 2 and N. The ear organs show higher stress resilience than other organs, which deserves our attention in future breeding programmes., (© 2023. The Author(s).)

Background: Tracheostomy tube capping is a commonly used test to determine if the tracheostomy tube can be removed. The success of the capping trial depends on the patient's ability to maintain sufficient spontaneous breathing with an occluded tracheostomy tube. The impact of an occluded tracheotomy tube on airway resistance is currently unknown. The aim of this study was to investigate tracheal pressure during capping or stoma button insertion and potential determinants concerning cuff., Methods: Eight cuffed and uncuffed tracheostomy tubes and three stoma buttons of various manufacturers and sizes were inserted into the trachea model. Cuffs were completely deflated or contained atmospheric pressure. The trachea was ventilated bidirectional with a respirator in volume-controlled mode and volume flows 15-60 L/min. Tracheal pressure drop during inspiration as a parameter of pressure required to move gas through the airway was measured., Results: Tracheal pressure drops occurred linearly or irregularly during capping trials to a maximum of 4.2 kPa at flow rates of 60 L/min for atmospheric pressure cuffs. In tracheostomy tubes with completely deflated cuffs, pressure drop in the trachea reaches a maximum of 3.4 kPa at a flow rate of 60 L/min. For tracheostomy tubes with cuff smaller inner or outer diameters do not regularly result in lower tracheal pressure drop. The pressure drop varies between different tracheostomy tubes depending on the manufacturer. In cuffed tracheostomy tubes, we observed three phenomena: sail-like positioning, folding over, and tightening of the cuff during flow. The maximum tracheal pressure drop during stoma button insertion reaches 0.014 kPa., Conclusions: The cuff is a central element for the pressure drop in the airway and thus airway resistance during spontaneous translaryngeal breathing with a capped TT. Complete deflation reduces the pressure drop in the trachea. Due to deformation of the cuff, measured pressures are irregular as the volume flow is increased. Incomplete deflated cuffs and material characteristics of tracheostomy tubes and cuffs in addition to anatomical and clinical variables may cause unsuccessful capping trials due to increased airway resistance. All stoma buttons showed that pressure drop and thus airway resistance due to stoma buttons has no clinical relevance., (© 2022. The Author(s).)

Amine transaminases (ATAs) are powerful biocatalysts for the stereoselective synthesis of chiral amines. However, wild-type ATAs usually show pH optima at slightly alkaline values and exhibit low catalytic activity under physiological conditions. For efficient asymmetric synthesis ATAs are commonly used in combination with lactate dehydrogenase (LDH, optimal pH: 7.5) and glucose dehydrogenase (GDH, optimal pH: 7.75) to shift the equilibrium towards the synthesis of the target chiral amine and hence their pH optima should fit to each other. Based on a protein structure alignment, variants of ( R )-selective transaminases were rationally designed, produced in E. coli , purified and subjected to biochemical characterization. This resulted in the discovery of the variant E49Q of the ATA from Aspergillus fumigatus , for which the pH optimum was successfully shifted from pH 8.5 to 7.5 and this variant furthermore had a two times higher specific activity than the wild-type protein at pH 7.5. A possible mechanism for this shift of the optimal pH is proposed. Asymmetric synthesis of ( R )-1-phenylethylamine from acetophenone in combination with LDH and GDH confirmed that the variant E49Q shows superior performance at pH 7.5 compared to the wild-type enzyme.

Fast screening of enzyme variants is crucial for tailoring biocatalysts for the asymmetric synthesis of non-natural chiral chemicals, such as amines. However, most existing screening methods either are limited by the throughput or require specialized equipment. Herein, we report a simple, high-throughput, low-equipment dependent, and generally applicable growth selection system for engineering amine-forming or converting enzymes and apply it to improve biocatalysts belonging to three different enzyme classes. This results in (i) an amine transaminase variant with 110-fold increased specific activity for the asymmetric synthesis of the chiral amine intermediate of Linagliptin; (ii) a 270-fold improved monoamine oxidase to prepare the chiral amine intermediate of Cinacalcet by deracemization; and (iii) an ammonia lyase variant with a 26-fold increased activity in the asymmetric synthesis of a non-natural amino acid. Our growth selection system is adaptable to different enzyme classes, varying levels of enzyme activities, and thus a flexible tool for various stages of an engineering campaign., (© 2022. The Author(s).)

Introduction: Genetic disorders are among the most prevalent causes leading to progressive glomerular disease and, ultimately, end-stage renal disease (ESRD) in children and adolescents. Identification of underlying genetic causes is indispensable for targeted treatment strategies and counseling of affected patients and their families., Methods: Here, we report on a boy who presented at 4 years of age with proteinuria and biopsy-proven focal segmental glomerulosclerosis (FSGS) that was temporarily responsive to treatment with ciclosporin A. Molecular genetic testing identified a novel mutation in alpha-actinin-4 (p.M240T). We describe a feasible and efficient experimental approach to test its pathogenicity by combining in silico , in vitro , and in vivo analyses., Results: The de novo p.M240T mutation led to decreased alpha-actinin-4 stability as well as protein mislocalization and actin cytoskeleton rearrangements. Transgenic expression of wild-type human alpha-actinin-4 in Drosophila melanogaster nephrocytes was able to ameliorate phenotypes associated with the knockdown of endogenous actinin. In contrast, p.M240T, as well as other established disease variants p.W59R and p.K255E, failed to rescue these phenotypes, underlining the pathogenicity of the novel alpha-actinin-4 variant., Conclusion: Our data highlight that the newly identified alpha-actinin-4 mutation indeed encodes for a disease-causing variant of the protein and promote the Drosophila model as a simple and convenient tool to study monogenic kidney disease in vivo ., (© 2022 Published by Elsevier Inc. on behalf of the International Society of Nephrology.)

Background and Purpose: Many patients with stroke cannot receive intravenous thrombolysis because the time of symptom onset is unknown. We tested whether a simple method of computed tomography (CT)-based quantification of water uptake in the ischemic tissue can identify patients with stroke onset within 4.5 hours., Methods: This retrospective analysis of the MissPerfeCT study (August 2009 to November 2017) includes consecutive patients with known onset of symptoms from seven tertiary stroke centers. We developed a simplified algorithm based on region of interest (ROI) measurements to quantify water uptake of the ischemic lesion and thereby quantify time of symptom onset within and beyond 4.5 hours. Perfusion CT was used to identify ischemic brain tissue, and its density was measured in non-contrast CT and related to the density of the corresponding area of the contralateral hemisphere to quantify lesion water uptake., Results: Of 263 patients, 204 (77.6%) had CT within 4.5 hours. Water uptake was significantly lower in patients with stroke onset within (6.7%; 95% confidence interval [CI], 6.0% to 7.4%) compared to beyond 4.5 hours (12.7%; 95% CI, 10.7% to 14.7%). The area under the curve for distinguishing these patient groups according to percentage water uptake was 0.744 with an optimal cut-off value of 9.5%. According to this cut-off the positive predictive value was 88.8%, sensitivity was 73.5%, specificity 67.8%, negative predictive value was 42.6%., Conclusions: Ischemic stroke patients with unknown time of symptom onset can be identified as being within a timeframe of 4.5 hours using a ROI-based method to assess water uptake on admission non-contrast head CT.

Acute kidney injury is a frequent complication in the clinical setting and associated with significant morbidity and mortality. Preconditioning with short-term caloric restriction is highly protective against kidney injury in rodent ischemia reperfusion injury models. However, the underlying mechanisms are unknown hampering clinical translation. Here, we examined the molecular basis of caloric restriction-mediated protection to elucidate the principles of kidney stress resistance. Analysis of an RNAseq dataset after caloric restriction identified Cyp4a12a, a cytochrome exclusively expressed in male mice, to be strongly downregulated after caloric restriction. Kidney ischemia reperfusion injury robustly induced acute kidney injury in male mice and this damage could be markedly attenuated by pretreatment with caloric restriction. In females, damage was significantly less pronounced and preconditioning with caloric restriction had only little effect. Tissue concentrations of the metabolic product of Cyp4a12a, 20-hydroxyeicosatetraenoic acid (20-HETE), were found to be significantly reduced by caloric restriction. Conversely, intraperitoneal supplementation of 20-HETE in preconditioned males partly abrogated the protective potential of caloric restriction. Interestingly, this effect was accompanied by a partial reversal of caloric restriction--induced changes in protein but not RNA expression pointing towards inflammation, endoplasmic reticulum stress and lipid metabolism. Thus, our findings provide an insight into the mechanisms underlying kidney protection by caloric restriction. Hence, understanding the mediators of preconditioning is an important prerequisite for moving towards translation to the clinical setting., (Copyright © 2022 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Amine transaminases (ATA) convert ketones into optically active amines and are used to prepare active pharmaceutical ingredients and building blocks. Novel ATA can be identified in protein databases due to the extensive knowledge of sequence-function relationships. However, predicting thermo- and operational stability from the amino acid sequence is a persisting challenge and a vital step towards identifying efficient ATA biocatalysts for industrial applications. In this study, we performed a database mining and characterized selected putative enzymes of the β-alanine:pyruvate transaminase cluster (3N5M) - a subfamily with so far only a few described members, whose tetrameric structure was suggested to positively affect operational stability. Four putative transaminases (TA-1: Bilophilia wadsworthia, TA-5: Halomonas elongata, TA-9: Burkholderia cepacia, and TA-10: Burkholderia multivorans) were obtained in a soluble form as tetramers in E. coli. During comparison of these tetrameric with known dimeric transaminases we found that indeed novel ATA with high operational stabilities can be identified in this protein subfamily, but we also found exceptions to the hypothesized correlation that a tetrameric assembly leads to increased stability. The discovered ATA from Burkholderia multivorans features a broad substrate specificity, including isopropylamine acceptance, is highly active (6 U/mg) in the conversion of 1-phenylethylamine with pyruvate and shows a thermostability of up to 70 °C under both, storage and operating conditions. In addition, 50% (v/v) of isopropanol or DMSO can be employed as co-solvents without a destabilizing effect on the enzyme during an incubation time of 16 h at 30 °C. KEY POINTS: • Database mining identified a thermostable amine transaminase in the β-alanine:pyruvate transaminase subfamily. • The tetrameric transaminase tolerates 50% DMSO and isopropanol under operating conditions at 30 °C. • A tetrameric structure is not necessarily associated with a higher operational stability., (© 2022. The Author(s).)

Chiral and enantiopure amines can be produced by enantioselective transaminases via kinetic resolution of amine racemates. This transamination reaction requires stoichiometric amounts of co-substrate. A dual-enzyme recycling system overcomes this limitation: l-amino acid oxidases (LAAO) recycle the accumulating co-product of (S)-selective transaminases in the kinetic resolution of racemic amines to produce pure (R)-amines. However, availability of suitable LAAOs is limited. Here we use the heterologously produced, highly active fungal hcLAAO4 with broad substrate spectrum. H 2 O 2 as byproduct of hcLAAO4 is detoxified by a catalase. The final system allows using sub-stoichiometric amounts of 1 mol% of the transaminase co-substrate as well as the initial application of l-amino acids instead of α-keto acids. With an optimized protocol, the synthetic potential of this kinetic resolution cascade was proven at the preparative scale (>90 mg) by the synthesis of highly enantiomerically pure (R)-methylbenzylamine (>99 %ee) at complete conversion (50 %)., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Many plants, including Arabidopsis (Arabidopsis thaliana), accumulate starch in the daytime and remobilize it to support maintenance and growth at night. Starch accumulation is increased when carbon is in short supply, for example, in short photoperiods. Mobilization is paced to exhaust starch around dawn, as anticipated by the circadian clock. This diel pattern of turnover is largely robust against loss of day, dawn, dusk, or evening clock components. Here, we investigated diel starch turnover in the triple circadian clock mutant lhy cca1 elf3, which lacks the LATE ELONGATED HYPOCOTYL and the CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) dawn components and the EARLY FLOWERING3 (ELF3) evening components of the circadian clock. The diel oscillations of transcripts for the remaining clock components and related genes like REVEILLE and PHYTOCHROME-INTERACING FACTOR family members exhibited attenuated amplitudes and altered peak time, weakened dawn dominance, and decreased robustness against changes in the external light-dark cycle. The triple mutant was unable to increase starch accumulation in short photoperiods. However, it was still able to pace starch mobilization to around dawn in different photoperiods and growth irradiances and to around 24 h after the previous dawn in T17 and T28 cycles. The triple mutant was able to slow down starch mobilization after a sudden low-light day or a sudden early dusk, although in the latter case it did not fully compensate for the lengthened night. Overall, there was a slight trend to less linear mobilization of starch. Thus, starch mobilization can be paced rather robustly to dawn despite a major disruption of the transcriptional clock. It is proposed that temporal information can be delivered from clock components or a semi-autonomous oscillator., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The integration of high-throughput phenotyping and metabolic approaches is a suitable strategy to study the genotype-by-environment interaction and identify novel traits for crop improvement from canopy to an organ level. Our aims were to study the phenotypic and metabolic traits that are related to grain yield and quality at canopy and organ levels, with a special focus on source-sink coordination under contrasting N supplies. Four modern durum wheat varieties with contrasting grain yield were grown in field conditions under two N fertilization levels in north-eastern Spain. We evaluated canopy vegetation indices taken throughout the growing season, physiological and metabolic traits in different photosynthetic organs (flag leaf blade, sheath, peduncle, awn, glume, and lemma) at anthesis and mid-grain filling stages, and agronomic and grain quality traits at harvest. Low N supply triggered an imbalance of C and N coordination at the whole plant level, leading to a reduction of grain yield and nutrient composition. The activities of key enzymes in C and N metabolism as well as the levels of photoassimilates showed that each organ plays an important role during grain filling, some with a higher photosynthetic capacity, others for nutrient storage for later stages of grain filling, or N assimilation and recycling. Interestingly, the enzyme activities and sucrose content of the ear organs were positively associated with grain yield and quality, suggesting, together with the regression models using isotope signatures, the potential contribution of these organs during grain filling. This study highlights the use of holistic approaches to the identification of novel targets to improve grain yield and quality in C 3 cereals and the key role of non-foliar organs at late-growth stages., Competing Interests: RM-P and NA were employed by Junta de Castilla y León. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Martínez-Peña, Schlereth, Höhne, Encke, Morcuende, Nieto-Taladriz, Araus, Aparicio and Vicente.)

Glomerular diseases are a major cause for chronic kidney disorders. In most cases podocyte injury is causative for disease development. Cytoskeletal rearrangements and morphological changes are hallmark features of podocyte injury and result in dedifferentiation and loss of podocytes. Here, we establish a link between the Par3 polarity complex and actin regulators necessary to establish and maintain podocyte architecture by utilizing mouse and Drosophila models to characterize the functional role of Par3A and Par3B and its fly homologue Bazooka in vivo. Only simultaneous inactivation of both Par3 proteins caused a severe disease phenotype. Rescue experiments in Drosophila nephrocytes revealed atypical protein kinase C (aPKC)-Par6 dependent and independent effects. While Par3A primarily acts via aPKC-Par6, Par3B function was independent of Par6. Actin-associated synaptopodin protein levels were found to be significantly upregulated upon loss of Par3A/B in mouse podocytes. Tropomyosin2, which shares functional similarities with synaptopodin, was also elevated in Bazooka depleted nephrocytes. The simultaneous depletion of Bazooka and Tropomyosin2 resulted in a partial rescue of the Bazooka knockdown phenotype and prevented increased Rho1-GTP, a member of a GTPase protein family regulating the cytoskeleton. The latter contribute to the nephrocyte phenotype observed upon loss of Bazooka. Thus, we demonstrate that Par3 proteins share a high functional redundancy but also have specific functions. Par3A acts in an aPKC-Par6 dependent way and regulates RhoA-GTP levels, while Par3B exploits Par6 independent functions influencing synaptopodin localization. Hence, Par3A and Par3B link elements of polarity signaling and actin regulators to maintain podocyte architecture., (Copyright © 2021 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Diseases of the kidney's glomerular filtration barrier are a leading cause of end stage renal failure. Despite a growing understanding of genes involved in glomerular disorders in children, the vast majority of adult patients lack a clear genetic diagnosis. The protein podocin p.R229Q, which results from the most common missense variant in NPHS2 , is enriched in cohorts of patients with FSGS. However, p.R229Q has been proposed to cause disease only when transassociated with specific additional genetic alterations, and population-based epidemiologic studies on its association with albuminuria yielded ambiguous results., Methods: To test whether podocin p.R229Q may also predispose to the complex disease pathogenesis in adults, we introduced the exact genetic alteration in mice using CRISPR/Cas9-based genome editing ( Pod R231Q ). We assessed the phenotype using super-resolution microscopy and albuminuria measurements and evaluated the stability of the mutant protein in cell culture experiments., Results: Heterozygous Pod R231Q/wild-type mice did not present any overt kidney disease or proteinuria. However, homozygous Pod R231Q/R231Q mice developed increased levels of albuminuria with age, and super-resolution microscopy revealed preceding ultrastructural morphologic alterations that were recently linked to disease predisposition. When injected with nephrotoxic serum to induce glomerular injury, heterozygous Pod R231Q/wild-type mice showed a more severe course of disease compared with Pod wild-type/wild-type mice. Podocin protein levels were decreased in Pod R231Q/wild-type and Pod R231Q/R231Q mice as well as in human cultured podocytes expressing the podocin R231Q variant. Our in vitro experiments indicate an underlying increased proteasomal degradation., Conclusions: Our findings demonstrate that podocin R231Q exerts a pathogenic effect on its own, supporting the concept of podocin R229Q contributing to genetic predisposition in adult patients., (Copyright © 2022 by the American Society of Nephrology.)

Photosynthesis-related pathways are regarded as a promising avenue for crop improvement. Whilst empirical studies have shown that photosynthetic efficiency is higher in microalgae than in C 3 or C 4 crops, the underlying reasons remain unclear. Using a tailor-made microfluidics labelling system to supply 13 CO 2 at steady state, we investigated in vivo labelling kinetics in intermediates of the Calvin Benson cycle and sugar, starch, organic acid and amino acid synthesis pathways, and in protein and lipids, in Chlamydomonas reinhardtii, Chlorella sorokiniana and Chlorella ohadii, which is the fastest growing green alga on record. We estimated flux patterns in these algae and compared them with published and new data from C 3 and C 4 plants. Our analyses identify distinct flux patterns supporting faster growth in photosynthetic cells, with some of the algae exhibiting faster ribulose 1,5-bisphosphate regeneration and increased fluxes through the lower glycolysis and anaplerotic pathways towards the tricarboxylic acid cycle, amino acid synthesis and lipid synthesis than in higher plants., (© 2021. The Author(s).)

Background: Diseases of the glomeruli, the renal filtration units, are a leading cause of progressive kidney disease. Assessment of the ultrastructure of podocytes at the glomerular filtration barrier is essential for diagnosing diverse disease entities, providing insight into the disease pathogenesis, and monitoring treatment responses., Methods: Here we apply previously published sample preparation methods together with stimulated emission depletion and confocal microscopy for resolving nanoscale podocyte substructure. The protocols are modified and optimized in order to be applied to formalin-fixed paraffin-embedded (FFPE) samples., Results: We successfully modified our protocols to allow for deep three-dimensional stimulated emission depletion and confocal imaging of FFPE kidney tissue with similar staining and image quality compared with our previous approaches. We further show that quantitative analysis can be applied to extract morphometrics from healthy and diseased samples from both mice and humans., Conclusions: The results from this study could increase the feasibility of implementing optical kidney imaging protocols in clinical routines because FFPE is the gold-standard method for storage of patient samples., Competing Interests: T. Benzing reports advisory activity for Otsuka in the field of cystic kidney disease and hyponatremia; support for ADPKD registry by Otsuka; speaker honoraria and travel support from Amgen, Hexal, Novartis, Otsuka, Roche, and Sanofi-Genzyme. He is also on the editorial board of the Journal of the American Society of Nephrology, Nephrology Dialysis Transplantation, and Science Signaling. H. Blom reports other interests/relationships with MedTechLabs, BioClinicum, Karolinska University Hospital, Solna, Sweden. P. Hoyer reports consultancy agreements with Boehringer Ingelheim and is a scientific advisor for Archives of Disease in Childhood. F. Koehler reports other interests/relationships with Else Kröner-FreseniusStiftung, German Research Foundation under Germany’s Excellence Strategy EXC 2030: CECAD—Excellent in Aging Research and Koeln Fortune program/Faculty of Medicine, University of Cologne, Germany. R. Mueller reports consultancy agreements with Alnylam and Sanofi; research funding from Otsuka Pharmaceuticals and Thermo Fisher Scientific, which was paid to the employer (Department II of Internal Medicine); and honoraria from Alnylam and Sanofi. He is also a board member of the WGIKD (ERA-EDTA), Scientific Advisory Board Santa Barbara Nutrients, Editorial Board “Kidney and Dialysis.” All remaining authors have nothing to disclose., (Copyright © 2022 by the American Society of Nephrology.)

SNF1-related Kinase 1 (SnRK1) is an evolutionarily conserved protein kinase with key functions in energy management during stress responses in plants. To address a potential role of SnRK1 under favorable conditions, we performed a metabolomic and transcriptomic characterization of rosettes of 20-d-old Arabidopsis (Arabidopsis thaliana) plants of SnRK1 gain- and loss-of-function mutants during the regular diel cycle. Our results show that SnRK1 manipulation alters the sucrose and trehalose 6-phosphate (Tre6P) relationship, influencing how the sucrose content is translated into Tre6P accumulation and modulating the flux of carbon to the tricarboxylic acid cycle downstream of Tre6P signaling. On the other hand, daily cycles of Tre6P accumulation were accompanied by changes in SnRK1 signaling, leading to a maximum in the expression of SnRK1-induced genes at the end of the night, when Tre6P levels are lowest, and to a minimum at the end of the day, when Tre6P levels peak. The expression of SnRK1-induced genes was strongly reduced by transient Tre6P accumulation in an inducible Tre6P synthase (otsA) line, further suggesting the involvement of Tre6P in the diel oscillations in SnRK1 signaling. Transcriptional profiling of wild-type plants and SnRK1 mutants also uncovered defects that are suggestive of an iron sufficiency response and of a matching induction of sulfur acquisition and assimilation when SnRK1 is depleted. In conclusion, under favorable growth conditions, SnRK1 plays a role in sucrose homeostasis and transcriptome remodeling in autotrophic tissues and its activity is influenced by diel fluctuations in Tre6P levels., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Loss of podocytes, possibly through the detachment of viable cells, is a hallmark of progressive glomerular disease. Podocytes are exposed to considerable physical forces due to pressure and flow resulting in circumferential wall stress and tangential shear stress exerted on the podocyte cell body, which have been proposed to contribute to podocyte depletion. However, estimations of in vivo alterations of physical forces in glomerular disease have been hampered by a lack of quantitative functional and morphological data. Here, we used ultra-resolution data and computational analyses in a mouse model of human disease, hereditary late-onset focal segmental glomerular sclerosis, to calculate increased mechanical stress upon podocyte injury. Transversal shear stress on the lateral walls of the foot processes was prominently increased during the initial stages of podocyte detachment. Thus, our study highlights the importance of targeting glomerular hemodynamics to treat glomerular disease., (Copyright © 2021. Published by Elsevier Inc.)

Biocatalysis has undergone revolutionary progress in the past century. Benefited by the integration of multidisciplinary technologies, natural enzymatic reactions are constantly being explored. Protein engineering gives birth to robust biocatalysts that are widely used in industrial production. These research achievements have gradually constructed a network containing natural enzymatic synthesis pathways and artificially designed enzymatic cascades. Nowadays, the development of artificial intelligence, automation, and ultra-high-throughput technology provides infinite possibilities for the discovery of novel enzymes, enzymatic mechanisms and enzymatic cascades, and gradually complements the lack of remaining key steps in the pathway design of enzymatic total synthesis. Therefore, the research of biocatalysis is gradually moving towards the era of novel technology integration, intelligent manufacturing and enzymatic total synthesis.

In recent years, many light-microscopy protocols have been published for visualization of nanoscale structures in the kidney. These protocols present researchers with new tools to evaluate both foot process anatomy and effacement, as well as protein distributions in foot processes, the slit diaphragm and in the glomerular basement membrane. However, these protocols either involve the application of different complicated super resolution microscopes or lengthy sample preparation protocols. Here, we present a fast and simple, five-hour long procedure for three-dimensional visualization of kidney morphology on all length scales. The protocol combines optical clearing and tissue expansion concepts to produce a mild swelling, sufficient for resolving nanoscale structures using a conventional confocal microscope. We show that the protocol can be applied to visualize a wide variety of pathologic features in both mouse and human kidneys. Thus, our fast and simple protocol can be beneficial for conventional microscopic evaluation of kidney tissue integrity both in research and possibly in future clinical routines., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Mammalian kidneys constantly filter large amounts of liquid, with almost complete retention of albumin and other macromolecules in the plasma. Breakdown of the three-layered renal filtration barrier results in loss of albumin into urine (albuminuria) across the wall of small renal capillaries, and is a leading cause of chronic kidney disease. However, exactly how the renal filter works and why its permeability is altered in kidney diseases is poorly understood. Here we show that the permeability of the renal filter is modulated through compression of the capillary wall. We collect morphometric data prior to and after onset of albuminuria in a mouse model equivalent to a human genetic disease affecting the renal filtration barrier. Combining quantitative analyses with mathematical modelling, we demonstrate that morphological alterations of the glomerular filtration barrier lead to reduced compressive forces that counteract filtration pressure, thereby resulting in capillary dilatation, and ultimately albuminuria. Our results reveal distinct functions of the different layers of the filtration barrier and expand the molecular understanding of defective renal filtration in chronic kidney disease.

The enzymatic transamination of ketones into ( R )-amines represents an important route for accessing a range of pharmaceuticals or building blocks. Although many publications have dealt with enzyme discovery, protein engineering, and the application of ( R )-selective amine transaminases [( R )-ATA] in biocatalysis, little is known about the actual in vivo role and how these enzymes have evolved from the ubiquitous α-amino acid transaminases (α-AATs). Here, we show the successful introduction of an ( R )-transaminase activity in an α-amino acid aminotransferase with one to six amino acid substitutions in the enzyme's active site. Bioinformatic analysis combined with computational redesign of the d-amino acid aminotransferase (DATA) led to the identification of a sextuple variant having a specific activity of 326 milliunits mg -1 in the conversion of ( R )-phenylethylamine and pyruvate to acetophenone and d-alanine. This value is similar to those of natural ( R )-ATAs, which typically are in the range of 250 milliunits mg -1 . These results demonstrate that ( R )-ATAs can evolve from α-AAT as shown here for the DATA scaffold.

AATF is a central regulator of the cellular outcome upon p53 activation, a finding that has primarily been attributed to its function as a transcription factor. Recent data showed that AATF is essential for ribosome biogenesis and plays a role in rRNA maturation. AATF has been implicated to fulfil this role through direct interaction with rRNA and was identified in several RNA-interactome capture experiments. Here, we provide a first comprehensive analysis of the RNA bound by AATF using CLIP-sequencing. Interestingly, this approach shows predominant binding of the 45S pre-ribosomal RNA precursor molecules. Furthermore, AATF binds to mRNAs encoding for ribosome biogenesis factors as well as snoRNAs. These findings are complemented by an in-depth analysis of the protein interactome of AATF containing a large set of proteins known to play a role in rRNA maturation with an emphasis on the protein-RNA-complexes known to be required for the generation of the small ribosomal subunit (SSU). In line with this finding, the binding sites of AATF within the 45S rRNA precursor localize in close proximity to the SSU cleavage sites. Consequently, our multilayer analysis of the protein-RNA interactome of AATF reveals this protein to be an important hub for protein and RNA interactions involved in ribosome biogenesis.

Diel starch turnover responds rapidly to changes in the light regime. We investigated if these responses require changes in the temporal dynamics of the circadian clock. Arabidopsis ( Arabidopsis thaliana ) was grown in a 12-h photoperiod for 19 d, shifted to three different reduced light levels or to low CO 2 for one light period, and returned to growth conditions. The treatments produced widespread changes in clock transcript abundance. However, almost all of the changes were restricted to extreme treatments that led to carbon starvation and were small compared to the magnitude of the circadian oscillation. Changes included repression of EARLY FLOWERNG 4 , slower decay of dusk components, and a slight phase delay at the next dawn, possibly due to abrogated Evening Complex function and sustained expression of PHYTOCHROME INTERACTING FACTORs and REVEILLEs during the night. Mobilization of starch in the night occurred in a linear manner and was paced to dawn, both in moderate treatments that did not alter clock transcripts and in extreme treatments that led to severe carbon starvation. We conclude that pacing of starch mobilization to dawn does not require retrograde carbon signaling to the transcriptional clock. On the following day, growth decreased, sugars rose, and starch accumulation was stimulated in low-light-treated plants compared to controls. This adaptive response was marked after moderate treatments and occurred independently of changes in the transcriptional clock. It is probably a time-delayed response to low-C signaling in the preceding 24-h cycle, possibly including changes in PHYTOCHROME INTERACTING FACTOR and REVEILLE expression., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Background: RNA-binding proteins (RBPs) are fundamental regulators of cellular biology that affect all steps in the generation and processing of RNA molecules. Recent evidence suggests that regulation of RBPs that modulate both RNA stability and translation may have a profound effect on the proteome. However, regulation of RBPs in clinically relevant experimental conditions has not been studied systematically., Methods: We used RNA interactome capture, a method for the global identification of RBPs to characterize the global RNA-binding proteome (RBPome) associated with polyA-tailed RNA species in murine ciliated epithelial cells of the inner medullary collecting duct. To study regulation of RBPs in a clinically relevant condition, we analyzed hypoxia-associated changes of the RBPome., Results: We identified >1000 RBPs that had been previously found using other systems. In addition, we found a number of novel RBPs not identified by previous screens using mouse or human cells, suggesting that these proteins may be specific RBPs in differentiated kidney epithelial cells. We also found quantitative differences in RBP-binding to mRNA that were associated with hypoxia versus normoxia., Conclusions: These findings demonstrate the regulation of RBPs through environmental stimuli and provide insight into the biology of hypoxia-response signaling in epithelial cells in the kidney. A repository of the RBPome and proteome in kidney tubular epithelial cells, derived from our findings, is freely accessible online, and may contribute to a better understanding of the role of RNA-protein interactions in kidney tubular epithelial cells, including the response of these cells to hypoxia., (Copyright © 2019 by the American Society of Nephrology.)

Acute kidney injury (AKI) leads to significant morbidity and mortality; unfortunately, strategies to prevent or treat AKI are lacking. In recent years, several preconditioning protocols have been shown to be effective in inducing organ protection in rodent models. Here, we characterized two of these interventions-caloric restriction and hypoxic preconditioning-in a mouse model of cisplatin-induced AKI and investigated the underlying mechanisms by acquisition of multi-layered omic data (transcriptome, proteome, N-degradome) and functional parameters in the same animals. Both preconditioning protocols markedly ameliorated cisplatin-induced loss of kidney function, and caloric restriction also induced lipid synthesis. Bioinformatic analysis revealed mRNA-independent proteome alterations affecting the extracellular space, mitochondria, and transporters. Interestingly, our analyses revealed a strong dissociation of protein and RNA expression after cisplatin treatment that showed a strong correlation with the degree of damage. N-degradomic analysis revealed that most posttranscriptional changes were determined by arginine-specific proteolytic processing. This included a characteristic cisplatin-activated complement signature that was prevented by preconditioning. Amyloid and acute-phase proteins within the cortical parenchyma showed a similar response. Extensive analysis of disease-associated molecular patterns suggested that transcription-independent deposition of amyloid P-component serum protein may be a key component in the microenvironmental contribution to kidney damage. This proof-of-principle study provides new insights into the pathogenesis of cisplatin-induced AKI and the molecular mechanisms underlying organ protection by correlating phenotypic and multi-layered omics data., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Plants accumulate reserves in the daytime to support growth at night. Circadian regulation of diel reserve turnover was investigated by profiling starch, sugars, glucose 6-phosphate, organic acids, and amino acids during a light-dark cycle and after transfer to continuous light in Arabidopsis wild types and in mutants lacking dawn (lhy cca1), morning (prr7 prr9), dusk (toc1, gi), or evening (elf3) clock components. The metabolite time series were integrated with published time series for circadian clock transcripts to identify circadian outputs that regulate central metabolism. (a) Starch accumulation was slower in elf3 and prr7 prr9. It is proposed that ELF3 positively regulates starch accumulation. (b) Reducing sugars were high early in the T-cycle in elf3, revealing that ELF3 negatively regulates sucrose recycling. (c) The pattern of starch mobilization was modified in all five mutants. A model is proposed in which dawn and dusk/evening components interact to pace degradation to anticipated dawn. (d) An endogenous oscillation of glucose 6-phosphate revealed that the clock buffers metabolism against the large influx of carbon from photosynthesis. (e) Low levels of organic and amino acids in lhy cca1 and high levels in prr7 prr9 provide evidence that the dawn components positively regulate the accumulation of amino acid reserves., (© 2018 John Wiley & Sons Ltd.)

Background: Viruses are the leading cause of acute gastroenteritis in children worldwide. Understanding of the occurrence and genetic diversity of these viruses can help to prevent infections., Objectives: The present study describes the presence, genetic diversity and possible recombination of five enteric viruses in children with gastroenteritis in Southwestern Nigeria., Study Design: From August 2012 to December 2013, stool samples and sociodemographic data of 103 diarrheic children <5 years were collected to detect and characterize rotavirus A, norovirus, human astrovirus, aichivirus and sapovirus using PCR techniques followed by sequencing and phylogenetic analyses., Results: At least one virus was identified in 58.3% (60/103) of the stool samples. Rotavirus, norovirus and astrovirus were detected in 39.8% (41/103), 10.7% (11/103), and 6.8% (7/103) respectively. Notably, aichivirus was detected for the first time in Nigeria (1/103; 0.97%). Sapovirus was not detected in the study. Co-infections with rotavirus were observed in eight samples either with norovirus or astrovirus or aichivirus. Phylogenetic analyses of different genome regions of norovirus positive samples provided indication for recombinant norovirus strains. A novel astrovirus strain closely related to canine astrovirus was identified and further characterized for the first time., Conclusions: Viruses are the common cause of acute gastroenteritis in Nigerian infants with rotavirus as most frequently detected pathogen. New norovirus recombinants and a not yet detected zoonotic astrovirus were circulating in Southwestern Nigeria, providing new information about emerging and unusual strains of viruses causing diarrhea., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this study, we combined photo-organo redox catalysis and biocatalysis to achieve asymmetric C-H bond functionalization of simple alkane starting materials. The photo-organo catalyst anthraquinone sulfate (SAS) was employed to oxyfunctionalise alkanes to aldehydes and ketones. We coupled this light-driven reaction with asymmetric enzymatic functionalisations to yield chiral hydroxynitriles, amines, acyloins and α-chiral ketones with up to 99 % ee. In addition, we demonstrate functional group interconversion to alcohols, esters and carboxylic acids. The transformations can be performed as concurrent tandem reactions. We identified the degradation of substrates and inhibition of the biocatalysts as limiting factors affecting compatibility, due to reactive oxygen species generated in the photocatalytic step. These incompatibilities were addressed by reaction engineering, such as applying a two-phase system or temporal and spatial separation of the catalysts. Using a selection of eleven starting alkanes, one photo-organo catalyst and 8 diverse biocatalysts, we synthesized 26 products and report for the model compounds benzoin and mandelonitrile > 97 % ee at gram scale.

Marine bacteria catabolize carbohydrate polymers of algae, which synthesize these structurally diverse molecules in ocean surface waters. Although algal glycans are an abundant carbon and energy source in the ocean, the molecular details that enable specific recognition between algal glycans and bacterial degraders remain largely unknown. Here we characterized a surface protein, GMSusD from the planktonic Bacteroidetes-Gramella sp. MAR&#95;2010&#95;102 that thrives during algal blooms. Our biochemical and structural analyses show that GMSusD binds glucose polysaccharides such as branched laminarin and linear pustulan. The 1.8 Å crystal structure of GMSusD indicates that three tryptophan residues form the putative glycan-binding site. Mutagenesis studies confirmed that these residues are crucial for laminarin recognition. We queried metagenomes of global surface water datasets for the occurrence of SusD-like proteins and found sequences with the three structurally conserved residues in different locations in the ocean. The molecular selectivity of GMSusD underscores that specific interactions are required for laminarin recognition. In conclusion, our findings provide insight into the molecular details of β-glucan binding by GMSusD and our bioinformatic analysis reveals that this molecular interaction may contribute to glucan cycling in the surface ocean., (© 2018 Federation of European Biochemical Societies.)

Cells and organelles are not homogeneous but include microcompartments that alter the spatiotemporal characteristics of cellular processes. The effects of microcompartmentation on metabolic pathways are however difficult to study experimentally. The pyrenoid is a microcompartment that is essential for a carbon concentrating mechanism (CCM) that improves the photosynthetic performance of eukaryotic algae. Using Chlamydomonas reinhardtii , we obtained experimental data on photosynthesis, metabolites, and proteins in CCM-induced and CCM-suppressed cells. We then employed a computational strategy to estimate how fluxes through the Calvin-Benson cycle are compartmented between the pyrenoid and the stroma. Our model predicts that ribulose-1,5-bisphosphate (RuBP), the substrate of Rubisco, and 3-phosphoglycerate (3PGA), its product, diffuse in and out of the pyrenoid, respectively, with higher fluxes in CCM-induced cells. It also indicates that there is no major diffusional barrier to metabolic flux between the pyrenoid and stroma. Our computational approach represents a stepping stone to understanding microcompartmentalized CCM in other organisms., Competing Interests: AK, FS, LY, LM, MH, SG, MJ, MS, MS, ZN, TM No competing interests declared, (© 2018, Küken et al.)

Ciliopathies comprise a large number of hereditary human diseases and syndromes caused by mutations resulting in dysfunction of either primary or motile cilia. Both types of cilia share a similar architecture. While primary cilia are present on most cell types, expression of motile cilia is limited to specialized tissues utilizing ciliary motility. We characterized protein complexes of ciliopathy proteins and identified the conserved AAA-ATPase Ruvbl1 as a common novel component. Here, we demonstrate that Ruvbl1 is crucial for the development and maintenance of renal tubular epithelium in mice: both constitutive and inducible deletion in tubular epithelial cells result in renal failure with tubular dilatations and fewer ciliated cells. Moreover, inducible deletion of Ruvbl1 in cells carrying motile cilia results in hydrocephalus, suggesting functional relevance in both primary and motile cilia. Cilia of Ruvbl1-negative cells lack crucial proteins, consistent with the concept of Ruvbl1-dependent cytoplasmic pre-assembly of ciliary protein complexes.

In diseases of many parenchymatous organs, heterogeneous deterioration of individual functional units determines the clinical prognosis. However, the molecular characterization at the level of such individual subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Proteinuric glomerular kidney diseases are frequent and assorted diseases affecting a fraction of glomeruli and their draining tubules to variable extents, and for which no specific treatment exists. Here, we developed and applied a mass spectrometry-based methodology to investigate heterogeneity of proteomes from individually isolated nephron segments from mice with proteinuric kidney disease. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins while genetic ablation of LAMP1-correlated lysosomal proteases could ameliorate glomerular damage in vivo. Furthermore, proteomic analyses of individual glomeruli from patients with genetic sclerotic and non-sclerotic proteinuric diseases revealed increased abundance of lysosomal proteins, in combination with a decreased abundance of mutated gene products. Thus, altered protein homeostasis (proteostasis) is a conserved key mechanism in proteinuric kidney diseases. Moreover, our technology can capture intra-individual variability in diseases of the kidney and other tissues at a sub-biopsy scale., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Objectives: In order to analyze the molecular epidemiology of human astroviruses (HAstV) in Germany, a retrospective long-term study was performed to characterize circulating human astrovirus in patients with acute gastroenteritis in Germany., Methods: A total of 2877 stool samples, collected between January 2010 and December 2015 from sporadic cases and outbreaks of acute gastroenteritis were retrospectively analyzed for astrovirus. A two-step PCR algorithm was developed and used to identify and characterize human astrovirus infections., Results: Overall, 143 samples were astrovirus-positive (5.0%). Astrovirus infection was most frequently detectable in samples from children of 3-4 years (15%) followed by children of 1-2 years (8.6%), detection rates in adults were lower (1%-3.6%). A high number (71.3%) of co-infections, mainly with noro- or rotaviruses, were identified. Genotyping revealed that at least ten genotypes from all four human MAstV species were circulating in the study population. HAstV-1 was predominant in different age groups. Novel HAstV (MLB and VA genotypes) were also circulating in Germany., Conclusion: Our findings give new insights into the circulation and genetic diversity of human astroviruses in patients with acute gastroenteritis. The novel HAstV-MLB and -VA genotypes could be characterized firstly in Germany while the analysis showed that these viruses have been dispersed in Germany since 2011 as a causative agent of acute gastroenteritis., (Copyright © 2018 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

A fundamental principle in malignant tranformation is the ability of cancer cells to escape the naturally occurring cell-intrinsic responses to DNA damage. Tumors progress despite the accumulation of DNA lesions. However, the underlying mechanisms of this tolerance to genotoxic stress are still poorly characterized. Here, we show that replication stress occurs in Kras-driven murine lung adenocarcinomas, as well as in proliferating murine embryonic and adult tissues. We identify the transcriptional regulator AATF/CHE-1 as a key molecule to sustain proliferative tissues and tumor progression in parts by inhibiting p53-driven apoptosis in vivo. In an autochthonous Kras-driven lung adenocarcinoma model, deletion of Aatf delayed lung cancer formation predominantly in a p53-dependent manner. Moreover, targeting Aatf in existing tumors through a dual recombinase strategy caused a halt in tumor progression. Taken together, these data suggest that AATF may serve as a drug target to treat KRAS-driven malignancies.