Abstract
Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique ...insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients, the genetic cause remains unknown.
We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology.
We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation.
In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema.
Passchier et al. identify novel pathogenic variants in two genes, AQP4 and GPRC5B, in patients with genetic brain oedema. They provide the first description of a genetic disease linked to dysfunction of the water channel aquaporin-4, and identify GPRC5B as a potentially druggable target for the treatment of brain oedema.
See Oegema (https://doi.org/10.1093/brain/awad230) for a scientific commentary on this article.
Enterovirus D68 (EV-D68) is an emerging pathogen that can cause severe respiratory disease and is associated with cases of paralysis, especially among children. Heretofore, information on host factor ...requirements for EV-D68 infection is scarce. Haploid genetic screening is a powerful tool to reveal factors involved in the entry of pathogens. We performed a genome-wide haploid screen with the EV-D68 prototype Fermon strain to obtain a comprehensive overview of cellular factors supporting EV-D68 infection. We identified and confirmed several genes involved in sialic acid (Sia) biosynthesis, transport, and conjugation to be essential for infection. Moreover, by using knockout cell lines and gene reconstitution, we showed that both α2,6- and α2,3-linked Sia can be used as functional cellular EV-D68 receptors. Importantly, the screen did not reveal a specific protein receptor, suggesting that EV-D68 can use multiple redundant sialylated receptors. Upon testing recent clinical strains, we identified strains that showed a similar Sia dependency, whereas others could infect cells lacking surface Sia, indicating they can use an alternative, nonsialylated receptor. Nevertheless, these Sia-independent strains were still able to bind Sia on human erythrocytes, raising the possibility that these viruses can use multiple receptors. Sequence comparison of Sia-dependent and Sia-independent EV-D68 strains showed that many changes occurred near the canyon that might allow alternative receptor binding. Collectively, our findings provide insights into the identity of the EV-D68 receptor and suggest the possible existence of Sia-independent viruses, which are essential for understanding tropism and disease.
To describe the number, specifics and co-occurrence of shoulder pathologies detected by MRI in manual wheelchair users with spinal cord injury and to evaluate the association between shoulder ...pathologies and presence of shoulder pain.
Cross-sectional observation study.
Community.
Fifty-one wheelchair-dependent persons with spinal cord injury (44 males, 7 females, median age 50 years (IQR 14), median time since injury 24 years (IQR 16)) were allocated to pain or no-pain group based on the Wheelchair User Shoulder Pain Index.
Not applicable
All persons underwent shoulder MRI. Pathologies were scored blinded by two experienced radiologists. Participant characteristics, number and severity of shoulder pathologies were analyzed descriptively. Logistic regression was performed to evaluate the association between MRI findings and shoulder pain.
The median number of co-occurring MRI findings per person ranged from 0 to 19 (out of 31 possible findings). The cluster of MRI findings occurring most often together were tendon tears of supraspinatus (present in 84%), subscapularis (69%) and biceps (67%) and osteoarthritis of acromioclavicular joint (80%). When correcting for age and time since injury, the logistic regression showed no statistically significant correlation between the individual pathologies and shoulder pain.
MRI findings of shoulder pathology are very frequent in persons with and without shoulder pain. Therefore, when diagnosing the cause of shoulder pain and planning interventions, health care professionals should keep this finding in mind and MRI should not be interpreted without careful consideration of clinical history and functional testing.
Calcium imaging is a versatile experimental approach capable of resolving single neurons with single-cell spatial resolution in the brain. Electrophysiological recordings provide high temporal, but ...limited spatial resolution, because of the geometrical inaccessibility of the brain. An approach that integrates the advantages of both techniques could provide new insights into functions of neural circuits. Here, we report a transparent, flexible neural electrode technology based on graphene, which enables simultaneous optical imaging and electrophysiological recording. We demonstrate that hippocampal slices can be imaged through transparent graphene electrodes by both confocal and two-photon microscopy without causing any light-induced artefacts in the electrical recordings. Graphene electrodes record high-frequency bursting activity and slow synaptic potentials that are hard to resolve by multicellular calcium imaging. This transparent electrode technology may pave the way for high spatio-temporal resolution electro-optic mapping of the dynamic neuronal activity.
Guinea fowl coronavirus (GfCoV) causes fulminating enteritis that can result in a daily death rate of 20% in guinea fowl flocks. Here, we studied GfCoV diversity and evaluated its phenotypic ...consequences. Over the period of 2014 to 2016, affected guinea fowl flocks were sampled in France, and avian coronavirus presence was confirmed by PCR on intestinal content and immunohistochemistry of intestinal tissue. Sequencing revealed 89% amino acid identity between the viral attachment protein S1 of GfCoV/2014 and that of the previously identified GfCoV/2011. To study the receptor interactions as a determinant for tropism and pathogenicity, recombinant S1 proteins were produced and analyzed by glycan and tissue arrays. Glycan array analysis revealed that, in addition to the previously elucidated biantennary di-
-acetyllactosamine (diLacNAc) receptor, viral attachment S1 proteins from GfCoV/2014 and GfCoV/2011 can bind to glycans capped with alpha-2,6-linked sialic acids. Interestingly, recombinant GfCoV/2014 S1 has an increased affinity for these glycans compared to that of GfCoV/2011 S1, which was in agreement with the increased avidity of GfCoV/2014 S1 for gastrointestinal tract tissues. Enzymatic removal of receptors from tissues before application of spike proteins confirmed the specificity of S1 tissue binding. Overall, we demonstrate that diversity in GfCoV S1 proteins results in differences in glycan and tissue binding properties.
Avian coronaviruses cause major global problems in the poultry industry. As causative agents of huge economic losses, the detection and understanding of the molecular determinants of viral tropism are of ultimate importance. Here, we set out to study those parameters and obtained in-depth insight into the virus-host interactions of guinea fowl coronavirus (GfCoV). Our data indicate that diversity in GfCoV viral attachment proteins results in differences in degrees of affinity for glycan receptors, as well as altered avidity for intestinal tract tissues, which might have consequences for GfCoV tissue tropism and pathogenesis in guinea fowls.
Much remains to be learned about the biology of mushroom-forming fungi, which are an important source of food, secondary metabolites and industrial enzymes. The wood-degrading fungus Schizophyllum ...commune is both a genetically tractable model for studying mushroom development and a likely source of enzymes capable of efficient degradation of lignocellulosic biomass. Comparative analyses of its 38.5-megabase genome, which encodes 13,210 predicted genes, reveal the species's unique wood-degrading machinery. One-third of the 471 genes predicted to encode transcription factors are differentially expressed during sexual development of S. commune. Whereas inactivation of one of these, fst4, prevented mushroom formation, inactivation of another, fst3, resulted in more, albeit smaller, mushrooms than in the wild-type fungus. Antisense transcripts may also have a role in the formation of fruiting bodies. Better insight into the mechanisms underlying mushroom formation should affect commercial production of mushrooms and their industrial use for producing enzymes and pharmaceuticals.
Regulatory T cells (Tregs) are thought to inhibit antitumor immune responses, and their depletion could therefore have a synergistic effect with therapeutic cancer vaccines. We investigated the ...impact of three medications on blood Treg frequency in vaccinated cancer patients.
To date, the most specific marker for human Tregs is demethylation in the DNA that encodes the transcription factor FOXP3. Thus, we used a FOXP3 methylation-specific quantitative PCR assay (MS-qPCR) to measure Treg frequencies in the peripheral blood mononuclear cells (PBMCs) of melanoma patients. The patients participated in three clinical trials that combined tumor vaccines with potential Treg-depleting agents: low-dose cyclophosphamide, anti-CD25 monoclonal antibody daclizumab, and the IL-2/diphtheria toxin fusion protein denileukin diftitox.
In the nine control patients, blood Treg frequencies varied over time; there was a 46% reduction in one patient. In treated patients, a more than 2-fold decrease in Tregs was observed in one out of 11 patients receiving cyclophosphamide and in four out of 13 receiving daclizumab, but there was no such Treg decrease in any of the six patients who received denileukin diftitox. As a positive control, a more than 2-fold increase in blood Tregs was detected in four out of nine patients who were treated with interleukin-2.
We used a MS-qPCR method that detects Tregs but not other activated T lymphocytes; however, none of the Treg-depleting strategies that we tested led, in the majority of patients, to a conservative 50% reduction in blood Tregs.
Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two ...thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanisms in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.
For deep partial-thickness burns no consensus on the optimal treatment has been reached due to conflicting study outcomes with low quality evidence. Treatment options in high- and middle-income ...countries include conservative treatment with delayed excision and grafting if needed; and early excision and grafting. The majority of timing of surgery studies focus on survival rather than on quality of life. This study protocol describes a study that aims to compare long-term scar quality, clinical outcomes, and patient-reported outcomes between the treatment options. A multicentre prospective study will be conducted in the three Dutch burn centres (Rotterdam, Beverwijk, and Groningen). All adult patients with acute deep-partial thickness burns, based on healing potential with Laser Doppler Imaging, are eligible for inclusion. During a nine-month baseline period, standard practice will be monitored. This includes conservative treatment with dressings and topical agents, and excision and grafting of residual defects if needed 14-21 days post-burn. The subsequent nine months, early surgery is advocated, involving excision and grafting in the first week to ten days post-burn. The primary outcome compared between the two groups is long-term scar quality assessed by the Patient and Observer Scar Assessment Scale 3.0 twelve months after discharge. Secondary outcomes include clinical outcomes and patient-reported outcomes like quality of life and return to work. The aim of the study is to assess long-term scar quality in deep partial-thickness burns after conservative treatment with delayed excision and grafting if needed, compared to early excision and grafting. Adding to the ongoing debate on the optimal treatment of these burns. The broad range of studied outcomes will be used for the development of a decision aid for deep partial-thickness burns, to fully inform patients at the point of consent to surgery and support optimal person-centred care.
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