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The thick filament is a key component of sarcomeres, the basic units of striated muscle 1 . Alterations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases 2 . Despite the central importance of the thick filament, its molecular organization remains unclear. Here we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by cryo-electron tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin, titin and myosin-binding protein C (MyBP-C). The arrangement of myosin molecules is dependent on their position along the filament, suggesting specialized capacities in terms of strain susceptibility and force generation. Three pairs of titin-α and titin-β chains run axially along the filament, intertwining with myosin tails and probably orchestrating the length-dependent activation of the sarcomere. Notably, whereas the three titin-α chains run along the entire length of the thick filament, titin-β chains do not. The structure also demonstrates that MyBP-C bridges thin and thick filaments, with its carboxy-terminal region binding to the myosin tails and directly stabilizing the OFF state of the myosin heads in an unforeseen manner. These results provide a foundation for future research investigating muscle disorders involving sarcomeric components., (© 2023. The Author(s).)

Objectives: The study aimed to determine the allergen, endotoxin and β-(1,3)-glucan concentrations at various areas on a university campus of veterinary medicine., Methods: Dust samples were collected four times a year for three years using electrostatic dust collectors (EDC) at 25 different locations on a campus of veterinary medicine and in laboratories of inorganic chemistry as a control area representing animal-free environment. Major animal allergens from dog, cat, horse, cattle and mouse, domestic mite (DM) allergens, and β-(1,3)-glucan were measured using enzyme immunoassays and endotoxin using the limulus amoebocyte lysate (LAL) assay. Seasonal, annual and local influences on exposure levels were analyzed using Bayesian mixed models., Results: With the exception of mouse allergens, all other determinants were found in almost all locations on the campus and in the control area, but in up to 10.000-fold variable concentrations. By far the highest levels of feline, canine, equine and bovine allergens were detected in buildings where the respective species were examined. The highest levels of mouse and DM allergens, β-(1,3)-glucan and endotoxin occurred together and were associated with locations where large animals were present. In buildings without animals, allergen levels were considerably lower but still elevated at several locations compared to the control area, especially for dog and horse allergens, and β-(1,3)-glucan. Significant seasonal effects were observed for dog, cat, horse and DM allergens, and β-(1,3)-glucan. Variations between years were less apparent than between seasons (except for β-(1,3)-glucan)., Conclusions: The strongest influencing factor on the concentration of mammalian allergens was the presence of the corresponding animal at the collection site. Seasonal influence on allergen concentrations was observed, while the overall exposure remained constant over the years. At locations with horses, elevated levels of mite allergens, endotoxin, and β-(1,3)-glucan can be expected, probably due to passive transfer from stable environment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Zahradnik et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The presence of meningeal ectopic lymphoid structures (ELS) in a subgroup of patients diagnosed with secondary progressive multiple sclerosis (SPMS) corresponds to a pronounced cortical inflammation and an aggravated disease course. In MP4-induced experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS), B cell aggregates develop in the central nervous system (CNS) in the chronic stage of the disease. Therefore, the model is suitable for studying key molecules of ELS development and maintenance. Here, we investigated whether there is a specific cytokine and chemokine signature in paired cerebrospinal fluid (CSF) and serum samples associated with the presence of cerebellar B cell and T cell pathology and B cell aggregates of MP4-immunized mice., Methods: Paired CSF and serum samples were collected from the cisterna magna and periphery of MP4-immunized mice at the chronic stage of disease. A control group with mice immunized only with the adjuvant (vehicle) was included in the study. A selected panel of 34 cytokines and chemokines were measured by MAGPIX® for both cohorts. For the assessment of B cell and T cell infiltration, immunohistochemical staining was performed and analyzed using light microscopy. To detect specific chemokine receptors additional staining was conducted., Results: While we detected several upregulated cytokines and chemokines in the CSF of MP4-immunized mice independent of the extent of B cell and T cell pathology compared to vehicle-immunized mice, C-C motif chemokine ligand (CCL)-1 was associated with high B cell and T cell infiltration. Furthermore, the level of certain chemokines, including CCL1, CCL5, CCL7, CCL12, CCL22 and C-X-C motif chemokine ligand (CXCL)-13, was significantly increased (p < 0.05) in MP4-immunized mice showing a high number of B cell aggregates. While C-C motif chemokine receptor (CCR)5 had a ubiquitous expression independent of the extent of B cell and T cell pathology, C-X-C motif chemokine receptor (CXCR)-5 and CXCR6 expression was specifically associated with high B cell and T cell pathology., Conclusion: Our data suggest that multiple cytokines and chemokines are involved in the pathophysiology of MP4-induced EAE. Furthermore, the presence of B cell aggregates was associated with a specific chemokine profile in the CSF, which might be useful for predicting the presence of these aggregates without the necessity to histologically screen the CNS tissue., (© 2023. The Author(s).)

Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging., Competing Interests: DB is an employee of Delmic B.V, JH, Sd has a financial interest in Delmic B.V, AJ, GJ, MK, AK, Cd, JP, SR, ST, ZW, Ev, RW No competing interests declared, CJ, ML were employees of Delmic B.V, (© 2022, Boltje et al.)

Intra-amniotic infection (IAI) is one major driver for preterm birth and has been demonstrated by clinical studies to exert both beneficial and injurious effects on the premature lung, possibly due to heterogeneity in the microbial type, timing, and severity of IAI. Due to the inaccessibility of the intra-amniotic cavity during pregnancies, preclinical animal models investigating pulmonary consequences of IAI are indispensable to elucidate the pathogenesis of bronchopulmonary dysplasia (BPD). It is postulated that on one hand imbalanced inflammation, orchestrated by lung immune cells such as macrophages, may impact on airway epithelium, vascular endothelium, and interstitial mesenchyme, resulting in abnormal lung development. On the other hand, excessive suppression of inflammation may as well cause pulmonary injury and a certain degree of inflammation is beneficial. So far, effective strategies to prevent and treat BPD are scarce. Therapeutic options targeting single mediators in signaling cascades and mesenchymal stromal cells (MSCs)-based therapies with global regulatory capacities have demonstrated efficacy in preclinical animal models and warrant further validation in patient populations. Ante-, peri- and postnatal exposome analysis and therapeutic investigations using multiple omics will fundamentally dissect the black box of IAI and its effect on the premature lung, contributing to precisely tailored and individualized therapies.

OCT1 and OCT2 are polyspecific membrane transporters that are involved in hepatic and renal drug clearance in humans and mice. In this study, we cloned dog OCT1 and OCT2 and compared their function to the human and mouse orthologs. We used liver and kidney RNA to clone dog OCT1 and OCT2. The cloned and the publicly available RNA-Seq sequences differed from the annotated exon-intron structure of OCT1 in the dog genome CanFam3.1. An additional exon between exons 2 and 3 was identified and confirmed by sequencing in six additional dog breeds. Next, dog OCT1 and OCT2 were stably overexpressed in HEK293 cells and the transport kinetics of five drugs were analyzed. We observed strong differences in the transport kinetics between dog and human orthologs. Dog OCT1 transported fenoterol with 12.9-fold higher capacity but 14.3-fold lower affinity (higher K M ) than human OCT1. Human OCT1 transported ipratropium with 5.2-fold higher capacity but 8.4-fold lower affinity than dog OCT1. Compared to human OCT2, dog OCT2 showed 10-fold lower transport of fenoterol and butylscopolamine. In conclusion, the functional characterization of dog OCT1 and OCT2 reported here may have implications when using dogs as pre-clinical models as well as for drug therapy in dogs.

Successful therapy with anti-CD20 monoclonal antibodies (mAbs) has reinforced the key role of B cells in the immunopathology of multiple sclerosis (MS). This study aimed to determine the effects of a novel class of anti-CD20 mAbs on vascular and extravascular central nervous system (CNS)-infiltrating B cells in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Male hCD20xhIgR3 mice and wild-type C57BL/6 (B6) mice were immunized with human myelin oligodendrocyte glycoprotein (MOG) 1-125 to induce EAE. While hCD20xhIgR3 mice were injected intravenously with an anti-human CD20 mAb (5 mg/kg) (rituximab (a type I anti-CD20 mAb) or obinutuzumab (a type II anti-CD20 mAb), B6 mice received the anti-mouse CD20 antibody 18B12. Neither mAb affected clinical disease or serum antibody levels. Obinutuzumab and rituximab had an impact on splenic and CNS-infiltrated B cells with slightly differential depletion efficacy. Additionally, obinutuzumab had beneficial effects on spinal cord myelination. B cell depletion rates in the 18B12/B6 model were comparable with those observed in obinutuzumab-treated hCD20xhIgR3 mice. Our results demonstrate the usefulness of anti-CD20 mAbs for the modulation of B cell-driven peripheral immune response and CNS pathology, with type II antibodies potentially being superior to type I in the depletion of tissue-infiltrating B cells.

Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease of the central nervous system (CNS) with a high socioeconomic relevance. The pathophysiology of MS, which is both complex and incompletely understood, is believed to be influenced by various environmental determinants, including diet. Since the 1990s, a correlation between the consumption of bovine milk products and MS prevalence has been debated. Here, we show that C57BL/6 mice immunized with bovine casein developed severe spinal cord pathology, in particular, demyelination, which was associated with the deposition of immunoglobulin G. Furthermore, we observed binding of serum from casein-immunized mice to mouse oligodendrocytes in CNS tissue sections and in culture where casein-specific antibodies induced complement-dependent pathology. We subsequently identified myelin-associated glycoprotein (MAG) as a cross-reactive antigenic target. The results obtained from the mouse model were complemented by clinical data showing that serum samples from patients with MS contained significantly higher B cell and antibody reactivity to bovine casein than those from patients with other neurologic diseases. This reactivity correlated with the B cell response to a mixture of CNS antigens and could again be attributed to MAG reactivity. While we acknowledge disease heterogeneity among individuals with MS, we believe that consumption of cow’s milk in a subset of patients with MS who have experienced a previous loss of tolerance to bovine casein may aggravate the disease. Our data suggest that patients with antibodies to bovine casein might benefit from restricting dairy products from their diet.

Cryo-electron tomography (cryo-ET) is an emerging technique to study the cellular architecture and the structure of proteins at high resolution in situ. Most biological specimens are too thick to be directly investigated and are therefore thinned by milling with a focused ion beam under cryogenic conditions (cryo-FIB). This procedure is prone to contaminations, which makes it a tedious process, often leading to suboptimal results. Here, we present new hardware that overcomes the current limitations. We developed a new glove box and a high vacuum cryo transfer system and installed a stage heater, a cryo-shield and a cryo-shutter in the FIB milling microscope. This reduces the ice contamination during the transfer and milling process and simplifies the handling of the sample. In addition, we tested a new software application that automates the key milling steps. Together, these improvements allow for high-quality, high-throughput cryo-FIB milling. This paves the way for new types of experiments, which have been previously considered infeasible., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The effects of an injectable anesthesia with 0.05 mg/kg medetomidine, 5 mg/kg ketamine, and 0.5 mg/kg butorphanol administered together intramuscularly were evaluated in 22 captive Humboldt penguins ( Spheniscus humboldti , 10 male and 12 female), with a mean age of 8.5 ± 8.23 years. The birds fasted for18-24 hours prior to the procedure. Induction was followed by 4 distinct progressive responses of the birds to the anesthetic effect, including onset of initial effects at 2.0 ± 1.7 minutes (x̄ ± SD), sternal recumbency with the head still elevated at 2.2 ± 1.6 minutes, lowering and placing the beak tip to the ground at 3.6 ± 3.4 minutes, and lateral positioning of the neck and head at 4.2 ± 3.4 minutes. A general state of sedation, muscle relaxation, and analgesia were noted 10.0 ± 2.8 minutes postinjection. However, according to an established scoring system for the assessment of anesthetic depth in avian patients, a surgical plane of anesthesia was not achieved. Muscle relaxation determined by the same scoring system lasted for 31.4 ± 17.1 minutes. The penguins' mean respiratory rate did not demonstrate significant change and spontaneous ventilation was present throughout the procedure. Relative peripheral arterial oxygen saturation decreased significantly from 92.83 ± 5.77% at 10 minutes to 90.91 ± 5.77% at 40 minutes following induction. The birds' heart rate also decreased significantly from 112.55 ± 23.97 beats/min at 10 minutes to 101.65 ± 25.42 beats/min at 40 minutes. The measured cloacal temperatures were maintained within normal range despite ambient temperatures of up to 28.3°C (82.9°F). Reversal of medetomidine with 0.25 mg/kg atipamezole was conducted after 45.1 ± 7.3 minutes. Recovery was smooth but of variable duration with patients being able or willing to stand steadily in an upright position after 50.1 ± 34.6 minutes. One penguin died during recovery from a ruptured left ventricle and consecutive pericardial tamponade, but no predisposing factors were identified. The anesthetic protocol proved to be effective for noninvasive and minor painful procedures (eg, diagnostic imaging, blood collection). Disadvantages to the administration of the combined anesthetic agents in the penguins included a short period of muscle relaxation and smooth but potentially prolonged recovery. The safety of the anesthetic protocol described for Humboldt penguins in this report has to be evaluated critically against the the death of 1 penguin during recovery.

Voltage gated sodium (Nav) channels contribute to axonal damage following demyelination in experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis (MS). The Nav1.6 isoform has been implicated as a primary contributor in this process. However, the role of Nav1.6 in immune processes, critical to the pathology of both MS and EAE, has not been extensively studied. EAE was induced with myelin oligodendrocyte (MOG 35-55 ) peptide in Scn8a dmu/+ mice, which have reduced Nav1.6 levels. Scn8a dmu/+ mice demonstrated improved motor capacity during the recovery and early chronic phases of EAE relative to wild-type animals. In the optic nerve, myeloid cell infiltration and the effects of EAE on the axonal ultrastructure were also significantly reduced in Scn8a dmu/+ mice. Analysis of innate immune parameters revealed reduced plasma IL-6 levels and decreased percentages of Gr-1 high /CD11b + and Gr-1 int /CD11b + myeloid cells in the blood during the chronic phase of EAE in Scn8a dmu/+ mice. Elevated levels of the anti-inflammatory cytokines IL-10, IL-13, and TGF-β1 were also observed in the brains of untreated Scn8a dmu/+ mice. A lipopolysaccharide (LPS) model was used to further evaluate inflammatory responses. Scn8a dmu/+ mice displayed reduced inflammation in response to LPS challenge. To further evaluate if this was an immune cell-intrinsic difference or the result of changes in the immune or hormonal environment, mast cells were derived from the bone marrow of Scn8a dmu/+ mice. These mast cells also produced lower levels of IL-6, in response to LPS, compared with those from wild type mice. Our results demonstrate that in addition to its recognized impact on axonal damage, Nav1.6 impacts multiple aspects of the innate inflammatory response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Alrashdi, Dawod, Tacke, Kuerten, Côté and Marshall.)

Objective: We investigated the predictive value of the enzyme-linked immunospot technique (ELISPOT) in identifying patients with relapsing-remitting multiple sclerosis (RRMS) who will respond to treatment with glatiramer acetate (GA) or interferon-β (IFN-β), based on the brain-reactive B-cell activity of peripheral blood cells., Methods: In this retrospective, cross-sectional, real-world multicenter study, we identified patients with RRMS in the NeuroTransData MS registry and stratified them based on their documented treatment response (relapse-free in the first 12 months of treatment) to GA or IFN-β. The GA group comprised 73 patients who responded to GA and 35 nonresponders. The IFN-β group comprised 62 responders to IFN-β and 37 nonresponders. Patients with previous or current therapy affecting B-cell activity were excluded. We polyclonally stimulated mononuclear cells from peripheral blood samples (collected after participant selection) and investigated brain-reactive B-cell activity after incubation on brain tissue lysate-coated ELISPOT plates. Validity metrics of the ELISPOT testing results were calculated (Python 3.6.8) in relation to the clinical responsiveness in the 2 treatment groups., Results: The ELISPOT B-cell activity assay showed a sensitivity of 0.74, a specificity of 0.76, a positive predictive value of 0.78, a negative predictive value of 0.28, and a diagnostic OR of 8.99 in predicting clinical response to GA vs IFN-β therapy in patients with RRMS., Conclusion: Measurement of brain-reactive B-cell activity by ELISPOT provides clinically meaningful predictive probabilities of individual patients' treatment response to GA or IFN-β. The assay has the potential to improve the selection of optimal first-line treatment for individual patients with RRMS., Classification of Evidence: This study provides Class II evidence that in patients with RRMS, the brain reactivity of their peripheral-blood B cells predicts clinical response to GA and IFN-β., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

B cell-depleting therapies have recently proven to be clinically highly successful in the treatment of multiple sclerosis (MS). This study aimed to determine the effects of the novel type II anti-human CD20 (huCD20) monoclonal antibody (mAb) obinutuzumab (OBZ) on spinal cord degeneration in a B cell-dependent mouse model of MS. Double transgenic huCD20xHIGR3 (CD20dbtg) mice, which express human CD20, were immunised with the myelin fusion protein MP4 to induce experimental autoimmune encephalomyelitis (EAE). Both light and electron microscopy were used to assess myelination and axonal pathology in mice treated with OBZ during chronic EAE. Furthermore, the effects of the already established murine anti-CD20 antibody 18B12 were assessed in C57BL/6 wild-type (wt) mice. In both models (18B12/wt and OBZ/CD20dbtg) anti-CD20 treatment significantly diminished the extent of spinal cord pathology. While 18B12 treatment mainly reduced the extent of axonal pathology, a significant decrease in demyelination and increase in remyelination were additionally observed in OBZ-treated mice. Hence, the data suggest that OBZ could have neuroprotective effects on the CNS, setting the drug apart from the currently available type I anti-CD20 antibodies.

Objective: Urethral calculi are a frequent cause of urinary disorders in male dogs. The aim of this study was to evaluate male dogs with urethral stones, which were relocated into the urinary bladder with the support of standardized epidural anesthesia in addition to general anesthesia., Materials and Methods: Data of 83 male dogs with urethral calculi were evaluated regarding clinical signs, localization and number of urethral calculi, diagnostic imaging, surgical procedure and postoperative radiographs. Additionally, bacterial culture and stone type analysis were evaluated. Besides general anesthesia all dogs received an epidural anesthesia., Results: With one exception all dogs showed signs of urinary disorders, in 33 cases, these were chronic. In 66 cases, urethral stones were diagnosed radiographically and in 11 cases, radiolucent urethral concrements were detected via ultrasonography. In 6 dogs, diagnosis was reached by catheterization and subsequent evidence of stones in the urinary bladder. At the time of presentation, more than one third of the dogs showed urethral calculi only. In 53 % of the dogs (n = 44), 3 or more urethral stones were present. In 77 of 83 dogs (92.7 %), relocation of all urethral stones into the urinary bladder was achieved. During postoperative radiography 9 dogs were diagnosed with residual urethral calculi., Conclusion and Clinical Relevance: Due to a significant proportion of dogs with sole urethral stones reliable radiological diagnosis of urethral calculi requires precise patient positioning. In cases of radiolucent calculi, ultrasonography of the urethra may lead to a diagnosis, sonographic evaluation of the urinary bladder alone is not sufficient. The use of epidural anesthesia should in the least be considered in cases in which relocation of the urethral stones is not possible by flushing. Postoperative radiographs is advisable in patients with radiodense calculi., Competing Interests: Die Autoren bestätigen, dass kein Interessenkonflikt besteht., (© Georg Thieme Verlag KG Stuttgart · New York.)

Background: In multiple sclerosis (MS) and in the experimental autoimmune encephalomyelitis (EAE) model of MS, the Nav1.6 voltage-gated sodium (Nav) channel isoform has been implicated as a primary contributor to axonal degeneration. Following demyelination Nav1.6, which is normally co-localized with the Na + /Ca 2+ exchanger (NCX) at the nodes of Ranvier, associates with β-APP, a marker of neural injury. The persistent influx of sodium through Nav1.6 is believed to reverse the function of NCX, resulting in an increased influx of damaging Ca 2+ ions. However, direct evidence for the role of Nav1.6 in axonal degeneration is lacking., Methods: In mice floxed for Scn8a, the gene that encodes the α subunit of Nav1.6, subjected to EAE we examined the effect of eliminating Nav1.6 from retinal ganglion cells (RGC) in one eye using an AAV vector harboring Cre and GFP, while using the contralateral either injected with AAV vector harboring GFP alone or non-targeted eye as control., Results: In retinas, the expression of Rbpms, a marker for retinal ganglion cells, was found to be inversely correlated to the expression of Scn8a. Furthermore, the gene expression of the pro-inflammatory cytokines Il6 (IL-6) and Ifng (IFN-γ), and of the reactive gliosis marker Gfap (GFAP) were found to be reduced in targeted retinas. Optic nerves from targeted eyes were shown to have reduced macrophage infiltration and improved axonal health., Conclusion: Taken together, our results are consistent with Nav1.6 promoting inflammation and contributing to axonal degeneration following demyelination.

Dendronized polymers (DPs) are large and compact main-chain linear polymers with a cylindrical shape and cross-sectional diameters of up to ∼15 nm. They are therefore considered molecular objects, and it was of interest whether given their experimentally accessible, well-defined dimensions, the density of individual DPs could be determined. We present measurements on individual, deposited DP chains, providing molecular dimensions from scanning and transmission electron microscopy and mass-per-length values from quantitative scanning transmission electron microscopy. These results are compared with density values obtained from small-angle X-ray scattering on annealed bulk specimen and with classical envelope density measurements, obtained using hydrostatic weighing or a density gradient column. The samples investigated comprise a series of DPs with side groups of dendritic generations g = 1-8. The key findings are a very large spread of the density values over all samples and methods, and a consistent increase of densities with g over all methods. While this work highlights the advantages and limitations of the applied methods, it does not provide a conclusive answer to the question of which method(s) to use for the determination of densities of individual molecular objects. We are nevertheless confident that these first attempts to answer this challenging question will stimulate more research into this important aspect of polymer and soft matter science.

Selecting particles from digital micrographs is an essential step in single-particle electron cryomicroscopy (cryo-EM). As manual selection of complete datasets-typically comprising thousands of particles-is a tedious and time-consuming process, numerous automatic particle pickers have been developed. However, non-ideal datasets pose a challenge to particle picking. Here we present the particle picking software crYOLO which is based on the deep-learning object detection system You Only Look Once (YOLO). After training the network with 200-2500 particles per dataset it automatically recognizes particles with high recall and precision while reaching a speed of up to five micrographs per second. Further, we present a general crYOLO network able to pick from previously unseen datasets, allowing for completely automated on-the-fly cryo-EM data preprocessing during data acquisition. crYOLO is available as a standalone program under http://sphire.mpg.de/ and is distributed as part of the image processing workflow in SPHIRE., Competing Interests: Competing interestsThe authors declare no competing interests.

Recent studies with B-cell-depleting antibodies have demonstrated clinical success in the treatment of multiple sclerosis (MS) patients. While these antibodies efficiently target B cells in the blood, it is unclear how effective they are in the central nervous system (CNS), especially in the context of limited blood-brain barrier (BBB) permeability and the ongoing discussion on the relevance of B-cell aggregate formation in the brains of SP-MS patients. The aim of this study was to evaluate BBB integrity in the context of B-cell-dependent neuroinflammation in a mouse model of MS. C57BL/6 mice were actively immunized with either myelin oligodendrocyte glycoprotein peptide 35-55 to induce T-cell-dependent experimental autoimmune encephalomyelitis (EAE), or with the myelin basic protein-proteolipid protein fusion protein MP4 for additional B-cell dependence. BBB integrity was assessed using Evans Blue or fluorescein isothiocyanate-dextran injection, respectively, in combination with immunofluorescence staining for key components of the BBB. In both EAE models, tracer leakage into the CNS parenchyma was observed indicating BBB leakiness. Yet, intensity and distribution patterns of leakage differed between the two models. There was no difference in the severity of BBB damage comparing acute and chronic MP4-induced EAE, but the formation of B-cell aggregates was associated with local BBB impairment in this model. This study underscores that a leaky BBB is a characteristic feature of EAE, but it also suggests that extent and region specificity of BBB damage differs between individual EAE models that vary in the underlying immunopathology.

Robust molecular tool kits in model and industrial microalgae are key to efficient targeted manipulation of endogenous and foreign genes in the nuclear genome for basic research and, as importantly, for the development of algal strains to produce renewable products such as biofuels. While Cas9-mediated gene knockout has been demonstrated in a small number of algal species with varying efficiency, the ability to stack traits or generate knockout mutations in two or more loci are often severely limited by selectable agent availability. This poses a critical hurdle in developing production strains, which require stacking of multiple traits, or in probing functionally redundant gene families. Here, we combine Cas9 genome editing with an inducible Cre recombinase in the industrial alga Nannochloropsis gaditana to generate a strain, NgCas9 + Cre + , in which the potentially unlimited stacking of knockouts and addition of new genes is readily achievable. Cre-mediated marker recycling is first demonstrated in the removal of the selectable marker and GFP reporter transgenes associated with the Cas9/Cre construct in NgCas9 + Cre + Next, we show the proof-of-concept generation of a markerless knockout in a gene encoding an acyl-CoA oxidase ( Aco1 ), as well as the markerless recapitulation of a 2-kb insert in the ZnCys gene 5'-UTR, which results in a doubling of wild-type lipid productivity. Finally, through an industrially oriented process, we generate mutants that exhibit up to ∼50% reduction in photosynthetic antennae size by markerless knockout of seven genes in the large light-harvesting complex gene family., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Mechanical ventilation is a life-saving clinical treatment but it can induce or aggravate lung injury. New therapeutic strategies, aimed at reducing the negative effects of mechanical ventilation such as excessive production of reactive oxygen species, release of pro-inflammatory cytokines, and transmigration as well as activation of neutrophil cells, are needed to improve the clinical outcome of ventilated patients. Though the inhaled anesthetic sevoflurane is known to exert organ-protective effects, little is known about the potential of sevoflurane therapy in ventilator-induced lung injury. This study focused on the effects of delayed sevoflurane application in mechanically ventilated C57BL/6N mice. Lung function, lung injury, oxidative stress, and inflammatory parameters were analyzed and compared between non-ventilated and ventilated groups with or without sevoflurane anesthesia. Mechanical ventilation led to a substantial induction of lung injury, reactive oxygen species production, pro-inflammatory cytokine release, and neutrophil influx. In contrast, sevoflurane posttreatment time dependently reduced histological signs of lung injury. Most interestingly, increased production of reactive oxygen species was clearly inhibited in all sevoflurane posttreatment groups. Likewise, the release of the pro-inflammatory cytokines interleukin-1β and MIP-1β and neutrophil transmigration were completely prevented by sevoflurane independent of the onset of sevoflurane administration. In conclusion, sevoflurane posttreatment time dependently limits lung injury, and oxidative and pro-inflammatory responses are clearly prevented by sevoflurane irrespective of the onset of posttreatment. These findings underline the therapeutic potential of sevoflurane treatment in ventilator-induced lung injury.

Macrophage filopodia, finger-like membrane protrusions, were first implicated in phagocytosis more than 100 years ago, but little is still known about the involvement of these actin-dependent structures in particle clearance. Using spinning disk confocal microscopy to image filopodial dynamics in mouse resident Lifeact-EGFP macrophages, we show that filopodia, or filopodia-like structures, support pathogen clearance by multiple means. Filopodia supported the phagocytic uptake of bacterial ( Escherichia coli ) particles by (i) capturing along the filopodial shaft and surfing toward the cell body, the most common mode of capture; (ii) capturing via the tip followed by retraction; (iii) combinations of surfing and retraction; or (iv) sweeping actions. In addition, filopodia supported the uptake of zymosan ( Saccharomyces cerevisiae ) particles by (i) providing fixation, (ii) capturing at the tip and filopodia-guided actin anterograde flow with phagocytic cup formation, and (iii) the rapid growth of new protrusions. To explore the role of filopodia-inducing Cdc42, we generated myeloid-restricted Cdc42 knock-out mice. Cdc42-deficient macrophages exhibited rapid phagocytic cup kinetics, but reduced particle clearance, which could be explained by the marked rounded-up morphology of these cells. Macrophages lacking Myo10, thought to act downstream of Cdc42, had normal morphology, motility, and phagocytic cup formation, but displayed markedly reduced filopodia formation. In conclusion, live-cell imaging revealed multiple mechanisms involving macrophage filopodia in particle capture and engulfment. Cdc42 is not critical for filopodia or phagocytic cup formation, but plays a key role in driving macrophage lamellipodial spreading., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)