Glucosylceramide in bunyavirus particles is essential for virus binding to host cells.

Hexosylceramides (HexCer) are implicated in the infection process of various pathogens. However, the molecular and cellular functions of HexCer in infectious cycles are poorly understood. Investigating the enveloped virus Uukuniemi (UUKV), a bunyavirus of the Phenuiviridae family, we performed a lipidomic analysis with mass spectrometry and determined the lipidome of both infected cells and derived virions. We found that UUKV alters the processing of HexCer to glycosphingolipids (GSL) in infected cells. The infection resulted in the overexpression of glucosylceramide (GlcCer) synthase (UGCG) and the specific accumulation of GlcCer and its subsequent incorporation into viral progeny. UUKV and several pathogenic bunyaviruses relied on GlcCer in the viral envelope for binding to various host cell types. Overall, our results indicate that GlcCer is a structural determinant of virions crucial for bunyavirus infectivity. This study also highlights the importance of glycolipids on virions in facilitating interactions with host cell receptors and infectious entry of enveloped viruses.

Model based process optimization of an industrial chromatographic process for separation of lactoferrin from bovine milk.

Model based process development using predictive mechanistic models is a powerful tool for in-silico downstream process development. It allows to obtain a thorough understanding of the process reducing experimental effort. While in pharma industry, mechanistic modeling becomes more common in the last years, it is rarely applied in food industry. This case study investigates risk ranking and possible optimization of the industrial process of purifying lactoferrin from bovine milk using SP Sepharose Big Beads with a resin particle diameter of 200 µm, based on a minimal number of lab-scale experiments combining traditional scale-down experiments with mechanistic modeling. Depending on the location and season, process water pH and the composition of raw milk can vary, posing a challenge for highly efficient process development. A predictive model based on the general rate model with steric mass action binding, extended for pH dependence, was calibrated to describe the elution behavior of lactoferrin and main impurities. The gained model was evaluated against changes in flow rate, step elution conditions, and higher loading and showed excellent agreement with the observed experimental data. The model was then used to investigate the critical process parameters, such as water pH, conductivity of elution steps, and flow rate, on process performance and purity. It was found that the elution behavior of lactoferrin is relatively consistent over the pH range of 5.5 to 7.6, while the elution behavior of the main impurities varies greatly with elution pH. As a result, a significant loss in lactoferrin is unavoidable to achieve desired purities at pH levels below pH 6.0. Optimal process parameters were identified to reduce water and salt consumption and increase purity, depending on water pH and raw milk composition. The optimal conductivity for impurity removal in a low conductivity elution step was found to be 43 mS/cm, while a conductivity of 95 mS/cm leads to the lowest overall salt usage during lactoferrin elution. Further increasing the conductivity during lactoferrin elution can only slightly lower the elution volume thus can also lead to higher total salt usage. Low flow rates during elution of 0.2 column volume per minute are beneficial compared to higher flow rates of 1 column volume per minute. The, on lab-scale, calibrated model allows predicting elution volume and impurity removal for large-scale experiments in a commercial plant processing over 106 liters of milk per day. The successful model extrapolation was possible without recalibration or detailed knowledge of the manufacturing plant. This study therefore provides a possible pathway for rapid process development of chromatographic purification in the food industries combining traditional scale-down experiments with mechanistic modeling.

Comparative Analysis of Six IRF Family Members in Alveolar Epithelial Cell-Intrinsic Antiviral Responses.

Host cell-intrinsic antiviral responses are largely mediated by pattern-recognition receptor (PRR) signaling and the interferon (IFN) system. The IFN regulatory factor (IRF) family of transcription factors takes up a central role in transcriptional regulation of antiviral innate immunity. IRF3 and IRF7 are known to be key players downstream of PRRs mediating the induction of type I and III IFNs. IFN signaling then requires IRF9 for the expression of the full array of interferon stimulated genes (ISGs) ultimately defining the antiviral state of the cell. Other members of the IRF family clearly play a role in mediating or modulating IFN responses, such as IRF1, IRF2 or IRF5, however their relative contribution to mounting a functional antiviral response is much less understood. In this study, we systematically and comparatively assessed the impact of six members of the IRF family on antiviral signaling in alveolar epithelial cells. We generated functional knockouts of IRF1, -2, -3, -5, -7, and -9 in A549 cells, and measured their impact on the expression of IFNs and further cytokines, ISGs and other IRFs, as well as on viral replication. Our results confirmed the vital importance of IRF3 and IRF9 in establishing an antiviral state, whereas IRF1, 5 and 7 were largely dispensable. The previously described inhibitory activity of IRF2 could not be observed in our experimental system.

From Sampling to Sequencing: A Liquid Biopsy Pre-Analytic Workflow to Maximize Multi-Layer Genomic Information from a Single Tube.

Liquid biopsies hold great promise for the management of cancer. Reliable liquid biopsy data depend on stable and reproducible pre-analytical protocols that comply with quality measures, irrespective of the sampling and processing site. We established a workflow for plasma preservation, followed by processing, cell-free nucleic acid isolation, quantification, and enrichment of potentially tumor-derived cell-free DNA and RNA. Employing the same input material for a direct comparison of different kits and protocols allowed us to formulate unbiased recommendations for sample collection, storage, and processing. The presented workflow integrates the stabilization in Norgen, PAX, or Streck tubes and subsequent parallel isolation of cell-free DNA and RNA with NucleoSnap and NucleoSpin. Qubit, Bioanalyzer, and TapeStation quantification and quality control steps were optimized for minimal sample use and high sensitivity and reproducibility. We show the efficiency of the proposed workflow by successful droplet digital PCR amplification of both cell-free DNA and RNA and by detection of tumor-specific alterations in low-coverage whole-genome sequencing and DNA methylation profiling of plasma-derived cell-free DNA. For the first time, we demonstrated successful parallel extraction of cell-free DNA and RNA from plasma samples. This workflow paves the road towards multi-layer genomic analysis from one single liquid biopsy sample.

Functional and Molecular Characterization of Mechanoinsensitive "Silent" Nociceptors.

Mechanical and thermal hyperalgesia (pain hypersensitivity) are cardinal signs of inflammation. Although the mechanism underlying thermal hyperalgesia is well understood, the cellular and molecular basis of mechanical hyperalgesia is poorly described. Here, we have identified a subset of peptidergic C-fiber nociceptors that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli when exposed to the inflammatory mediator nerve growth factor (NGF). Strikingly, NGF did not affect mechanosensitivity of other nociceptors. We show that these mechanoinsensitive "silent" nociceptors are characterized by the expression of the nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3) and that the mechanically gated ion channel PIEZO2 mediates NGF-induced mechanosensitivity in these neurons. Retrograde tracing revealed that CHRNA3+ nociceptors account for ∼50% of all peptidergic nociceptive afferents innervating visceral organs and deep somatic tissues. Hence, our data suggest that NGF-induced "un-silencing" of CHRNA3+ nociceptors significantly contributes to the development of mechanical hyperalgesia during inflammation.