Development of Antisense Tools to Study Bodo saltans and Its Intracellular Symbiont.

Obligate symbioses are common in nature and present a particular challenge for functional genetic analysis. In many cases, the host is a non-model species with poor tools for genetic manipulation, and the symbiont cannot be cultured or its gene expression manipulated to investigate function. Here, we investigated the potential for using antisense inhibition to analyze host and symbiont gene function within an obligate aquatic symbiosis.

Hypoxia Dependent Inhibition of Glioblastoma Cell Proliferation, Invasion, and Metabolism by the Choline-Kinase Inhibitor JAS239.

Elevated choline kinase alpha (ChoK) levels are observed in most solid tumors, including glioblastomas (GBM), and ChoK inhibitors have demonstrated limited efficacy in GBM models. Given that hypoxia is associated with resistance to GBM therapy, we hypothesized that tumor hypoxia could be responsible for the limited response. Therefore, we evaluated the effects of hypoxia on the function of JAS239, a potent ChoK inhibitor in four GBM cell lines.

Whole organism and tissue-specific analysis of pexophagy in .

Peroxisomes are essential organelles involved in critical metabolic processes in animals such as fatty acid oxidation, ether phospholipid production and reactive oxygen species detoxification. We have generated transgenic models expressing fluorescent reporters for the selective autophagy of peroxisomes, a process known as pexophagy. We show that these reporters are colocalized with a peroxisomal marker and that they can reflect pexophagy induction by iron chelation and inhibition by depletion of the core autophagy protein Atg5.

A biocompatible supramolecular hydrogel mesh for sample stabilization in light microscopy and nanoscopy.

Most embedding media for live and fixed samples were not designed for microscopy and have issues including long polymerization times, peak of toxicity toward the sample during the sol-gel transition, and irreversibility of this transition. Gels derived from biological sources are widely used in microscopy, but their precise composition is ill-defined and can vary between batches. Non-physiological temperatures and/or specific enzymatic solutions are often needed to revert the gel back to the sol state to allow sample recovery.

Pulmonary SARS-CoV-2 infection leads to para-infectious immune activation in the brain.

Neurological complications, including encephalopathy and stroke, occur in a significant proportion of COVID-19 cases but viral protein is seldom detected in the brain parenchyma. To model this situation, we developed a novel low-inoculum K18-hACE2 mouse model of SARS-CoV-2 infection during which active viral replication was consistently seen in mouse lungs but not in the brain. We found that several mediators previously associated with encephalopathy in clinical samples were upregulated in the lung, including CCL2, and IL-6.

The activation of the adaptor protein STING depends on its interactions with the phospholipid PI4P.

Activation of the endoplasmic reticulum (ER)-resident adaptor protein STING, a component of a cytosolic DNA-sensing pathway, induces the transcription of genes encoding type I interferons (IFNs) and other proinflammatory factors. Because STING is activated at the Golgi apparatus, control of the localization and activation of STING is important in stimulating antiviral and antitumor immune responses. Through a genome-wide CRISPR interference screen, we found that STING activation required the Golgi-resident protein ACBD3, which promotes the generation of phosphatidylinositol 4-phosphate (PI4P) at the trans-Golgi network, as well as other PI4P-associated proteins.

Inhibition of glioblastoma cell proliferation and invasion by the choline-kinase inhibitor JAS239 varies with cell type and hypoxia.

Background: Elevated choline kinase alpha (ChoK) is observed in most solid tumours including glioblastomas (GBM), yet until recently, inhibitors of ChoK have demonstrated limited efficacy in GBM models. Given that hypoxia is associated with GBM therapy resistance, we hypothesised that tumour hypoxia could be responsible for such limitations. We therefore evaluated in GBM cells, the effect of hypoxia on the function of JAS239, a potent ChoK inhibitor.

Para-infectious brain injury in COVID-19 persists at follow-up despite attenuated cytokine and autoantibody responses.

To understand neurological complications of COVID-19 better both acutely and for recovery, we measured markers of brain injury, inflammatory mediators, and autoantibodies in 203 hospitalised participants; 111 with acute sera (1-11 days post-admission) and 92 convalescent sera (56 with COVID-19-associated neurological diagnoses). Here we show that compared to 60 uninfected controls, tTau, GFAP, NfL, and UCH-L1 are increased with COVID-19 infection at acute timepoints and NfL and GFAP are significantly higher in participants with neurological complications. Inflammatory mediators (IL-6, IL-12p40, HGF, M-CSF, CCL2, and IL-1RA) are associated with both altered consciousness and markers of brain injury.

Community-developed checklists for publishing images and image analyses.

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However, for scientists wishing to publish obtained images and image-analysis results, there are currently no unified guidelines for best practices.

Community-developed checklists for publishing images and image analyses.

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However for scientists wishing to publish the obtained images and image analyses results, there are to date no unified guidelines.

Correction: Fmoc-diphenylalanine hydrogels: understanding the variability in reported mechanical properties.

Correction for 'Fmoc-diphenylalanine hydrogels: understanding the variability in reported mechanical properties' by Jaclyn Raeburn , , 2012, , 1168-1174, https://doi.org/10.1039/C1SM06929B.

Oxygen-dependent changes in binding partners and post-translational modifications regulate the abundance and activity of HIF-1α/2α.

Cellular adaptation to low-oxygen environments is mediated in part by the hypoxia-inducible factors (HIFs). Like other transcription factors, the stability and transcriptional activity of HIFs-and consequently, the hypoxic response-are regulated by post-translational modifications (PTMs) and changes in protein-protein interactions. Our current understanding of PTM-mediated regulation of HIFs is primarily based on in vitro protein fragment-based studies typically validated in fragment-expressing cells treated with hypoxia-mimicking compounds.

Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signalling networks using SILAC-based phosphoproteomics.

Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion.

Trypanosoma brucei colonizes the tsetse gut via an immature peritrophic matrix in the proventriculus.

The peritrophic matrix of blood-feeding insects is a chitinous structure that forms a protective barrier against oral pathogens and abrasive particles. Tsetse flies transmit Trypanosoma brucei, which is the parasite that causes human sleeping sickness and is also partially responsible for animal trypanosomiasis in Sub-Saharan Africa. For this parasite to establish an infection in flies, it must first colonize the area between the peritrophic matrix and gut epithelium called the ectoperitrophic space.

Decrease of Nibrin expression in chronic hypoxia is associated with hypoxia-induced chemoresistance in some brain tumour cells.

Background: Solid tumours are less oxygenated than normal tissues. This is called tumour hypoxia and leads to resistance to radiotherapy and chemotherapy. The molecular mechanisms underlying such resistance have been investigated in a range of tumour types, including the adult brain tumours glioblastoma, yet little is known for paediatric brain tumours.

Caught in a Trap? Proteomic Analysis of Neutrophil Extracellular Traps in Rheumatoid Arthritis and Systemic Lupus Erythematosus.

Neutrophil Extracellular Traps (NETs) are implicated in the development of auto-immunity in diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) through the externalization of intracellular neoepitopes e.g., dsDNA and nuclear proteins in SLE and citrullinated peptides in RA.

Efficacy of a Surfactant-Based Wound Dressing in the Prevention of Biofilms.

Objective: To assess the biofilm prevention action of two wound dressings, a concentrated surfactant gel preserved with antimicrobials and a concentrated surfactant gel with 1% silver sulfadiazine.

Methods: The microorganisms Staphylococcus aureus, methicillin-resistant S aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Enterococcus faecalis were used. Several biofilm models were used whereby the surfaces of each model were coated with either the concentrated surfactant gel preserved with antimicrobials or the concentrated surfactant gel with SSD before biofilm growth.

Live Imaging of Cell Invasion Using a Multicellular Spheroid Model and Light-Sheet Microscopy.

Three-dimensional cellular assays are becoming increasingly popular as a fundamental tool to bridge the gap between tissue culture systems and in vivo tissue. In particular, spheroids are recognised today as a necessary intermediate model between testing in monolayer cultures and testing in animals. This chapter describes a straightforward protocol, from sample preparation to image acquisition and initial post-processing, based on one of most widely used commercial light-sheet fluorescence microscopy platform, the Zeiss Lightsheet Z.

SERCA directs cell migration and branching across species and germ layers.

Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in air sac budding.

Evaluation of quantum dot conjugated antibodies for immunofluorescent labelling of cellular targets.

Semiconductor quantum dots (Qdots) have been utilised as probes in fluorescence microscopy and provide an alternative to fluorescent dyes and fluorescent proteins due to their brightness, photostability, and the possibility to excite different Qdots with a single wavelength. In spite of these attractive properties, their implemenation by biologists has been somewhat limited and only a few Qdot conjugates are commercially available for the labelling of cellular targets. Although many protocols have been reported for the specific labelling of proteins with Qdots, the majority of these relied on Qdot-conjugated antibodies synthesised specifically by the authors (and therefore not widely available), which limits the scope of applications and complicates replication.