Dynamic parametric MRI and deep learning: Unveiling renal pathophysiology through accurate kidney size quantification.

Renal pathologies often manifest as alterations in kidney size, providing a valuable avenue for employing dynamic parametric MRI as a means to derive kidney size measurements for the diagnosis, treatment, and monitoring of renal disease. Furthermore, this approach holds significant potential in supporting MRI data-driven preclinical investigations into the intricate mechanisms underlying renal pathophysiology. The integration of deep learning algorithms is crucial in achieving rapid and precise segmentation of the kidney from temporally resolved parametric MRI, facilitating the use of kidney size as a meaningful (pre)clinical biomarker for renal disease. To explore this potential, we employed dynamic parametric T2 mapping of the kidney in rats in conjunction with a custom-tailored deep dilated U-Net (DDU-Net) architecture. The architecture was trained, validated, and tested on manually segmented ground truth kidney data, with benchmarking against an analytical segmentation model and a self-configuring no new U-Net. Subsequently, we applied our approach to in vivo longitudinal MRI data, incorporating interventions that emulate clinically relevant scenarios in rats. Our approach achieved high performance metrics, including a Dice coefficient of 0.98, coefficient of determination of 0.92, and a mean absolute percentage error of 1.1% compared with ground truth. The DDU-Net enabled automated and accurate quantification of acute changes in kidney size, such as aortic occlusion (-8% ± 1%), venous occlusion (5% ± 1%), furosemide administration (2% ± 1%), hypoxemia (-2% ± 1%), and contrast agent-induced acute kidney injury (11% ± 1%). This approach can potentially be instrumental for the development of dynamic parametric MRI-based tools for kidney disorders, offering unparalleled insights into renal pathophysiology.

Influence of Polymer Side Chain Size and Backbone Length on the Self-Assembly of Supramolecular Polymer Bottlebrushes.

Hydrogen bonds are a versatile tool for creating fibrous, bottlebrush-like assemblies of polymeric building blocks. However, a delicate balance of forces exists between the steric repulsion of the polymer chains and these directed supramolecular forces. In this work we have systematically investigated the influence of structural parameters of the attached polymers on the assembly behaviour of benzene trisurea (BTU) and benzene tris(phenylalanine) (BTP) conjugates in water. Polymers with increasing main chain lengths and different side chain sizes were prepared by reversible addition-fragmentation chain-transfer (RAFT) polymerization of hydroxyethyl acrylate (HEA), tri(ethylene glycol) methyl ether acrylate (TEGA) and oligo(ethylene glycol) methyl ether acrylate (OEGA). The resulting structures were analyzed using small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM). Both BTU and BTP formed fibres with PHEA attached, but a transition to spherical morphologies was observed at degrees of polymerisation (DP) of 70 and above. Overall, the main chain length appeared to be a dominating factor in inducing morphology transitions. Increasing the side chain size generally had a similar effect but mainly impeded any aggregation as is the case of POEGA. Interestingly, BTP conjugates still formed fibres, suggesting that the stronger intermolecular interactions can compensate partially for the steric repulsion.

Transumbilical laparoscopic-assisted appendectomy in children and adolescents: what have we learnt in more than 1200 cases?

PURPOSE: TULAA combines the laparoscopic and open technique and is considered to be a safe, fast and cost-effective procedure. On the other hand, preparation is limited due to the single instrument, especially in complicated appendicitis. In this study we analyze the outcome of our TULAA patients, focusing on conversion and complication rates. METHODS: We performed a retrospective study including all patients treated with TULAA in our department between 2006 and 2016. We analyzed patient data, operative data, costs, complications, and conversion rate to standard laparoscopic or open appendectomy. RESULTS: 1275 children and adolescents were enrolled. Mean age was 10.2 years. TULAA was completed in 88% of cases. The overall mean operative time was 33 min. The overall complication rate was 5.7%. The most common complications were wound infection (2.7%), seroma (1.7%) and wound abscess (1.4%). Both the conversion rate and the complication rate were significantly higher in complicated appendicitis. Furthermore, the conversion rate is higher in overweight or obese patients. CONCLUSION: TULAA is a safe, quick and cost-effective treatment option for acute appendicitis in children and adolescents. The complication rate and conversion rate are significantly correlated with the degree of appendiceal inflammation and comparable other surgical procedures.

MRI of kidney size matters.

OBJECTIVE: To highlight progress and opportunities of measuring kidney size with MRI, and to inspire research into resolving the remaining methodological gaps and unanswered questions relating to kidney size assessment. MATERIALS AND METHODS: This work is not a comprehensive review of the literature but highlights valuable recent developments of MRI of kidney size. RESULTS: The links between renal (patho)physiology and kidney size are outlined. Common methodological approaches for MRI of kidney size are reviewed. Techniques tailored for renal segmentation and quantification of kidney size are discussed. Frontier applications of kidney size monitoring in preclinical models and human studies are reviewed. Future directions of MRI of kidney size are explored. CONCLUSION: MRI of kidney size matters. It will facilitate a growing range of (pre)clinical applications, and provide a springboard for new insights into renal (patho)physiology. As kidney size can be easily obtained from already established renal MRI protocols without the need for additional scans, this measurement should always accompany diagnostic MRI exams. Reconciling global kidney size changes with alterations in the size of specific renal layers is an important topic for further research. Acute kidney size measurements alone cannot distinguish between changes induced by alterations in the blood or the tubular volume fractions-this distinction requires further research into cartography of the renal blood and the tubular volumes.

S3 guideline: Diagnosis and treatment of epidermal necrolysis (Stevens-Johnson syndrome and toxic epidermal necrolysis) - Part 2: Supportive therapy of EN in the acute and post-acute stages.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are rare, predominantly drug-induced, acute life-threatening diseases of skin and mucosae. SJS and TEN are nowadays considered as variants of one disease entity with varying degrees of severity called epidermal necrolysis (EN). EN is associated with high morbidity and mortality and constitutes a major disease burden for affected patients. The guideline "Diagnosis and treatment of epidermal necrolysis (Stevens-Johnson syndrome and toxic epidermal necrolysis)" was developed under systematic consideration of existing scientific literature and in a formal consensus process according to regulations issued by the Association of Scientific Medical Societies in Germany (AWMF) to establish an evidence-based framework to support clinical decision-making. The interdisciplinary guideline commission consisted of representatives from various specialist societies and of patient representatives. The guideline is aimed at specialists in the fields of dermatology, ophthalmology, plastic surgery, intensive care, and pediatrics in hospitals and offices, as well as other medical specialties involved in the diagnosis and treatment of EN. The guideline is also aimed at patients, their relatives, insurance funds, and policymakers. The second part is concerned with the topics of supportive therapy in the acute phase of EN and outpatient follow-up treatment.

S3 guideline: Diagnosis and treatment of epidermal necrolysis (Stevens-Johnson syndrome and toxic epidermal necrolysis) - Part 1: Diagnosis, initial management, and immunomodulating systemic therapy.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are rare, predominantly drug-induced, acute, life-threatening diseases of skin and mucosae. SJS and TEN are nowadays considered variants of one disease entity with varying degrees of severity called epidermal necrolysis (EN). EN is associated with high morbidity and mortality and constitutes a major disease burden for affected patients. The guideline "Diagnosis and treatment of epidermal necrolysis (Stevens-Johnson syndrome and toxic epidermal necrolysis)" was developed under systematic consideration of existing scientific literature and in a formal consensus process according to regulations issued by the Association of Scientific Medical Societies in Germany (AWMF) to establish an evidence-based framework to support clinical decision-making. The interdisciplinary guideline commission consisted of representatives from various specialist societies and patient representatives. The guideline is aimed at specialists in the fields of dermatology, ophthalmology, plastic surgery, intensive care, and pediatrics in hospitals and offices, as well as other medical speciallved in the diagnosis and treatment of EN. The guideline is also aimed at patients, their relatives, insurance funds, and policymakers. This first part focuses on the diagnostic aspects, the initial management as well as the immunomodulating systemic therapy.

Scaling-up problem management plus for refugees in Switzerland - a qualitative study.

BACKGROUND: Refugees are at an increased risk of developing symptoms of mental disorders but face various structural and socio-cultural barriers to accessing mental health care. The SPIRIT project (Scaling-up Psychological Interventions in Refugees In SwiTzerland) seeks to promote the resilience of refugees and improve their access to mental health care. For this purpose, Problem Management Plus (PM+), an evidence-based low-intensity psychological intervention delivered by trained non-specialist "helpers", is being scaled-up in Switzerland. OBJECTIVE: To identify factors influencing the process of the large-scale implementation of PM + for refugees in Switzerland and to develop recommendations to guide the implementation process. METHODS: 22 semi-structured interviews were conducted with key informants (Syrian refugees who previously participated in PM+, PM + helpers, health professionals working with refugees and decision-makers from the migration, integration, social, and health sectors). The data were analyzed using thematic analysis, combining an inductive and deductive approach. RESULTS: The data revealed three major themes, which might have an impact for the longer-term implementation of PM + in Switzerland. First, preconditions for successful integration in the health system prior to scaling-up such as sustainable funding or the introduction of a stepped care approach. Second, the requirements for the PM + intervention supporting scale-up such as quality control during PM + delivery, PM + modality, time and setting when PM + is offered or the views on task sharing. Third, the perceived benefits of scaling-up PM + in Switzerland. CONCLUSIONS: Our results have shown that PM + must be scaled-up within a stepped care approach, including a functioning triage system and sustainable funding. Rather than selecting one modality or setting, it seemed preferable to offer a variety of formats and settings to achieve maximum reach and benefits. A successful scale-up of PM + in Switzerland might have various benefits. Communicating them to policy-makers and health providers, might enhance their acceptability of the intervention and their willingness to adopt PM + in regulatory structure and promote it.

Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates.

BACKGROUND: Substrate-limited fed-batch conditions have the favorable effect of preventing overflow metabolism, catabolite repression, oxygen limitation or inhibition caused by elevated substrate or osmotic concentrations. Due to these favorable effects, fed-batch mode is predominantly used in industrial production processes. In contrast, screening processes are usually performed in microtiter plates operated in batch mode. This leads to a different physiological state of the production organism in early screening and can misguide the selection of potential production strains. To close the gap between screening and production conditions, new techniques to enable fed-batch mode in microtiter plates have been described. One of these systems is the ready-to-use and disposable polymer-based controlled-release fed-batch microtiter plate (fed-batch MTP). In this work, the fed-batch MTP was applied to establish a glucose-limited fed-batch screening procedure for industrially relevant protease producing Bacillus licheniformis strains. RESULTS: To achieve equal initial growth conditions for different clones with the fed-batch MTP, a two-step batch preculture procedure was developed. Based on this preculture procedure, the standard deviation of the protease activity of glucose-limited fed-batch main culture cultivations in the fed-batch MTP was ± 10%. The determination of the number of replicates revealed that a minimum of 6 parallel cultivations were necessary to identify clones with a statistically significant increased or decreased protease activity. The developed glucose-limited fed-batch screening procedure was applied to 13 industrially-relevant clones from two B. licheniformis strain lineages. It was found that 12 out of 13 clones (92%) were classified similarly as in a lab-scale fed-batch fermenter process operated under glucose-limited conditions. When the microtiter plate screening process was performed in batch mode, only 5 out of 13 clones (38%) were classified similarly as in the lab-scale fed-batch fermenter process. CONCLUSION: The glucose-limited fed-batch screening process outperformed the usual batch screening process in terms of the predictability of the clone performance under glucose-limited fed-batch fermenter conditions. These results highlight that the implementation of glucose-limited fed-batch conditions already in microtiter plate scale is crucial to increase the precision of identifying improved protease producing B. licheniformis strains. Hence, the fed-batch MTP represents an efficient high-throughput screening tool that aims at closing the gap between screening and production conditions.

Validation of ion mobility spectrometry - mass spectrometry as a screening tool to identify type II kinase inhibitors of FGFR1 kinase.

RATIONALE: The protein kinase FGFR1 regulates cellular processes in human development. As over-activity of FGFR1 is implicated with cancer, effective inhibitors are in demand. Type I inhibitors, which bind to the active form of FGFR1, are less effective than type II inhibitors, which bind to the inactive form. Screening to distinguish between type I and type II inhibitors is required. METHODS: X-ray crystallography was used to indicate whether a range of potential inhibitors bind to the active or inactive FGFR1 kinase conformation. The binding affinity of each ligand to FGFR1 was measured using biochemical methods. Electrospray ionisation - ion mobility spectrometry - mass spectrometry (ESI-IMS-MS) in conjunction with collision-induced protein unfolding generated a conformational profile of each FGFR1-ligand complex. The results indicate that the protein's conformational profile depends on whether the inhibitor is type I or type II. RESULTS: X-ray crystallography confirmed which of the kinase inhibitors bind to the active or inactive form of FGFR1 kinase. Collision-induced unfolding combined with ESI-IMS-MS showed distinct differences in the FGFR1 folding landscape for type I and type II inhibitors. Biochemical studies indicated a similar range of FGFR1 affinities for both types of inhibitors, thus providing confidence that the conformational variations detected using ESI-IMS-MS can be interpretated unequivocally and that this is an effective screening method. CONCLUSIONS: A robust ESI-IMS-MS method has been implemented to distinguish between the binding mode of type I and type II inhibitors by monitoring the conformational unfolding profile of FGFR1. This rapid method requires low sample concentrations and could be used as a high-throughput screening technique for the characterisation of novel kinase inhibitors.

Overcoming the Necessity of a Lateral Aggregation in the Formation of Supramolecular Polymer Bottlebrushes in Water.

The assembly of supramolecular polymer bottlebrushes in aqueous systems is, in most cases, associated with a lateral aggregation of the supramolecular building blocks in addition to their axial stacking. Here, it is demonstrated that this limitation can be overcome by attaching three polymer chains to a central supramolecular unit that possesses a sufficiently high number of hydrogen bonding units to compensate for the increased steric strain. Therefore, a 1,3,5-benzenetrisurea-polyethylene oxide conjugate is modified with different peptide units located next to the urea groups which should facilitate self-assembly in water. For a single amino acid per arm, spherical micelles are obtained for all three tested amino acids (alanine, leucine, and phenylalanine) featuring different hydrophobicities. Only a slight increase in size and solution stability of spherical micelles is observed with increasing hydrophobicity of amino acid unit. In contrast, introducing two amino acid units per arm and thus increasing the number of hydrogen bonds per unimer molecule results in the formation of cylindrical structures, that is, supramolecular polymer bottlebrushes, despite a suppressed lateral aggregation. Consequently, it can be concluded that the number of hydrogen bonds has a more profound impact on the resulting solution morphology than the hydrophobicity of the amino acid unit.

Adjusting the length of supramolecular polymer bottlebrushes by top-down approaches.

Controlling the length of one-dimensional (1D) polymer nanostructures remains a key challenge on the way toward the applications of these structures. Here, we demonstrate that top-down processing facilitates a straightforward adjustment of the length of polyethylene oxide (PEO)-based supramolecular polymer bottlebrushes (SPBs) in aqueous solutions. These cylindrical structures self-assemble via directional hydrogen bonds formed by benzenetrisurea (BTU) or benzenetrispeptide (BTP) motifs located within the hydrophobic core of the fiber. A slow transition from different organic solvents to water leads first to the formation of µm-long fibers, which can subsequently be fragmented by ultrasonication or dual asymmetric centrifugation. The latter allows for a better adjustment of applied shear stresses, and thus enables access to differently sized fragments depending on time and rotation rate. Extended sonication and scission analysis further allowed an estimation of tensile strengths of around 16 MPa for both the BTU and BTP systems. In combination with the high kinetic stability of these SPBs, the applied top-down methods represent an easily implementable technique toward 1D polymer nanostructures with an adjustable length in the range of interest for perspective biomedical applications.

Liquid Viscosity and Surface Tension of n-Hexane, n-Octane, n-Decane, and n-Hexadecane up to 573 K by Surface Light Scattering (SLS).

In the present study, the simultaneous and accurate determination of liquid viscosity and surface tension of the n-alkanes n-hexane (n-C6H14), n-octane (n-C8H18), n-decane (n-C10H22), and n-hexadecane (n-C16H34) by surface light scattering (SLS) in thermodynamic equilibrium is demonstrated. Measurements have been performed over a wide temperature range from 283.15 K up to 473.15 K for n-C6H14, 523.15 K for n-C8H18, and 573.15 K for n-C10H22 as well as n-C16H34. The liquid dynamic viscosity and surface tension data with average total measurement uncertainties (k = 2) of (2.0 and 1.7) % agree with the available literature and contribute to a new database at high temperatures. Over the entire temperature range, a Vogel-type equation for the dynamic viscosity and a modified van der Waals equation for the surface tension represent the measured data for the four n-alkanes within experimental uncertainties. By also considering our former SLS data for n-dodecane (n-C12H26) and n-octacosane (n-C28H58), empirical models for the liquid viscosity and surface tension of n-alkanes were developed as a function of temperature and carbon number covering values between 6 and 28. Agreement between these models and reference correlations for further selected n-alkanes which were not included in the development procedure was found.

Cross-Cultural Work in Music Cognition: Challenges, Insights, and Recommendations.

Many foundational questions in the psychology of music require cross-cultural approaches, yet the vast majority of work in the field to date has been conducted with Western participants and Western music. For cross-cultural research to thrive, it will require collaboration between people from different disciplinary backgrounds, as well as strategies for overcoming differences in assumptions, methods, and terminology. This position paper surveys the current state of the field and offers a number of concrete recommendations focused on issues involving ethics, empirical methods, and definitions of "music" and "culture."

Introducing substrate limitations to overcome catabolite repression in a protease producing Bacillus licheniformis strain using membrane-based fed-batch shake flasks.

To overcome catabolite repression, industrial fermentation processes are usually operated in substrate-limited fed-batch mode. Therefore, the implementation of such an operating mode at small scale is crucial to maintain comparable process conditions. In this study, Bacillus licheniformis, a well-known producer of proteases, was cultivated with carbon (glucose)- and nitrogen (ammonium)-limited fed-batch conditions using the previously introduced membrane-based fed-batch shake flasks. A repression of protease production by glucose and ammonium was thus avoided and yields increased 1.5- and 2.1-fold relative to batch, respectively. An elevated feeding rate of glucose caused depletion of ammonium, which was recognizable within the oxygen transfer rate (OTR) signal measured with the Respiration Activity MOnitoring System (RAMOS). Ammonium limitation was prevented by feeding ammonium simultaneously with glucose. The OTR signal clearly indicated the initiation of the fed-batch phase and gave direct feedback on the nutrient release kinetics. Increased feeding rates of glucose and ammonium led to an elevated protease activity without affecting the protease yield (YP/Glu ). In addition to YP/Glu , protease yields were determined based on the metabolized amount of oxygen (YP/O2) . The results showed that the protease production correlated with the amount of consumed glucose as well as with the amount of consumed oxygen. The membrane-based fed-batch shake flask in combination with the RAMOS device is a powerful combination to investigate the effect of substrate-limited fed-batch conditions.

Glucose-containing polymer rings enable fed-batch operation in microtiter plates with parallel online measurement of scattered light, fluorescence, dissolved oxygen tension, and pH.

Bioprocesses operated in batch mode can induce adverse effects like overflow metabolism, substrate inhibition, osmotic inhibition, oxygen limitation, and catabolite repression. To avoid these adverse effects, fed-batch is the predominant operation mode in industrial production. Nevertheless, screening for optimal production strains is usually performed in microtiter plates and shake flasks operated in batch mode without any online monitoring. Recently, a polymer-based controlled-release fed-batch microtiter plate with stable glucose release characteristics was described. In this study, a glucose-containing polymer matrix was used to manufacture polymer rings that were placed at the bottom of a 48-well microtiter plate. Thereby, the liquid content of the well became accessible for optical measurement by the BioLector device. Reflections caused by the polymer ring were minimized by adjusting the scattered-light measurement position. Influences on the measurement of the dissolved oxygen tension and pH could be avoided by choosing appropriate polymer-ring geometries. These adjustments enabled parallel online measurement of scattered light, fluorescence, dissolved oxygen tension, and pH of Escherichia coli BL21 (DE3) fed-batch cultivations. The online monitoring and fed-batch operation capabilities of the fed-batch microtiter plate presented in this study finds optimal application in screenings and initial process development.

Exploring small-scale chemostats to scale up microbial processes: 3-hydroxypropionic acid production in S. cerevisiae.

BACKGROUND: The physiological characterization of microorganisms provides valuable information for bioprocess development. Chemostat cultivations are a powerful tool for this purpose, as they allow defined changes to one single parameter at a time, which is most commonly the growth rate. The subsequent establishment of a steady state then permits constant variables enabling the acquisition of reproducible data sets for comparing microbial performance under different conditions. We performed physiological characterizations of a 3-hydroxypropionic acid (3-HP) producing Saccharomyces cerevisiae strain in a miniaturized and parallelized chemostat cultivation system. The physiological conditions under investigation were various growth rates controlled by different nutrient limitations (C, N, P). Based on the cultivation parameters obtained subsequent fed-batch cultivations were designed. RESULTS: We report technical advancements of a small-scale chemostat cultivation system and its applicability for reliable strain screening under different physiological conditions, i.e. varying dilution rates and different substrate limitations (C, N, P). Exploring the performance of an engineered 3-HP producing S. cerevisiae strain under carbon-limiting conditions revealed the highest 3-HP yields per substrate and biomass of 16.6 %C-mol and 0.43 g gCDW-1, respectively, at the lowest set dilution rate of 0.04 h-1. 3-HP production was further optimized by applying N- and P-limiting conditions, which resulted in a further increase in 3-HP yields revealing values of 21.1 %C-mol and 0.50 g gCDW-1 under phosphate-limiting conditions. The corresponding parameters favoring an increased 3-HP production, i.e. dilution rate as well as C- and P-limiting conditions, were transferred from the small-scale chemostat cultivation system to 1-L bench-top fermenters operating in fed-batch conditions, revealing 3-HP yields of 15.9 %C-mol and 0.45 g gCDW-1 under C-limiting, as well as 25.6 %C-mol and 0.50 g gCDW-1 under phosphate-limiting conditions. CONCLUSIONS: Small-scale chemostat cultures are well suited for the physiological characterization of microorganisms, particularly for investigating the effect of changing cultivation parameters on microbial performance. In our study, optimal conditions for 3-HP production comprised (i) a low dilution rate of 0.04 h-1 under carbon-limiting conditions and (ii) the use of phosphate-limiting conditions. Similar 3-HP yields were achieved in chemostat and fed-batch cultures under both C- and P-limiting conditions proving the growth rate as robust parameter for process transfer and thus the small-scale chemostat system as powerful tool for process optimization.

Functional Brush Poly(2-ethyl-2-oxazine)s: Synthesis by CROP and RAFT, Thermoresponsiveness and Grafting onto Iron Oxide Nanoparticles.

Brush polymers are highly functional polymeric materials combining the properties of different polymer classes and have found numerous applications, for example, in nanomedicine. Here, the synthesis of functional phosphonate-ester-bearing brush polymers based on poly(2-oxazine)s is reported through a combination of cationic ring-opening polymerization (CROP) of 2-ethyl-2-oxazine and reversible addition-fragmentation chain transfer (RAFT) polymerization. In this way, a small library of well-defined (D ≤ 1.17) poly(oligo(2-ethyl-2-oxazine) methacrylate) P(OEtOzMA)n brushes with tunable lower critical solution temperature (LCST) behavior and negligible cell toxicity is prepared. Upon deprotection, the phosphonic acid end-group of the P(OEtOzMA)n brush enables the successful grafting-onto iron oxide nanoparticles (IONPs). Colloidal stability of the particle suspension in combination with suitable magnetic resonance imaging (MRI) relaxivities demonstrates the potential of these particles for future applications as negative MRI contrast agents.

Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion.

Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative proteome analysis is a powerful tool to identify targets for an efficient increase of protein production and secretion in S. pombe Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016.

Media photo-degradation in pharmaceutical biotechnology - impact of ambient light on media quality, cell physiology, and IgG production in CHO cultures.

BACKGROUND: Many vital components in bioprocess media are prone to photo-conversion or photo-degradation upon exposure to ambient light, with severe negative consequences for biomass yield and overall productivity. However, there is only limited awareness of light irradiation as a potential risk factor when working in transparent glass bioreactors, storage vessels or disposable bag systems. The chemical complexity of most media renders a root-cause analysis difficult. This study investigated in a novel, holistic approach how light-induced changes in media composition relate to alterations in radical burden, cell physiology, morphology, and product formation in industrial Chinese hamster ovary (CHO) bioprocesses. RESULTS: Two media formulations from proprietary and commercial sources were tested in a pre-hoc light exposure scenario prior to cultivation. Using fluorescence excitation/emission (EEM) matrix spectroscopy, a photo-sensitization of riboflavin was identified as a likely cause for drastically decreased IgG titers (up to -80%) and specific growth rates (-50% to -90%). Up to three-fold higher radical levels were observed in photo-degraded medium. On the biological side, this resulted in significant changes in cell morphology and aberrations in the normal IgG biosynthesis/secretion pathway. CONCLUSION: These findings clearly illustrate the underrated impact of room light after only short periods of exposure, occurring accidentally or knowingly during bioprocess development and scale- up. The detrimental effects, which may share a common mechanistic cause at the molecular level, correlate well with changes in spectroscopic properties. This offers new perspectives for online monitoring concepts, and improved detectability of such effects in future. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry.

Increased production of L-serine in Escherichia coli through Adaptive Laboratory Evolution.

L-serine is a promising building block biochemical with a high theoretical production yield from glucose. Toxicity of L-serine is however prohibitive for high-titer production in E. coli. Here, E. coli lacking L-serine degradation pathways was evolved for improved tolerance by gradually increasing L-serine concentration from 3 to 100g/L using adaptive laboratory evolution (ALE). Genome sequencing of isolated clones revealed multiplication of genetic regions, as well as mutations in thrA, thereby showing a potential mechanism of serine inhibition. Additional mutations were evaluated by MAGE combined with amplicon sequencing, revealing role of rho, lrp, pykF, eno, and rpoB on tolerance and fitness in minimal medium. Production using the tolerant strains resulted in 37g/L of L-serine with a 24% mass yield. The resulting titer is similar to the highest production reported for any organism thereby highlighting the potential of ALE for industrial biotechnology.