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Abstract Background Studying habitat use and vertical movement patterns of individual fish over continuous time and space is innately challenging and has therefore largely remained elusive for a wide range of species. Amongst sharks, this applies particularly to smaller-bodied and less wide-ranging species such as the spurdog (Squalus acanthias Linnaeus, 1758), which, despite its importance for fisheries, has received limited attention in biologging and biotelemetry studies, particularly in the North-East Atlantic. Methods To investigate seasonal variations in fine-scale niche use and vertical movement patterns in female spurdog, we used archival data from 19 pregnant individuals that were satellite-tagged for up to 365 days in Norwegian fjords. We estimated the realised niche space with kernel densities and performed continuous wavelet analyses to identify dominant periods in vertical movement. Triaxial acceleration data were used to identify burst events and infer activity patterns. Results Pregnant females frequently utilised shallow depths down to 300 m at temperatures between 8 and 14 °C. Oscillatory vertical moments revealed persistent diel vertical migration (DVM) patterns, with descents at dawn and ascents at dusk. This strict normal DVM behaviour dominated in winter and spring and was associated with higher levels of activity bursts, while in summer and autumn sharks predominantly selected warm waters above the thermocline with only sporadic dive and bursts events. Conclusions The prevalence of normal DVM behaviour in winter months linked with elevated likely foraging-related activity bursts suggests this movement behaviour to be foraging-driven. With lower number of fast starts exhibited in warm waters during the summer and autumn months, habitat use in this season might be rather driven by behavioural thermoregulation, yet other factors may also play a role. Individual and cohort-related variations indicate a complex interplay of movement behaviour and habitat use with the abiotic and biotic environment. Together with ongoing work investigating fine-scale horizontal movement as well as sex- and age-specific differences, this study provides vital information to direct the spatio-temporal distribution of a newly reopened fishery and contributes to an elevated understanding of the movement ecology of spurdog in the North-East Atlantic and beyond. Graphical Abstract

Abstract Objective The COVID-19 pandemic has had significant health and socioeconomic impacts worldwide. Extensive measures, including contact restrictions, were implemented to control the spread of the virus. This study aims to examine the factors that influenced private and professional contact behaviour during the COVID-19 pandemic. Results We used baseline data (January–April 2021) from the SeMaCo study (Serologische Untersuchungen bei Blutspendern des Großraums Magdeburg auf Antikörper gegen SARS-CoV-2), a longitudinal, regional cohort study assessing COVID-19 seroprevalence in blood donors from Magdeburg and surrounding areas in Germany. In the blood donor cohort (n = 2,195), there was a general reduction in private contacts (by 78.9%) and professional contacts (by 54.4%) after March 18, 2020. Individuals with higher education reduced both private (by 84.1%) and professional (by 70.1%) contacts more than those with lower education levels (private contacts 59.5%; professional contacts 37%). Younger age groups (18–30 years) reduced private contacts more frequently (by 85.4%) than older individuals (61–83 years, by 68.6%) and demonstrated a higher likelihood of private contact reduction compared to older age groups (51–60 years: odds ratio (OR) 0.45 [95% [CI] 0.32–0.65]; 61–83 years: OR 0.33 [95% [CI] 0.22–0.48]).

Although belowground invasive species are probably equally widespread and as important as their aboveground counterparts, they remain understudied, and their impacts are likely to be stronger when these invaders act as ecosystem engineers and differ functionally from native species. This is the case in regions historically devoid of native earthworms, such as parts of northern North America, which are now experiencing an invasion by European earthworms. Although invasive earthworms have been reported to have multiple consequences for native communities and ecosystem functioning, this knowledge is mostly based on observational studies, and the mechanisms underlying their cascading impacts need to be investigated. Here, we thus investigated the sequence of events, i.e., ecological cascades following earthworm invasion, that have rarely been studied before, in a two-year field experiment. We expected that the changes in soil abiotic properties observed following invasion would coincide with changes in plant community diversity and community trait composition, as well as in alterations in above- and belowground ecosystem functions. To test these hypotheses, we set up a field experiment that ran for two years in a forest in Alberta (Canada) to investigate soil properties and understory plant community composition in response to invasive earthworms. Our study shows that invasive European earthworms alter several soil abiotic properties (i.e., soil nutrient content, and pH) after two years of experiment. Invasive earthworm effects varied with soil depth for some soil properties (i.e., soil pH, water-stable aggregates, nitrogen, and microbial basal respiration), but we did not find any significant earthworm effect on soil water content, bulk density, or the total soil microbial biomass independently of the soil layer. Moreover, invasive earthworms did not affect plant community composition and only slightly affected community diversity in this short-term experiment. The minor changes observed in plant functional group composition are thus potentially the first signs of invasive-earthworm effects on plant communities. Our research provides experimental evidence that previously reported observational effects of invasive earthworms on soil properties are indeed causal and already significant after two years of invasion. These changes in soil properties are likely to have cascading effects on plant community composition, functional diversity, and ecosystem functioning, but such effects may take longer than two years to materialize.

Gremlin-1 has been implicated in liver fibrosis in metabolic dysfunction-associated steatohepatitis (MASH) via inhibition of bone morphogenetic protein (BMP) signalling and has thereby been identified as a potential therapeutic target. Using rat in vivo and human in vitro and ex vivo model systems of MASH fibrosis, we show that neutralisation of Gremlin-1 activity with monoclonal therapeutic antibodies does not reduce liver inflammation or liver fibrosis. Still, Gremlin-1 was upregulated in human and rat MASH fibrosis, but expression was restricted to a small subpopulation of COL3A1/THY1+ myofibroblasts. Lentiviral overexpression of Gremlin-1 in LX-2 cells and primary hepatic stellate cells led to changes in BMP-related gene expression, which did not translate to increased fibrogenesis. Furthermore, we show that Gremlin-1 binds to heparin with high affinity, which prevents Gremlin-1 from entering systemic circulation, prohibiting Gremlin-1-mediated organ crosstalk. Overall, our findings suggest a redundant role for Gremlin-1 in the pathogenesis of liver fibrosis, which is unamenable to therapeutic targeting.

Summary: Neuroblastoma exhibits significant inter- and intra-tumor genetic heterogeneity and varying clinical outcomes. Extrachromosomal DNAs (ecDNAs) may drive this heterogeneity by independently segregating during cell division, leading to rapid oncogene amplification. While ecDNA-mediated oncogene amplification is linked to poor prognosis in various cancers, the effects of ecDNA copy-number heterogeneity on intermediate phenotypes are poorly understood. Here, we leverage DNA and RNA sequencing from the same single cells in cell lines and neuroblastoma patients to investigate these effects. By analyzing ecDNA amplicon structures, we reveal extensive intercellular ecDNA copy-number heterogeneity. We also provide direct evidence of how this heterogeneity influences the expression of cargo genes, including MYCN and its downstream targets, and the overall transcriptional state of neuroblastoma cells. Our findings highlight the role of ecDNA copy number in promoting rapid adaptability of cellular states within tumors, underscoring the need for ecDNA-specific treatment strategies to address tumor formation and adaptation.

Summary: Regulatory T cells (Tregs) suppress pro-inflammatory conventional T cell (Tconv) responses. As lipids impact cell signaling and function, we compare the lipid composition of CD4+ thymus-derived (t)Tregs and Tconvs. Lipidomics reveal constitutive enrichment of neutral lipids in Tconvs and phospholipids in tTregs. TNFR2-co-stimulated effector tTregs and Tconvs are both glycolytic, but only in tTregs are glycolysis and the tricarboxylic acid (TCA) cycle linked to a boost in fatty acid (FA) synthesis (FAS), supported by relevant gene expression. FA chains in tTregs are longer and more unsaturated than in Tconvs. In contrast to Tconvs, tTregs effectively use either lactate or glucose for FAS and rely on this process for proliferation. FASN and SCD1, enzymes responsible for FAS and FA desaturation, prove essential for the ability of tTregs to suppress Tconvs. These data illuminate how effector tTregs can thrive in inflamed or cancerous tissues with limiting glucose but abundant lactate levels.

Although the addition of buffers provides improved stability to flucloxacillin (FLU) solutions, unbuffered solutions are often preferred in clinical practice. The first purpose of this study was to investigate whether a 50 mg/mL solution of FLU in normal saline is stable for 24 h at 33 °C so that it can be applied for outpatient parenteral antimicrobial therapy (OPAT) using portable elastomeric infusion pumps (PEIPs). When the PEIPs were stored in an oven at 33 °C and deflated over 24 h, the volume of the collected solution, pH, and FLU concentration were checked every 4 h. Obtaining better results than expected based on the literature data, other storage conditions, such as refrigeration, room temperature (RT), 37 °C, refrigeration followed by 24 h at 33 °C and 37 °C, and different batches/brands, were also tested. This study confirmed the pronounced effect of temperature on the stability of FLU and also showed the relationship between the stability of FLU and the initial pH of the solution. FLU was quite stable at refrigeration and RT conditions, with more than 99% and 95% remaining. After 24 h at 33 °C, more than 92% of FLU was still present in the solution, while this number decreased to less than 85% when the storage temperature reached 37 °C. The remaining percentage was found to be even lower when the solution was stored at 2–8 °C for 6 days, followed by 24 h storage at 33 °C or 37 °C, with losses of 17% and 30%, respectively. The stability of FLU became worse when the initial pH of the solution was lower than 5.9 since the concentration of FLU dropped to less than 90% after 24 h at 33 °C, and a precipitate started to form when the initial pH of the solution was around 5.3. Therefore, FLU in PEIPs could be employed for 24 h if the temperature was ideally not more than 33 °C, while the pH should be not less than 5.9 upon reconstituting the FLU solution.

Abstract Many Norwegian rivers and lakes are regulated for hydropower, which affects freshwater ecosystems and anadromous fish species, such as sea trout (Salmo trutta). Lakes are an important feature of many anadromous river systems. However, there is limited knowledge on the importance of lakes as habitat for sea trout and how hydropower affects the behaviour of sea trout in lakes. To investigate this, we conducted an acoustic telemetry study. A total of 31 adult sea trout (532 ± 93 mm total length) were captured by angling in river Aurlandselva, Norway, and tagged between July 20 and August 12, 2021. The tags were instrumented with accelerometer, temperature, and depth sensors, which provided information on the sea trout’s presence and behaviour in lake Vassbygdevatnet. Our results indicate that there was a large prevalence of sea trout in the lake during the spawning migration, and that the sea trout were less active in the lake compared to the riverine habitats. An increase in activity of sea trout in the lake during autumn might indicate that sea trout spawn in the lake. However, the discharge from the high-head storage plant into the lake did not affect the depth use or activity of sea trout in the lake. Furthermore, the large prevalence of spawners in the lake during autumn will likely cause an underestimation of the size of the sea trout population in rivers with lakes during annual stock assessment. In conclusion, our results could not find evidence of a large impact of the discharge on the behaviour of sea trout in the lake.

Antisense technology demonstrates significant potential for addressing inherited brain diseases, with over a dozen products already available and numerous others in the development pipeline. The versatility of differentiating induced pluripotent stem cells (iPSCs) into nearly all neural cell types proves invaluable for comprehending the mechanisms behind neurological diseases, replicating cellular phenotypes, and advancing the testing and development of new therapies, including antisense oligonucleotide therapeutics. While delivering antisense oligonucleotides (ASOs) to human iPSC-based neuronal models has posed challenges, this study explores various delivery methods, including lipid-based transfection, gymnotic uptake, Ca(2+)-enhanced medium (CEM)-based delivery, and electroporation, in 2D and 3D hiPSC-derived neuronal models. This study reveals that CEM-based delivery exhibits efficiency and low toxicity in both 2D neuronal cultures and 3D brain organoids. Furthermore, the findings indicate that CEM is slightly more effective in neurons than in astrocytes, suggesting promising avenues for further exploration and optimization of preclinical ASO strategies in the treatment of neurological disorders.

T cell activation is critical for an effective immune response against pathogens. However, dysregulation contributes to the pathogenesis of autoimmune diseases, including Juvenile Idiopathic Arthritis (JIA). The molecular mechanisms underlying T cell activation are still incompletely understood. T cell activation promotes the acetylation of histone 3 at Lysine 27 (H3K27ac) at enhancer and promoter regions of proinflammatory cytokines, thereby increasing the expression of these genes which is essential for T cell function. Co-activators E1A binding protein P300 (P300) and CREB binding protein (CBP), collectively known as P300/CBP, are essential to facilitate H3K27 acetylation. Presently, the role of P300/CBP in human CD4+ T cells activation remains incompletely understood. To assess the function of P300/CBP in T cell activation and autoimmune disease, we utilized iCBP112, a selective inhibitor of P300/CBP, in T cells obtained from healthy controls and JIA patients. Treatment with iCBP112 suppressed T cell activation and cytokine signaling pathways, leading to reduced expression of many proinflammatory cytokines, including IL-2, IFN-γ, IL-4, and IL-17A. Moreover, P300/CBP inhibition in T cells derived from the inflamed synovium of JIA patients resulted in decreased expression of similar pathways and preferentially suppressed the expression of disease-associated genes. This study underscores the regulatory role of P300/CBP in regulating gene expression during T cell activation while offering potential insights into the pathogenesis of autoimmune diseases. Our findings indicate that P300/CBP inhibition could potentially be leveraged for the treatment of autoimmune diseases such as JIA in the future.

Abstract There has recently been great interest in the use of accelerometers onboard electronic transmitters to characterise various aspects of the ecology of wild animals. We review use cases and outline how these tools can provide opportunities for studying activity and survival, exercise physiology of wild animals, the response to stressors, energy landscapes and conservation planning tools, and the means with which to identify behaviours remotely from transmitted data. Accelerometer transmitters typically send data summaries to receivers at fixed intervals after filtering out static acceleration and calculating root-mean square error or overall dynamic body action of 2- or 3-axis acceleration values (often at 5–12.5 Hz) from dynamic acceleration onboard the tag. Despite the popularity of these transmitters among aquatic ecologists, we note that there is wide variation in the sampling frequencies and windows used among studies that will potentially affect the ability to make comparisons in the future. Accelerometer transmitters will likely become increasingly popular tools for studying finer scale details about cryptic species that are difficult to recapture and hence not suitable for studies using data loggers. We anticipate that there will continue to be opportunities to adopt methods used for analysing data from loggers to datasets generated from acceleration transmitters, to generate new knowledge about the ecology of aquatic animals.

Biological pest control is successful in many agricultural systems, yet various pests can still not be controlled with natural enemies. Examples are invasive thrips species such as Echinothrips americanus Morgan and Thrips parvispinus (Karny) in Europe. The lack of biological control of these pests in several crops has been subscribed to their complex life cycle and cryptic behaviours, but their antipredator behaviours may prevent successful control and have not been studied extensively. One of these behaviours is the potential of thrips to kill the eggs of predatory mites, which we quantified. Furthermore, we observed the behaviour of first- and second-instar thrips larvae when attacked by starved predatory mites Amblyseius swirskii Athias-Henriot. For comparison, we included Frankliniella occidentalis (Pergande), which shows an array of defensive behaviours, but can be controlled successfully by several species of predatory mites. Second-instar larvae and adults of F. occidentalis and T. parvispinus killed predator eggs, but E. americanus did not. First- and second-instar larvae of all three species swung their abdomen to defend themselves, and F. occidentalis and T. parvispinus produced anal droplets, which are also involved in defence. Predators were not successful in attacking any second-instar larvae during 15-minute observations, which is sufficiently long for an attack. First-instar larvae of F. occidentalis and T. parvispinus were sometimes successfully attacked by the predatory mite before they could mount a defence, but first-instar E. americanus always defended themselves and were almost never preyed upon. Cooling thrips larvae to suppress antipredator behaviour increased predation for all species and stages, except for first instars of F. occidentalis. In conclusion, the lack of successful control of E. americanus is not caused by killing predator eggs, but by the efficient defensive behaviour of both larval instars. Our study furthermore suggests that T. parvispinus can potentially be controlled by A. swirskii.

Abstract Background A well-known method for evaluating plant resistance to insects is by measuring insect reproduction or oviposition. Whiteflies are vectors of economically important viral diseases and are, therefore, widely studied. In a common experiment, whiteflies are placed on plants using clip-on-cages, where they can lay hundreds of eggs on susceptible plants in a few days. When quantifying whitefly eggs, most researchers perform manual eye measurements using a stereomicroscope. Compared to other insect eggs, whitefly eggs are many and very tiny, usually 0.2 mm in length and 0.08 mm in width; therefore, this process takes a lot of time and effort with and without prior expert knowledge. Plant insect resistance experiments require multiple replicates from different plant accessions; therefore, an automated and rapid method for quantifying insect eggs can save time and human resources. Results In this work, a novel automated tool for fast quantification of whitefly eggs is presented to accelerate the determination of plant insect resistance and susceptibility. Leaf images with whitefly eggs were collected from a commercial microscope and a custom-built imaging system. A deep learning-based object detection model was trained using the collected images. The model was incorporated into an automated whitefly egg quantification algorithm, deployed in a web-based application called Eggsplorer. Upon evaluation on a testing dataset, the algorithm was able to achieve a counting accuracy as high as 0.94, r 2 of 0.99, and a counting error of ± 3 eggs relative to the actual number of eggs counted by eye. The automatically collected counting results were used to determine the resistance and susceptibility of several plant accessions and were found to yield significantly comparable results as when using the manually collected counts for analysis. Conclusion This is the first work that presents a comprehensive step-by-step method for fast determination of plant insect resistance and susceptibility with the assistance of an automated quantification tool.

Before the emergence of SARS-CoV-1, MERS-CoV, and most recently, SARS-CoV-2, four other coronaviruses (the alpha coronaviruses NL63 and 229E and the beta coronaviruses OC43 and HKU1) had already been circulating in the human population. These circulating coronaviruses all cause mild respiratory illness during the winter seasons, and most people are already infected in early life. Could antibodies and/or T cells, especially against the beta coronaviruses, have offered some form of protection against (severe) COVID-19 caused by infection with SARS-CoV-2? Related is the question of whether survivors of SARS-CoV-1 or MERS-CoV would be relatively protected against SARS-CoV-2. More importantly, would humoral and cellular immunological memory generated during the SARS-CoV-2 pandemic, either by infection or vaccination, offer protection against future coronaviruses? Or rather than protection, could antibody-dependent enhancement have taken place, a mechanism by which circulating corona antibodies enhance the severity of COVID-19? Another related phenomenon, the original antigenic sin, would also predict that the effectiveness of the immune response to future coronaviruses would be impaired because of the reactivation of memory against irrelevant epitopes. The currently available evidence indicates that latter scenarios are highly unlikely and that especially cytotoxic memory T cells directed against conserved epitopes of human coronaviruses could at least offer partial protection against future coronaviruses.

Aerococcus urinae is an aerobic Gram-positive coccus that grows as tiny alpha-hemolytic colonies. Actinotignum schaalii is a slow-growing facultative anaerobic Gram-positive rod. These bacteria are part of the urogenital microbiota of healthy patients, but can also be involved in urinary tract infections (UTIs), particularly in elderly men and young children. Because A. urinae and A. schaalii are fastidious and are difficult to identify with phenotypic methods, they are underestimated causes of UTIs. Their growth is slow and requires a blood-enriched medium incubated under an anaerobic or 5% CO2 atmosphere for 48 h and from 24 to 48 h for A. schaalii and A. urinae, respectively. Furthermore, accurate identification is only possible using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) or molecular-based methods. In rare cases, these bacteria can be responsible for invasive infections. We describe, here, an unusual case of bacteremic UTI caused by both A. schaalii and A. urinae in an 89-year-old woman. She presented with dyspnea, and bacteriuria was noted. This challenging clinical and microbiological diagnosis was made in our laboratory by Gram staining urine with a leucocyte count >50/μL and/or a bacterial count >14/μL urinary culture on a blood agar plate. After 10 days of antimicrobial treatment consisting of 2 g amoxicillin PO t.i.d., the patient was discharged with a complete clinical and biological recovery. A. schaalii and A. urinae are probably still underestimated causes of UTIs. Microbiologists could consider the presence of these two bacteria using appropriate culture and identification methods in cases where a positive direct examination of urine reveals small Gram-positive rods or cocci, where undocumented UTIs are present in elderly patients, but also where a urinary dipstick is negative for nitrites and is associated with leukocyturia.

Plants with innate disease and pest resistance can contribute to more sustainable agriculture. Natural defence compounds produced by plants have the potential to provide a general protective effect against pathogens and pests, but they are not a primary target in resistance breeding. Here, we identified a wild relative of potato, Solanum commersonii, that provides us with unique insight in the role of glycoalkaloids in plant immunity. We cloned two atypical resistance genes that provide resistance to Alternaria solani and Colorado potato beetle through the production of tetraose steroidal glycoalkaloids (SGA). Moreover, we provide in vitro evidence to show that these compounds have potential against a range of different (potato pathogenic) fungi. This research links structural variation in SGAs to resistance against potato diseases and pests. Further research on the biosynthesis of plant defence compounds in different tissues, their toxicity, and the mechanisms for detoxification, can aid the effective use of such compounds to improve sustainability of our food production.

An assessment of all commercially available antibodies targeting the G-protein-coupled receptor (GPCR), glucagon receptor (GCGR) is used to highlight the most reliable antibody in immunostaining, including an antibody-independent verification.

Nanodiamonds are increasingly popular in biomedical applications, including optical labelling, drug delivery and nanoscale sensing. Potential new applications are in studying infertility or labelling sperm cells. However, for these applications, it is necessary that nanodiamonds are inert and do not alter sperm properties. In this article, we assessed the biocompatibility of nanodiamonds in detail. We investigated different sizes and concentrations of nanodiamonds and sperm preparation methods. We evaluated if the metabolic activity, membrane integrity, morphology and formation of reactive oxygen species were altered. These parameters were tested for sperm cells in their uncapacitated and capacitated states. Unfortunately, FNDs are not universally biocompatible. Generally, cells in the capacitated state are more prone to stress. Additionally, larger particles and lower concentrations are tolerated better than smaller and higher concentrated particles.

Summary: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had a tremendous impact worldwide. Mapping virus-host interactions is critical to understand disease progression. MicroRNAs (miRNAs) are important RNA regulators, but their interaction with SARS-CoV-2 RNA was not experimentally investigated. Here, using Argonaute (AGO) cross-linking immunoprecipitation combined with RNA proximity ligation (CLEAR-CLIP), we provide unbiased mapping of SARS-CoV-2/miRNA interactions. We identified six main regions on the viral RNA bound primarily by one specific miRNA. Targeted mutagenesis and AGO1-3 knockdown demonstrated that these interactions are not critical for virus production. Moreover, we identified perturbed regulation of cellular miRNA interactions during infection, including non-compensated viral sequestration of the miR-15 family. Transcriptome analysis further showed that mRNAs targeted by this miRNA family are derepressed. This work delineates the interphase between miRNA regulation and SARS-CoV-2 infection and further contributes to deciphering the full molecular interactome of this virus.

Marfan syndrome (MFS) is a connective tissue disorder with pleiotropic manifestations in the ocular, skeletal and cardiovascular system; and is typically cause by pathogenic variants in the fibrillin-1 (FBN1) gene. We report a generated induced pluripotent cell (iPSC) line of a MFS patient with an FBN1 c.7754T > C (p.Ile2585Thr) variant. The cell line was generated from peripheral blood mononuclear cells (PBMCs) and after reprogramming the line showed a no relevant copy number alterations, expression of pluripotency markers and was able to differentiate into three germ layers while carrying the original genotype.

Establishment and maintenance of the primary cilium as a signaling-competent organelle requires a high degree of fine tuning, which is at least in part achieved by a variety of post-translational modifications. One such modification is ubiquitination. The small and highly conserved ubiquitin protein possesses a unique versatility in regulating protein function via its ability to build mono and polyubiquitin chains onto target proteins. We aimed to take an unbiased approach to generate a comprehensive blueprint of the ciliary ubiquitinome by deploying a multi-proteomics approach using both ciliary-targeted ubiquitin affinity proteomics, as well as ubiquitin-binding domain-based proximity labelling in two different mammalian cell lines. This resulted in the identification of several key proteins involved in signaling, cytoskeletal remodeling and membrane and protein trafficking. Interestingly, using two different approaches in IMCD3 and RPE1 cells, respectively, we uncovered several novel mechanisms that regulate cilia function. In our IMCD3 proximity labeling cell line model, we found a highly enriched group of ESCRT-dependent clathrin-mediated endocytosis-related proteins, suggesting an important and novel role for this pathway in the regulation of ciliary homeostasis and function. In contrast, in RPE1 cells we found that several structural components of caveolae (CAV1, CAVIN1, and EHD2) were highly enriched in our cilia affinity proteomics screen. Consistently, the presence of caveolae at the ciliary pocket and ubiquitination of CAV1 specifically, were found likely to play a role in the regulation of ciliary length in these cells. Cilia length measurements demonstrated increased ciliary length in RPE1 cells stably expressing a ubiquitination impaired CAV1 mutant protein. Furthermore, live cell imaging in the same cells revealed decreased CAV1 protein turnover at the cilium as the possible cause for this phenotype. In conclusion, we have generated a comprehensive list of cilia-specific proteins that are subject to regulation via ubiquitination which can serve to further our understanding of cilia biology in health and disease.

Abstract Background Precise gene dosage of the X chromosomes is critical for normal development and cellular function. In mice, XX female somatic cells show transcriptional X chromosome upregulation of their single active X chromosome, while the other X chromosome is inactive. Moreover, the inactive X chromosome is reactivated during development in the inner cell mass and in germ cells through X chromosome reactivation, which can be studied in vitro by reprogramming of somatic cells to pluripotency. How chromatin processes and gene regulatory networks evolved to regulate X chromosome dosage in the somatic state and during X chromosome reactivation remains unclear. Results Using genome-wide approaches, allele-specific ATAC-seq and single-cell RNA-seq, in female embryonic fibroblasts and during reprogramming to pluripotency, we show that chromatin accessibility on the upregulated mammalian active X chromosome is increased compared to autosomes. We further show that increased accessibility on the active X chromosome is erased by reprogramming, accompanied by erasure of transcriptional X chromosome upregulation and the loss of increased transcriptional burst frequency. In addition, we characterize gene regulatory networks during reprogramming and X chromosome reactivation, revealing changes in regulatory states. Our data show that ZFP42/REX1, a pluripotency-associated gene that evolved specifically in placental mammals, targets multiple X-linked genes, suggesting an evolutionary link between ZFP42/REX1, X chromosome reactivation, and pluripotency. Conclusions Our data reveal the existence of intrinsic compensatory mechanisms that involve modulation of chromatin accessibility to counteract X-to-Autosome gene dosage imbalances caused by evolutionary or in vitro X chromosome loss and X chromosome inactivation in mammalian cells.

The neuronal microtubule cytoskeleton is key to establish axon-dendrite polarity. Dendrites are characterized by the presence of minus-end out microtubules. However, the mechanisms that organize these microtubules with the correct orientation are still poorly understood. Using Caenorhabditis elegans as a model system for microtubule organization, we characterized the role of 2 microtubule minus-end related proteins in this process, the microtubule minus-end stabilizing protein calmodulin-regulated spectrin-associated protein (CAMSAP/PTRN-1), and the NINEIN homologue, NOCA-2 (noncentrosomal microtubule array). We found that CAMSAP and NINEIN function in parallel to mediate microtubule organization in dendrites. During dendrite outgrowth, RAB-11-positive vesicles localized to the dendrite tip to nucleate microtubules and function as a microtubule organizing center (MTOC). In the absence of either CAMSAP or NINEIN, we observed a low penetrance MTOC vesicles mislocalization to the cell body, and a nearly fully penetrant phenotype in double mutant animals. This suggests that both proteins are important for localizing the MTOC vesicles to the growing dendrite tip to organize microtubules minus-end out. Whereas NINEIN localizes to the MTOC vesicles where it is important for the recruitment of the microtubule nucleator γ-tubulin, CAMSAP localizes around the MTOC vesicles and is cotranslocated forward with the MTOC vesicles upon dendritic growth. Together, these results indicate that microtubule nucleation from the MTOC vesicles and microtubule stabilization are both important to localize the MTOC vesicles distally to organize dendritic microtubules minus-end out.

During renewal of the intestine, cells are continuously generated by proliferation. Proliferation and differentiation must be tightly balanced, as any bias toward proliferation results in uncontrolled exponential growth. Yet, the inherently stochastic nature of cells raises the question how such fluctuations are limited. We used time-lapse microscopy to track all cells in crypts of growing mouse intestinal organoids for multiple generations, allowing full reconstruction of the underlying lineage dynamics in space and time. Proliferative behavior was highly symmetric between sister cells, with both sisters either jointly ceasing or continuing proliferation. Simulations revealed that such symmetric proliferative behavior minimizes cell number fluctuations, explaining our observation that proliferating cell number remained constant even as crypts increased in size considerably. Proliferative symmetry did not reflect positional symmetry but rather lineage control through the mother cell. Our results indicate a concrete mechanism to balance proliferation and differentiation with minimal fluctuations that may be broadly relevant for other tissues.

Stable grasping is essential for assistive robots aiding individuals with severe motor–sensory disabilities in their everyday lives. Slip detection can prevent unstably grasped objects from falling out of the gripper and causing accidents. Recent research on slip detection focuses on tactile sensing; however, not every robot arm can be equipped with such sensors. In this paper, we propose a slip detection method solely based on data collected by a RealSense D435 Red Green Blue-Depth (RGBd) camera. By utilizing Farneback optical flow (OF) to estimate the motion field of the grasped object relative to the gripper, while also removing potential background noise, the algorithm can perform in a multitude of environments. The algorithm was evaluated on a dataset of 28 daily objects that were lifted 30 times each, resulting in a total of 840 frame sequences. Our proposed slip detection method achieves an accuracy of up to 82.38% and a recall of up to 87.14%, which is comparable to state-of-the-art approaches when only using camera data. When excluding objects for which movements are challenging for vision-based methods to detect, such as untextured or transparent objects, the proposed method performs even better, with an accuracy of up to 87.19% and a recall of up to 95.09%.

Osteopontin (OPN) is a bioactive integrin-binding protein found in high concentrations in milk, where it is present both as a full-length protein and as several N-terminally derived fragments. OPN resists gastric digestion, and via interaction with receptors in the gut or by crossing the intestinal barrier into circulation, ingested milk OPN may influence physiological processes. The aim of this study was to investigate OPN interaction with intestinal cells and its transport across models of the intestinal barrier. Immunodetection of OPN incubated with Caco-2 cells at 4 °C and 37 °C showed that OPN binds to the intestinal cells, but it is not internalised. Transepithelial transport was studied using mono- and co-cultures of Caco-2 cells and mucus-producing HT29-MTX cells in transwell membranes. OPN was shown to cross the barrier models in a time-, temperature-, and energy-dependent process inhibited by wortmannin, indicating that the transport takes place via the transcytosis pathway. Analyses of the naturally occurring milk mixture of full-length and N-terminal fragments showed that the N-terminal fragments of OPN bound intestinal cells most effectively and that the fragments were transported across the intestinal membrane models. This suggests that proteolytic processing of OPN increases its biological activity after ingestion.

Polyploid cells contain more than 2 copies of the genome and are found in many plant and animal tissues. Different types of polyploidy exist, in which the genome is confined to either 1 nucleus (mononucleation) or 2 or more nuclei (multinucleation). Despite the widespread occurrence of polyploidy, the functional significance of different types of polyploidy is largely unknown. Here, we assess the function of multinucleation in Caenorhabditis elegans intestinal cells through specific inhibition of binucleation without altering genome ploidy. Through single-worm RNA sequencing, we find that binucleation is important for tissue-specific gene expression, most prominently for genes that show a rapid up-regulation at the transition from larval development to adulthood. Regulated genes include vitellogenins, which encode yolk proteins that facilitate nutrient transport to the germline. We find that reduced expression of vitellogenins in mononucleated intestinal cells leads to progeny with developmental delays and reduced fitness. Together, our results show that binucleation facilitates rapid up-regulation of intestine-specific gene expression during development, independently of genome ploidy, underscoring the importance of spatial genome organization for polyploid cell function. Why do some cells contain more than one nucleus? By comparing mononucleated and multinucleated polyploid cells in C. elegans, this study shows that having multiple nuclei is important for optimal transcriptional upregulation of developmentally controlled genes.

The centrosome is the main microtubule-organizing center of animal cells, and is composed of two barrel-shaped microtubule-based centrioles embedded in protein dense pericentriolar material. Compositional and architectural re-organization of the centrosome drives its duplication, and enables its microtubule-organizing activity and capability to form the primary cilium, which extends from the mature (mother) centriole, as the cell exits the cell cycle. Centrosomes and primary cilia are essential to human health, signified by the causal role of centrosome- and cilia-aberrations in numerous congenic disorders, as well as in the etiology and progression of cancer. The list of disease-associated centrosomal proteins and their proximitomes is steadily expanding, emphasizing the need for high resolution mapping of such proteins to specific substructures of the organelle. Here, we provide a detailed 3D-structured illumination microscopy (3D-SIM) protocol for comparative localization analysis of fluorescently labeled proteins at the centrosome in fixed human cell lines, at approximately 120 nm lateral and 300 nm axial resolution. The procedure was optimized to work with primary antibodies previously known to depend on more disruptive fixation reagents, yet largely preserves centriole and centrosome architecture, as shown by transposing acquired images of landmark proteins on previously published transmission electron microscopy (TEM) images of centrosomes. Even more advantageously, it is compatible with fluorescent protein tags. Finally, we introduce an internal reference to ensure correct 3D channel alignment. This protocol hence enables flexible, swift, and information-rich localization and interdependence analyses of centrosomal proteins, as well as their disorder-associated mutations.

Abstract Background Many cancers arise from mutations in cells within epithelial tissues. Mutations manifesting at the subcellular level influence the structure and function of the tissue resulting in cancer. Previous work has proposed how cell level properties can lead to mutant cell invasion, but has not incorporated detailed subcellular modelling Results We present a framework that allows the straightforward integration and simulation of SBML representations of subcellular dynamics within multiscale models of epithelial tissues. This allows us to investigate the effect of mutations in subcellular pathways on the migration of cells within the colorectal crypt. Using multiple models we find that mutations in APC, a key component in the Wnt signalling pathway, can bias neutral drift and can also cause downward invasion of mutant cells in the crypt. Conclusions Our framework allows us to investigate how subcellular mutations, i.e. knockouts and knockdowns, affect cell-level properties and the resultant migration of cells within epithelial tissues. In the context of the colorectal crypt, we see that mutations in APC can lead directly to mutant cell invasion.

CEP78 is a centrosomal protein implicated in ciliogenesis and ciliary length control, and mutations in the CEP78 gene cause retinal cone-rod dystrophy associated with hearing loss. However, the mechanism by which CEP78 affects cilia formation is unknown. Based on a recently discovered disease-causing CEP78 p.L150S mutation, we identified the disease-relevant interactome of CEP78. We confirmed that CEP78 interacts with the EDD1-DYRK2-DDB1VPRBP E3 ubiquitin ligase complex, which is involved in CP110 ubiquitination and degradation, and identified a novel interaction between CEP78 and CEP350 that is weakened by the CEP78L150S mutation. We show that CEP350 promotes centrosomal recruitment and stability of CEP78, which in turn leads to centrosomal recruitment of EDD1. Consistently, cells lacking CEP78 display significantly increased cellular and centrosomal levels of CP110, and depletion of CP110 in CEP78-deficient cells restored ciliation frequency to normal. We propose that CEP78 functions downstream of CEP350 to promote ciliogenesis by negatively regulating CP110 levels via an EDD1-dependent mechanism.

Myasthenia Gravis (MG) is characterised by muscle weakness and increased fatigability. The aim of this pilot study was to investigate if patients with MG demonstrate different functional chewing patterns and report more complaints related to mastication as compared with healthy controls. Twelve patients (median 60 years Q1–Q3: 46–70) with generalised MG and nine healthy controls (median 57 years Q1–Q3: 55–63) participated. All participants underwent dental and oral examination and were asked to fill in a questionnaire concerning oral health. Static maximum bite force was measured with a bite force transducer, electromyography in the masseter, temporalis, and suprahyoid muscles were recorded, and jaw movement was tracked, during a 5-minute gum chewing test. The patients had more oral complaints (oral health impact profile total score 22.6 vs 7.5 P

Enterobacterales bloodstream infections are life-threatening and require rapid, targeted antibiotherapy based on antibiotic susceptibility testing (AST). A new method using Muller-Hinton Rapid-SIR (MHR-SIR) agar (i2a, Montpellier, France) allows complete direct AST (dAST) to be read from positive blood culture bottles (BCBs) for all Enterobacterales species after 6–8 h of incubation. We evaluated (i) the performance of dAST from positive BCBs on MHR-SIR agar using two different inoculum protocols; (ii) the categorical agreement between dAST results obtained with MHR-SIR agar vs. those obtained with Muller-Hinton (MH) agar; and (iii) the ability of the MHR-SIR medium to detect β-lactam resistant Enterobacterales. Finally, we estimated the saved turnaround time (TAT) with MHR-SIR compared with MH agar in our 24/7 laboratory. Our results showed that the most suitable inoculation protocol for dAST on MHR-SIR agar was 1 drop of BCB/5 mL H2O. For monomicrobial Enterobacterales BCBs, dAST performed on MHR-SIR medium showed 99.3% categorical agreement with AST on MH agar. Furthermore, MHR-SIR agar allows early detection of β-lactam resistance mechanisms, including AmpC hyperproduction, extended-spectrum β-lactamase, and carbapenemase. Finally, TAT reduction in our 24/7 laboratory was 16 h, enabling a significantly faster provision of antibiotic advice.

Spontaneous bacterial peritonitis (SBP) is a severe infection that requires fast and accurate antibiotic therapy to improve the patient outcome. Direct bacterial identification using MALDI-TOF mass spectrometry from ascitic fluid inoculated in blood culture bottles (BCBs) could therefore improve patients’ management. We evaluated the impact of the implementation of this method for the treatment of patients. Our identification protocol was performed on 136 positive BCBs collected from 61 patients between December 2018 and December 2020. The therapeutic impact of our protocol was evaluated using a before (2015–2016) and after (2019–2020) case–control study in two populations of 41 patients diagnosed with SBP and treated with antibiotics. The decrease in time to first identification and the optimization of antibiotic therapy following communication of the identification result were evaluated. Our protocol allowed us to identify 78% of bacteria in ascitic fluids. The transmission of the direct identification allowed the introduction or adaption of the antibiotic therapy early in 37% of SBP, with a mean decrease in time to first antibiotic change of 17 h. Our direct identification protocol for positive inoculated ascitic fluids is fast, reliable and inexpensive. Its routine integration into a microbiology laboratory allows the early introduction of appropriate antibiotic therapy and improves the management of patients with SBP.

Cell division site positioning is precisely regulated but the underlying mechanisms are incompletely understood. In the social bacterium Myxococcus xanthus, the ~15 MDa tripartite PomX/Y/Z complex associates with and translocates across the nucleoid in a PomZ ATPase-dependent manner to directly position and stimulate formation of the cytokinetic FtsZ-ring at midcell, and then undergoes fission during division. Here, we demonstrate that PomX consists of two functionally distinct domains and has three functions. The N-terminal domain stimulates ATPase activity of the ParA/MinD ATPase PomZ. The C-terminal domain interacts with PomY and forms polymers, which serve as a scaffold for PomX/Y/Z complex formation. Moreover, the PomX/PomZ interaction is important for fission of the PomX/Y/Z complex. These observations together with previous work support that the architecturally diverse ATPase activating proteins of ParA/MinD ATPases are highly modular and use the same mechanism to activate their cognate ATPase via a short positively charged N-terminal extension.

Lymphocyte numbers need to be quite tightly regulated. It is generally assumed that lymphocyte production and lifespan increase homeostatically when lymphocyte numbers are low and, vice versa, return to normal once cell numbers have normalized. This widely accepted concept is largely based on experiments in mice, but is hardly investigated in vivo in humans. Here we quantified lymphocyte production and loss rates in vivo in patients 0.5–1 year after their autologous hematopoietic stem cell transplantation (autoHSCT). We indeed found that the production rates of most T- and B-cell subsets in autoHSCT-patients were two to eight times higher than in healthy controls, but went hand in hand with a threefold to ninefold increase in cell loss rates. Both rates also did not normalize when cell numbers did. This shows that increased lymphocyte production and loss rates occur even long after autoHSCT and can persist in the face of apparently normal cell numbers.

The mechanisms and design principles of regulatory systems establishing stable polarized protein patterns within cells are well studied. However, cells can also dynamically control their cell polarity. Here, we ask how an upstream signaling system can switch the orientation of a polarized pattern. We use a mathematical model of a core polarity system based on three proteins as the basis to study different mechanisms of signal-induced polarity switching. The analysis of this model reveals four general classes of switching mechanisms with qualitatively distinct behaviors: the transient oscillator switch, the reset switch, the prime-release switch, and the push switch. Each of these regulatory mechanisms effectively implements the function of a spatial toggle switch, however with different characteristics in their nonlinear and stochastic dynamics. We identify these characteristics and also discuss experimental signatures of each type of switching mechanism.

Circular RNAs (circRNAs) represent an abundant and conserved entity of non-coding RNAs; however, the principles of biogenesis are currently not fully understood. Here, we identify two factors, splicing factor proline/glutamine rich (SFPQ) and non-POU domain-containing octamer-binding protein (NONO), to be enriched around circRNA loci. We observe a subclass of circRNAs, coined DALI circRNAs, with distal inverted Alu elements and long flanking introns to be highly deregulated upon SFPQ knockdown. Moreover, SFPQ depletion leads to increased intron retention with concomitant induction of cryptic splicing, premature transcription termination, and polyadenylation, particularly prevalent for long introns. Aberrant splicing in the upstream and downstream regions of circRNA producing exons are critical for shaping the circRNAome, and specifically, we identify missplicing in the immediate upstream region to be a conserved driver of circRNA biogenesis. Collectively, our data show that SFPQ plays an important role in maintaining intron integrity by ensuring accurate splicing of long introns, and disclose novel features governing Alu-independent circRNA production.

Summary: E2F transcription factors control the expression of cell-cycle genes. Cancers often demonstrate enhanced E2F target gene expression, which can be explained by increased percentages of replicating cells. However, we demonstrate in human cancer biopsy specimens that individual neoplastic cells display abnormally high levels of E2F-dependent transcription. To mimic this situation, we delete the atypical E2F repressors (E2F7/8) or overexpress the E2F3 activator in untransformed cells. Cells with elevated E2F activity during S/G2 phase fail to exit the cell cycle after DNA damage and undergo mitosis. In contrast, wild-type cells complete S phase and then exit the cell cycle by activating the APC/CCdh1 via repression of the E2F target Emi1. Many arrested wild-type cells eventually inactivate APC/CCdh1 to execute a second round of DNA replication and mitosis, thereby becoming tetraploid. Cells with elevated E2F transcription fail to exit the cell cycle after DNA damage, which potentially causes genomic instability, promotes malignant progression, and reduces drug sensitivity.

Mast cells (MCs) are distributed in tissues throughout the body and are highly involved in many physiological and pathophysiological processes. The potential and involvement of different MC phenotypes are still not well understood. MCs are present in blood vessel walls, but their specific phenotypic features are unknown. We aimed at characterizing MCs from human saphenous veins for localization, mediator content, and receptor expression. This was done in MCs from both healthy and varicose human saphenous veins (hSV and vSV, respectively). For both vSV and hSV, we found that vein MCs are mainly present in the tunica adventitia (99% MCs in adventitia) and that the population consists of both MCT and MCTC phenotypes (vSV: 55% MCT, hSV: 64% MCT). The vein MCs contained high levels of histamine (vSV: 27 pg/MC, hSV: 55 pg/MC) and tryptase (vSV: 98 pg/MC, hSV: 111 pg/MC), indicating a strong potential for regulatory effects on blood vessels. The receptor expression of FcεRI, MRGPRX2, PTAFR, C3aR, and C5aR was found, even though the percentage of positive cells differed between vSV and hSV MCs. We conclude that vein MCs from the blood vessel wall have a high potential to affect the tissue around them.

Human cells lacking RIF1 are highly sensitive to replication inhibitors, but the reasons for this sensitivity have been enigmatic. Here, we show that RIF1 must be present both during replication stress and in the ensuing recovery period to promote cell survival. Of two isoforms produced by alternative splicing, we find that RIF1-Long alone can protect cells against replication inhibition, but RIF1-Short is incapable of mediating protection. Consistent with this isoform-specific role, RIF1-Long is required to promote the formation of the 53BP1 nuclear bodies that protect unrepaired damage sites in the G1 phase following replication stress. Overall, our observations show that RIF1 is needed at several cell cycle stages after replication insult, with the RIF1-Long isoform playing a specific role during the ensuing G1 phase in damage site protection.

Intracellular transport relies on multiple kinesins, but it is poorly understood which kinesins are present on particular cargos, what their contributions are and whether they act simultaneously on the same cargo. Here, we show that Rab6-positive secretory vesicles are transported from the Golgi apparatus to the cell periphery by kinesin-1 KIF5B and kinesin-3 KIF13B, which determine the location of secretion events. KIF5B plays a dominant role, whereas KIF13B helps Rab6 vesicles to reach freshly polymerized microtubule ends, to which KIF5B binds poorly, likely because its cofactors, MAP7-family proteins, are slow in populating these ends. Sub-pixel localization demonstrated that during microtubule plus-end directed transport, both kinesins localize to the vesicle front and can be engaged on the same vesicle. When vesicles reverse direction, KIF13B relocates to the middle of the vesicle, while KIF5B shifts to the back, suggesting that KIF5B but not KIF13B undergoes a tug-of-war with a minus-end directed motor.

The differentiation of neuronal stem cells into polarized neurons is a well-coordinated process which has mostly been studied in classical non-human model systems, but to what extent these findings are recapitulated in human neurons remains unclear. To study neuronal polarization in human neurons, we cultured hiPSC-derived neurons, characterized early developmental stages, measured electrophysiological responses, and systematically profiled transcriptomic and proteomic dynamics during these steps. The neuron transcriptome and proteome shows extensive remodeling, with differential expression profiles of ~1100 transcripts and ~2200 proteins during neuronal differentiation and polarization. We also identified a distinct axon developmental stage marked by the relocation of axon initial segment proteins and increased microtubule remodeling from the distal (stage 3a) to the proximal (stage 3b) axon. This developmental transition coincides with action potential maturation. Our comprehensive characterization and quantitative map of transcriptome and proteome dynamics provides a solid framework for studying polarization in human neurons.

Mounting evidence indicates that the presence of cardiovascular disease (CVD) and risk factors elevates the incidence of cognitive impairment (CI) and dementia. CVD and associated decline in cardiovascular function can impair cerebral blood flow (CBF) regulation, leading to the disruption of oxygen and nutrient supply in the brain where limited intracellular energy storage capacity critically depends on CBF to sustain proper neuronal functioning. During hypertension and acute as well as chronic CVD, cerebral hypoperfusion and impaired cerebrovascular function are often associated with neurodegeneration and can lead to CI and dementia. Currently, all forms of neurodegeneration associated to CVD lack effective treatments, which highlights the need to better understand specific mechanisms linking cerebrovascular dysfunction and CBF deficits to neurodegeneration. In this review, we discuss vascular targets that have already shown attenuation of neurodegeneration or CI associated to hypertension, heart failure (HF) and stroke by improving cerebrovascular function or CBF deficits.

Muscle synergy theory assumes that the central nervous system generates a wide range of complex motor outputs by recruiting muscle synergies with different strengths and timings. The current understanding is that a common set of muscle synergies underlies unperturbed as well as perturbed walking at self-selected speeds. However, it is not known whether this is the case for substantially slower walking. The aim of this study was to investigate whether a shared set of muscle synergies underlies balance recovery responses following inward- and outward-directed perturbations in the mediolateral direction at various perturbation onsets and walking speeds. Twelve healthy subjects walked at three walking speeds (0.4, 0.6, and 0.8 m/s) on a treadmill while perturbations were applied to the pelvis using the balance assessment robot. A set of sixteen EMG signals, i.e., eight muscles per leg, was measured and decomposed into muscle synergies and weighting curves using non-negative matrix factorization. The muscles included were left and right tibialis anterior, soleus, gastrocnemius medialis, gastrocnemius lateralis, rectus femoris, hamstring, gluteus medius, and gluteus maximus. In general, four muscle synergies were needed to adequately reconstruct the data. Muscle synergies were similar for unperturbed and perturbed walking at a high walking speed (0.8 m/s). However, the number of similar muscle synergies between perturbed and unperturbed walking was significantly lower for low walking speeds (0.4 and 0.6 m/s). These results indicate that shared muscle synergies underlying perturbed and unperturbed walking are less present during slow walking compared to fast walking.

Wheelchair mounted upper limb exoskeletons offer an alternative way to support disabled individuals in their activities of daily living (ADL). Key challenges in exoskeleton technology include innovative mechanical design and implementation of a control method that can assure a safe and comfortable interaction between the human upper limb and exoskeleton. In this article, we present a mechanical design of a four degrees of freedom (DOF) wheelchair mounted upper limb exoskeleton. The design takes advantage of non-backdrivable mechanism that can hold the output position without energy consumption and provide assistance to the completely paralyzed users. Moreover, a PD-based trajectory tracking control is implemented to enhance the performance of human exoskeleton system for two different tasks. Preliminary results are provided to show the effectiveness and reliability of using the proposed design for physically disabled people.

Ischemic stroke is caused by a thromboembolic occlusion of a major cerebral artery, with the impaired blood flow triggering neuroinflammation and subsequent neuronal damage. Both the innate immune system (e.g., neutrophils, monocytes/macrophages) in the acute ischemic stroke phase and the adaptive immune system (e.g., T cells, B cells) in the chronic phase contribute to this neuroinflammatory process. Considering that the available therapeutic strategies are insufficiently successful, there is an urgent need for novel treatment options. It has been shown that increasing cAMP levels lowers neuroinflammation. By inhibiting cAMP-specific phosphodiesterases (PDEs), i.e., PDE4, 7, and 8, neuroinflammation can be tempered through elevating cAMP levels and, thereby, this can induce an improved functional recovery. This review discusses recent preclinical findings, clinical implications, and future perspectives of cAMP-specific PDE inhibition as a novel research interest for the treatment of ischemic stroke. In particular, PDE4 inhibition has been extensively studied, and is promising for the treatment of acute neuroinflammation following a stroke, whereas PDE7 and 8 inhibition more target the T cell component. In addition, more targeted PDE4 gene inhibition, or combined PDE4 and PDE7 or 8 inhibition, requires more extensive research.

Marine sponges and soft corals have yielded novel compounds with antineoplastic and antimicrobial activities. Their mechanisms of action are poorly understood, and in most cases, little relevant experimental evidence is available on this topic. In the present study, we investigated whether agelasine D (compound 1) and three agelasine analogs (compound 2−4) as well as malonganenone J (compound 5), affect the physical properties of a simple lipid model system, consisting of dioleoylphospahtidylcholine and dioleoylphosphatidylethanolamine. The data indicated that all the tested compounds increased stored curvature elastic stress, and therefore, tend to deform the bilayer which occurs without a reduction in the packing stress of the hexagonal phase. Furthermore, lower concentrations (1%) appear to have a more pronounced effect than higher ones (5−10%). For compounds 4 and 5, this effect is also reflected in phospholipid headgroup mobility assessed using 31P chemical shift anisotropy (CSA) values of the lamellar phases. Among the compounds tested, compound 4 stands out with respect to its effects on the membrane model systems, which matches its efficacy against a broad spectrum of pathogens. Future work that aims to increase the pharmacological usefulness of these compounds could benefit from taking into account the compound effects on the fluid lamellar phase at low concentrations.

Scope: Theobromine is a major active compound in cocoa with allegedly beneficial effect on high-density-lipoprotein-cholesterol (HDL-CH). We have investigated the effect of theobromine (TB) consumption on the concentrations of triglyceride (TG) and cholesterol (CH) in various lipoprotein (LP) subclasses.Methods: In a randomized, double-blind, placebo-controlled, cross-over study, 44 apparently healthy women and men (age: 60 ± 6 years, BMI: 29 ± 3 kg/m2) with low baseline HDL-CH concentrations consumed a drink supplemented with 500 mg/d theobromine for 4 weeks. TG and CH concentrations in 15 LP subclasses were predicted from diffusion-edited 1H NMR spectra of fasting serum.Results: The LP phenotype of the subjects was characterized by low CH concentrations in the large HDL particles and high TG concentrations in large VLDL and chylomicron (CM) particles, which clearly differed from a LP phenotype of subjects with normal HDL-CH. TB only reduced CH concentrations in the LDL particles by 3.64 and 6.79%, but had no effect on TG and CH in any of the HDL, VLDL and CM subclasses.Conclusion: TB was not effective on HDL-CH in subjects with a LP phenotype characterized by low HDL-CH and high TG in VLDL.

Infection with the helminth Schistosoma (S.) mansoni drives the development of interleukin (IL)-10-producing regulatory B (Breg) cells in mice and man, which have the capacity to reduce experimental allergic airway inflammation and are thus of high therapeutic interest. However, both the involved antigen and cellular mechanisms that drive Breg cell development remain to be elucidated. Therefore, we investigated whether S. mansoni soluble egg antigens (SEA) directly interact with B cells to enhance their regulatory potential, or act indirectly on B cells via SEA-modulated macrophage subsets. Intraperitoneal injections of S. mansoni eggs or SEA significantly upregulated IL-10 and CD86 expression by marginal zone B cells. Both B cells as well as macrophages of the splenic marginal zone efficiently bound SEA in vivo, but macrophages were dispensable for Breg cell induction as shown by macrophage depletion with clodronate liposomes. SEA was internalized into acidic cell compartments of B cells and induced a 3-fold increase of IL-10, which was dependent on endosomal acidification and was further enhanced by CD40 ligation. IPSE/alpha-1, one of the major antigens in SEA, was also capable of inducing IL-10 in naïve B cells, which was reproduced by tobacco plant-derived recombinant IPSE. Other major schistosomal antigens, omega-1 and kappa-5, had no effect. SEA depleted of IPSE/alpha-1 was still able to induce Breg cells indicating that SEA contains more Breg cell-inducing components. Importantly, SEA- and IPSE-induced Breg cells triggered regulatory T cell development in vitro. SEA and recombinant IPSE/alpha-1 also induced IL-10 production in human CD1d+ B cells. In conclusion, the mechanism of S. mansoni-induced Breg cell development involves a direct targeting of B cells by SEA components such as the secretory glycoprotein IPSE/alpha-1.

TFE-fusion renal cell carcinomas (TFE-fusion RCCs) are caused by chromosomal translocations that lead to overexpression of the TFEB and TFE3 genes (Kauffman et al., 2014). The mechanisms leading to kidney tumor development remain uncharacterized and effective therapies are yet to be identified. Hence, the need to model these diseases in an experimental animal system (Kauffman et al., 2014). Here, we show that kidney-specific TFEB overexpression in transgenic mice, resulted in renal clear cells, multi-layered basement membranes, severe cystic pathology, and ultimately papillary carcinomas with hepatic metastases. These features closely recapitulate those observed in both TFEB- and TFE3-mediated human kidney tumors. Analysis of kidney samples revealed transcriptional induction and enhanced signaling of the WNT β-catenin pathway. WNT signaling inhibitors normalized the proliferation rate of primary kidney cells and significantly rescued the disease phenotype in vivo. These data shed new light on the mechanisms underlying TFE-fusion RCCs and suggest a possible therapeutic strategy based on the inhibition of the WNT pathway.