Unraveling the potential and constraints associated with corn steep liquor as a nutrient source for industrial fermentations.

Costly complex media components such as yeast extract and peptone are still widely used in industrial bioprocesses, despite their ill-defined composition. Side stream products such as corn steep liquor (CSL) present a compelling economical alternative that contains valuable nutrients required for microbial growth, that is, nitrogen and amino acids, but also vitamins, trace elements, and other minerals. However, as a side stream product, CSL may be subject to batch-to-batch variations and compositional heterogeneity. In this study, the Respiration Activity MOnitoring System designed for shake flasks (RAMOS) and 96-well microtiter plates (µTOM) were applied to investigate the potential and constraints of CSL utilization for two model microorganisms: E. coli and B. subtilis. Considering the dry substance content of complex nutrients involved, CSL-based media are more efficient in biomass production than the common lysogeny broth (LB) medium, containing 5 g/L yeast extract, 10 g/L peptone, and 5 g/L NaCl. At a glucose to CSL (glucose/CSL, g/g) ratio of 1/1 (g/g) and 2/1 (g/g), a secondary substrate limitation occurred in E. coli and B. subtilis cultivations, respectively. The study sheds light on differences in the metabolic activity of the two applied model organisms between varying CSL batches, which relate to CSL origin and production process, as well as the effect of targeted nutrient supplementation. Through a targeted nutrient supplementation, the most limiting component of the CSL-glucose medium used for these applied model microorganisms was identified to be ammonium nitrogen. This study proves the suitability of CSL as an alternative nutrient source for E. coli and B. subtilis. The RAMOS and µTOM technique detected differences between CSL batches, allowing easy and early identification of varying batches. A consistent performance of the CSL batches in E. coli and B. subtilis cultivations was demonstrated.

Reducing water uptake into BY-2 cells by systematically optimizing the cultivation parameters increases product yields achieved by transient expression in plant cell packs.

Plant-based production systems are inexpensive and easy to handle, allowing them to complement existing platforms for the production of protein-based vaccines, therapeutics and diagnostic reagents. However, screening product candidates in whole plants requires a large facility footprint and is challenging due to natural variations in recombinant protein accumulation. In contrast, plant cell packs (PCPs) allow more than 1000 samples to be screened per day in microtiter plates. PCPs enable rapid development cycles based on transient expression in as little as 3 days, and yield milligram quantities of product for initial quality assessment and functional testing. However, this requires high-level expression in BY-2 cells and consistent cell quality across batches. We therefore used a statistical design of experiments (DoE) approach to systematically assess factors that contribute to consistent high yields of recombinant proteins in PCPs. Specifically, we tested the osmolality, pH, carbon source, light source, and additives during cell cultivation, as well as cell and PCP harvest times. The careful adjustment of these factors increased overall productivity by approximately fourfold. Remarkably all cultivation conditions leading to high productivities during transient expression in PCPs were associated with a reduced water uptake into the central vacuole. The universal presence of a vacuole in plant cells indicates that our results should be transferrable to other cells lines. Our findings therefore support the broad application of PCPs for screening and product analysis during the development of protein-based pharmaceuticals and reagents in plants.

Investigating the influence of excipient batch variation on the structure, consistency and physical stability of polysorbate 60-based topical vehicles.

Fatty alcohol-polysorbate 60-water ternary systems were used as models to represent the continuous phases of the respective semisolid oil-in-water emulsions for topical delivery of cosmetic and medicinal agents. The influence of batch variation of polysorbate 60 and fatty alcohol on structure and consistency of these systems was investigated using microscopy, rheology, differential scanning calorimetry and X-ray scattering techniques. The polysorbate 60 : cetostearyl alcohol mixed emulsifying wax showed swelling in water, that is, the lamellar repeat distance continually augmented from 93 to 125 Å with water percentage 20-90%. Cetostearyl alcohol ternary systems were thicker than cetyl alcohol ones independently of polysorbate 60 batches used. All the ternary systems showed an initial increase in consistency over the first 2 weeks of storage, which was followed by slight changes in consistency (cetostearyl alcohol systems) due to the re-allocation of polysorbate 60 molecules in the gel network or significant breakdown of structure (cetyl alcohol systems) due to the transformation of swollen α-lamellar gel phase into ß, γ crystals on 25°C storage. With all fatty alcohols, the consistency of polysorbate 60 ternary system was directly dependent upon interlamellar water thickness as governed by the length and distribution of polyoxyethylene groups within polysorbate 60 molecules. In relation with the composition of polysorbate 60 batches used, the consistency of ternary systems was higher when prepared with the polysorbate 60 batch containing a greater amount of sorbitan polyoxyethylene monoesters. It was proposed that the swollen α-crystalline gel phase could be better formed by sorbitan polyoxyethylene monoesters rather than sorbitan polyoxyethylene diesters.

Des systemes ternaires alcool gras-polysorbate 60-eau ont été utilisés comme modèles pour représenter les phases continues des émulsions huile-dans-eau semi-solides respectives pour l'administration topique d'agents cosmétiques et médicinaux. L'influence de la variation des lots de polysorbate 60 et d'alcool gras sur la structure et la consistance de ces systèmes a été étudiée en utilisant la microscopie, la rhéologie et la calorimétrie différentielle à balayage et la diffusion des rayons X. La cire émulsifiante mixte polysorbate 60 : alcool cétostearylique a montré un gonflement dans l'eau, c'est-à-dire que la distance de répétition du motif lamellaire a continuellement augmentée de 93 à 125 A° avec un pourcentage d'eau de 20-90%. Les systèmes ternaires d'alcool cétostearylique étaient plus épais que ceux d'alcool cétylique indépendamment des lots de polysorbate 60 utilisés. Tous les systèmes ternaires ont montré une augmentation initiale de la consistance au cours des 2 premières semaines de stockage, qui a été suivie par de légers changements de consistance (systèmes d'alcool cétostearylique) en raison de la re-affectation des molécules de polysorbate 60 dans le réseau de gel ou d'une rupture significative de structure (systèmes d'alcool cétylique) en raison de la transformation des phases lamellaires gonfles de type α-gel en cristaux ß, γ conservés a 25°C. Avec tous les alcools gras, la consistance du système ternaire polysorbate 60 dépendait directement de l'épaisseur inter lamellaire de l'eau car gouverné par la longueur et la distribution des groupes de polyoxyethylène au sein des molécules de polysorbate 60. En relation avec la composition des lots de polysorbate 60 utilisés, la consistance des systèmes ternaires était plus élevée lorsque préparé avec le lot de polysorbate 60 contenant une plus grande quantité de monoesters de polyoxyethylene sorbitane. Il a été proposé que la phase de gel α-cristallin gonflé pourrait être mieux formée par des monoesters de polyoxyethylene sorbitane plutôt que des diesters de polyoxyethylene sorbitane.

A novel G-quadruplex aptamer-based spike trimeric antigen test for the detection of SARS-CoV-2.

The recent SARS-CoV-2 outbreak has been declared a global health emergency. It will take years to vaccinate the whole population to protect them from this deadly virus, hence the management of SARS-CoV-2 largely depends on the widespread availability of an accurate diagnostic test. Toward addressing the unmet need of a reliable diagnostic test in the current work by utilizing the power of Systematic Evolution of Ligands by EXponential enrichment, a 44-mer G-quadruplex-forming DNA aptamer against spike trimer antigen of SARS-CoV-2 was identified. The lead aptamer candidate (S14) was characterized thoroughly for its binding, selectivity, affinity, structure, and batch-to-batch variability by utilizing various biochemical, biophysical, and in silico techniques. S14 has demonstrated a low nanomolar KD, confirming its tight binding to a spike antigen of SARS-CoV-2. S14 can detect as low as 2 nM of antigen. The clinical evaluation of S14 aptamer on nasopharyngeal swab specimens (n = 232) has displayed a highly discriminatory response between SARS-CoV-2 infected individuals from the non-infected one with a sensitivity and specificity of ∼91% and 98%, respectively. Importantly, S14 aptamer-based test has evinced a comparable performance with that of RT-PCR-based assay. Altogether, this study established the utility of aptamer technology for the detection of SARS-CoV-2.

Repeatability and reproducibility assessment in a large-scale population-based microbiota study: case study on human milk microbiota.

BACKGROUND: Quality control including assessment of batch variabilities and confirmation of repeatability and reproducibility are integral component of high throughput omics studies including microbiome research. Batch effects can mask true biological results and/or result in irreproducible conclusions and interpretations. Low biomass samples in microbiome research are prone to reagent contamination; yet, quality control procedures for low biomass samples in large-scale microbiome studies are not well established. RESULTS: In this study, we have proposed a framework for an in-depth step-by-step approach to address this gap. The framework consists of three independent stages: (1) verification of sequencing accuracy by assessing technical repeatability and reproducibility of the results using mock communities and biological controls; (2) contaminant removal and batch variability correction by applying a two-tier strategy using statistical algorithms (e.g. decontam) followed by comparison of the data structure between batches; and (3) corroborating the repeatability and reproducibility of microbiome composition and downstream statistical analysis. Using this approach on the milk microbiota data from the CHILD Cohort generated in two batches (extracted and sequenced in 2016 and 2019), we were able to identify potential reagent contaminants that were missed with standard algorithms and substantially reduce contaminant-induced batch variability. Additionally, we confirmed the repeatability and reproducibility of our results in each batch before merging them for downstream analysis. CONCLUSION: This study provides important insight to advance quality control efforts in low biomass microbiome research. Within-study quality control that takes advantage of the data structure (i.e. differential prevalence of contaminants between batches) would enhance the overall reliability and reproducibility of research in this field. Video abstract.

Open Problems in Extracellular RNA Data Analysis: Insights From an ERCC Online Workshop.

We now know RNA can survive the harsh environment of biofluids when encapsulated in vesicles or by associating with lipoproteins or RNA binding proteins. These extracellular RNA (exRNA) play a role in intercellular signaling, serve as biomarkers of disease, and form the basis of new strategies for disease treatment. The Extracellular RNA Communication Consortium (ERCC) hosted a two-day online workshop (April 19-20, 2021) on the unique challenges of exRNA data analysis. The goal was to foster an open dialog about best practices and discuss open problems in the field, focusing initially on small exRNA sequencing data. Video recordings of workshop presentations and discussions are available (https://exRNA.org/exRNAdata2021-videos/). There were three target audiences: experimentalists who generate exRNA sequencing data, computational and data scientists who work with those groups to analyze their data, and experimental and data scientists new to the field. Here we summarize issues explored during the workshop, including progress on an effort to develop an exRNA data analysis challenge to engage the community in solving some of these open problems.

Interpretive discrepancies caused by target values inter-batch variations in chemiluminescence immunoassay for SARS-CoV-2 IgM/IgG by MAGLUMI.

Plasma specimens from coronavirus disease 2019 patients were double-tested for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies by two different batches of MAGLUMI 2019-nCov immunoglobulin M/immunoglobulin G (IgM/IgG) assays to evaluate IgM/IgG levels, qualitative interpretation, antibody kinetics, and linearity of diluted specimen. Here we show that (i) high-level IgM specimens need to be diluted with negative human plasma but not kit diluents and (ii) measured anti-SARS-CoV-2 IgM/IgG concentrations are substantially higher with later marketed immunoassay batch leading to (iii) the change of qualitative interpretation (positive vs. negative) in 12.3% of specimens measured for IgM, (iv) the informative time-course pattern of antibody production only when data from different immunoassay batches are not combined.

Optimal inter-batch normalization method for GC/MS/MS-based targeted metabolomics with special attention to centrifugal concentration.

This study investigated the optimal inter-batch normalization method for gas chromatography/tandem mass spectrometry (GC/MS/MS)-based targeted metabolome analysis of rodent blood samples. The effect of centrifugal concentration on inter-batch variation was also investigated. Six serum samples prepared from a mouse and 2 quality control (QC) samples from pooled mouse serum were assigned to each batch, and the 3 batches were analyzed by GC/MS/MS at different days. The following inter-batch normalization methods were applied to metabolome data: QC-based methods with quadratic (QUAD)- or cubic spline (CS)-fitting, total signal intensity (TI)-based method, median signal intensity (MI)-based method, and isotope labeled internal standard (IS)-based method. We revealed that centrifugal concentration was a critical factor to cause inter-batch variation. Unexpectedly, neither the QC-based normalization methods nor the IS-based method was able to normalize inter-batch variation, though MI- or TI-based normalization methods were effective in normalizing inter-batch variation. For further validation, 6 disease model rat and 6 control rat plasma were evenly divided into 3 batches, and analyzed as different batches. Same as the results above, MI- or TI-based methods were able to normalize inter-batch variation. In particular, the data normalized by TI-based method showed similar metabolic profiles obtained from their intra-batch analysis. In conclusion, the TI-based normalization method is the most effective to normalize inter-batch variation for GC/MS/MS-based metabolome analysis. Graphical abstract.

Glutaraldehyde - A Subtle Tool in the Investigation of Healthy and Pathologic Red Blood Cells.

Glutaraldehyde is a well-known substance used in biomedical research to fix cells. Since hemolytic anemias are often associated with red blood cell shape changes deviating from the biconcave disk shape, conservation of these shapes for imaging in general and 3D-imaging in particular, like confocal microscopy, scanning electron microscopy or scanning probe microscopy is a common desire. Along with the fixation comes an increase in the stiffness of the cells. In the context of red blood cells this increased rigidity is often used to mimic malaria infected red blood cells because they are also stiffer than healthy red blood cells. However, the use of glutaraldehyde is associated with numerous pitfalls: (i) while the increase in rigidity by an application of increasing concentrations of glutaraldehyde is an analog process, the fixation is a rather digital event (all or none); (ii) addition of glutaraldehyde massively changes osmolality in a concentration dependent manner and hence cell shapes can be distorted; (iii) glutaraldehyde batches differ in their properties especially in the ratio of monomers and polymers; (iv) handling pitfalls, like inducing shear artifacts of red blood cell shapes or cell density changes that needs to be considered, e.g., when working with cells in flow; (v) staining glutaraldehyde treated red blood cells need different approaches compared to living cells, for instance, because glutaraldehyde itself induces a strong fluorescence. Within this paper we provide documentation about the subtle use of glutaraldehyde on healthy and pathologic red blood cells and how to deal with or circumvent pitfalls.

Therapeutic Potential of Enoxaparin in Lichen Planus: Exploring Reasons for Inconsistent Reports.

Lichen planus (LP) is an uncommon mucocutaneous inflammatory condition, that is immunologically mediated, typically pruritic and often recurs. The currently advocated therapies are either not highly effective or associated with severe side effects. Enoxaparin, a widely used anticoagulant, is composed of both anticoagulant and non-anticoagulant fragments. Enoxaparin is reported to have anti-inflammatory properties and it was found to be effective in LP. However, the results from clinical studies have varied substantially and, therefore, the clinical role of enoxaparin in LP remains uncertain. This review focuses on potential reasons for the reported inconsistent outcomes, as well as proposing solutions; these include identifying batch-to-batch inconsistency in the composition of enoxaparin. The potential therapeutic value of enoxaparin in LP must be explored using well-designed clinical trials, combined with experimental studies that focus on identifying the anti-inflammatory fragments of enoxaparin and elucidating the mechanism of action of these non-anticoagulant fragments.

Simultaneous patch testing with fragrance mix I, fragrance mix II and their ingredients in southern Sweden between 2009 and 2015.

BACKGROUND: Fragrance mix I (FM I) and fragrance mix II (FM II) are included in the European baseline series as screening substances for fragrance contact allergy. OBJECTIVES: To investigate the frequency of allergic reactions to FM I, FM II and their ingredients in consecutively patch tested patients. MATERIALS AND METHODS: A retrospective analysis of data from 4430 patients patch tested between 2009 and 2015 was performed. RESULTS: Of the patients, 6.5% were FM I-positive and 3.2% were FM II-positive. Forty-five per cent of FM I-positive patients did not have positive reactions to FM I ingredients. Thirty-five per cent of those who were FM II-positive did not have positive reactions to FM II ingredients. Twenty-seven per cent of those with positive reactions to one or more of the FM I ingredients were FM I-negative, and 36% of those who had positive reactions to one or more of the FM II ingredients were FM II-negative. The allergens with the highest pick-up rates were Evernia prunastri (1.8%), cinnamal (1.3%), citral (1.2%), and hydroxyisohexyl 3-cyclohexene carboxaldehyde (1.2%). Significant differences were observed in the proportions of positive reactions to FM I, FM II, eugenol, isoeugenol, and farnesol when results from patch testing with materials from different suppliers were compared. CONCLUSIONS: There is a risk of missing fragrance contact allergy when testing with only the fragrance mixes is performed. The use of preparations from different suppliers may affect the patch test results.

Effect of long-term storage in biobanks on cerebrospinal fluid biomarker Aß1-42, T-tau, and P-tau values.

INTRODUCTION: We studied the effect of long-term storage at -80°C on cerebrospinal fluid (CSF) biomarker levels. Our approach assumed consistency of mean biomarker levels in a homogenous Alzheimer's disease patient cohort over time. METHODS: We selected 148 Alzheimer's disease samples that had inclusion dates equally distributed over the years 2001 to 2013 from our biobank. The concentrations of CSF biomarkers, amyloid ß1-42 (Aß1-42), total tau (T-tau), and phosphorylated tau181 (P-tau), were measured with one enzyme-linked immunosorbent assay lot. Results were compared with historical results obtained at biobank inclusion. RESULTS: Linear regression analyses showed that the levels of CSF biomarkers, Aß1-42, T-tau, and P-tau, were not related to storage time at -80°C (ß = 0.015, 0.048, and 0.0016 pg/mL per day, not significant). However, the differences between remeasured concentrations of Aß1-42 and concentrations at biobank inclusion measured for more than 30 assay batches increased with increasing time difference. DISCUSSION: The levels of CSF biomarkers, Aß1-42, T-tau, and P-tau, did not significantly change during the maximum period of 12 years of storage at -80°C. Batch variation for Aß1-42 is a factor that should be controlled for when using historical cohorts.

Derivation of Mesenchymal Stem Cells from Embryonic Stem Cells: A Non-Variable and Inexhaustive Source of Adult Stem Cells.

Mesenchymal stem cells (MSCs) provide an opportunity to bring the field of regenerative medicine to realization. A lot of clinical trials are presently trying to establish their applicability in real-world scenarios. Some of the biggest challenges encountered in bringing MSCs from bench to bedside are the number of MSCs required, their procurement from various sources, and the batch-to-batch variability. This often leads to inconclusive results within and between different studies. Therefore, we have hereby proposed a simple protocol to source mesenchymal stem cells through differentiation of embryonic stem cells.

Nanomechanical testing technique for millimeter-sized and smaller molecular crystals.

Large crystals are used as a control for the development of a mounting and nanoindentation testing technique for millimeter-sized and smaller molecular crystals. Indentation techniques causing either only elastic or elastic-plastic deformation produce similar results in assessing elastic modulus, however, the elastic indents are susceptible to surface angle and roughness effects necessitating larger sample sizes for similar confidence bounds. Elastic-plastic indentations give the most accurate results and could be used to determine the different elastic constants for anisotropic materials by indenting different crystal faces, but not by rotating the indenter about its axis and indenting the same face in a different location. The hardness of small and large crystals is similar, suggesting that defect content probed in this study is similar, and that small crystals can be compared directly to larger ones. The Young's modulus and hardness of the model test material, griseofulvin, are given for the first time to be 11.5GPa and 0.4GPa respectively.

A modified data normalization method for GC-MS-based metabolomics to minimize batch variation.

The goal of metabolomics data pre-processing is to eliminate systematic variation, such that biologically-related metabolite signatures are detected by statistical pattern recognition. Although several methods have been developed to tackle the issue of batch-to-batch variation, each method has its advantages and disadvantages. In this study, we used a reference sample as a normalization standard for test samples within the same batch, and each metabolite value is expressed as a ratio relative to its counterpart in the reference sample. We then applied this approach to a large multi-batch data set to facilitate intra- and inter-batch data integration. Our results demonstrate that normalization to a single reference standard has the potential to minimize batch-to-batch data variation across a large, multi-batch data set.