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Abstract In this paper, we put in dialogue the local dimension of a nation-state with the global challenges faced by universities worldwide. We focus on the case of Denmark, a nation that was exceptionally active in implementing international university reforms and where digitalisation is a high priority of the public sector governance. The article seeks to contribute to speculative research and critical studies by presenting a mixed-methods study that explores higher education teachers’ views about desirable and realistic future scenarios in Denmark. The study draws on data from a survey applied at a large Danish university, analysed both quantitatively (N = 755) and qualitatively (N = 53). The findings show that teachers share clusters of concern about uncontrolled digitalisation and teaching automation, commodification of education, and modularisation of university courses, the latter being a contested but realistic perspective in the latest political reforms. They reaffirm the mission of the university as preparing students for solving real problems and contributing to the challenges of the present time. The paper concludes with a call for university management to recalibrate future imaginaries to the values expressed by the teachers, and the university they wish to create.

This study investigates Danish university teachers’ agency in using digital technologies in teaching. Furthermore, it explores how teachers’ conduct and experienced constraints are connected to different dimensions of teachers’ agency. The paper is based on survey responses from 344 teachers about their agentic will and power to influence aspects of technology usage in teaching. Using an exploratory factor analysis, we identify three distinct factors and their relations: 1) wanting to control digital data and having the power to do so is distinct from 2) teachers’ power and will to control the technologies adopted and 3) how technologies are applied in teaching. Furthermore, all factors correlate with the teachers’ conduct (extent to which technology facilitates education interactions), while there are factor-specific patterns to the constraints (e.g., time, experience). The results provide a basis for revisiting teachers’ role in university decision-making. We include a discussion on the entangledness of agency across institutional levels.

ObjectiveTo evaluate the value of the thyrotropin-releasing hormone (TRH) test in the diagnosis of central hypothyroidism (CH) in patients with pituitary disease.MethodsSystematic evaluation of 359 TRH tests in patients with pituitary disease including measurements of thyroxine (T4), TBG-corrected T4 (T4corr), baseline TSH (TSH0) and relative or absolute TSH increase (TSHfold, TSHabsolute).ResultsPatients diagnosed with CH (n=39) show comparable TSH0 (p-value 0.824) but lower T4corr (p-value

Recycling endosomes maintain plasma membrane homeostasis and are important for cell polarity, migration, and cytokinesis. Yet, the molecular machineries that drive endocytic recycling remain largely unclear. The CORVET complex is a multi-subunit tether required for fusion between early endosomes. Here we show that the CORVET-specific subunits Vps3 and Vps8 also regulate vesicular transport from early to recycling endosomes. Vps3 and Vps8 localise to Rab4-positive recycling vesicles and co-localise with the CHEVI complex on Rab11-positive recycling endosomes. Depletion of Vps3 or Vps8 does not affect transferrin recycling, but delays the delivery of internalised integrins to recycling endosomes and their subsequent return to the plasma membrane. Consequently, Vps3/8 depletion results in defects in integrin-dependent cell adhesion and spreading, focal adhesion formation, and cell migration. These data reveal a role for Vps3 and Vps8 in a specialised recycling pathway important for integrin trafficking. [ABSTRACT FROM AUTHOR]

Plain English summary Adolescents with anorexia nervosa (AN) who receive care from Child and Adolescent Mental Health Services (CAMHS) often need further treatment from Adult Mental Health Services (AHMS). This study explores the transition between CAMHS and AHMS for parents of adolescents with AN in a naturalistic public mental health-care setting. Our study identified six themes concerning the transition between CAMHS and AMHS, based on parents’ experiences of the process and what they considered influenced the transition. In general, parents and patients are both unprepared for the sudden discharge from CAMHS when the patient turns 18 years old. The transition is often characterized by a lack of continuity, leading to deterioration and relapses among patients. The lack of involvement and information parents experience in the transition causes distress, and parents have overwhelming multifaceted responsibilities in the transition period. Parents view knowledge to be the key to a successful transition, and they are stressed and need support in the transition.

Abstract Background The transition period between child and adolescent mental health services (CAMHS) and adult mental health services (AMHS) has been identified as an especially critical time for patients with anorexia nervosa. In the present study, to better facilitate patients’ recovery process, we explored the experiences of professionals concerning the transition from CAMHS to AMHS. Method A qualitative explorative study was carried out based on recorded interviews from one multi-step focus group and two individual interviews with eight experienced health care professionals. Together they had experience with treating patients with AN and the transition from CAMHS to AMHS, both from specialized eating disorder units, specialized mental health care units, and from a school nurse context. Service users with parents` perspectives and patients’ perspectives were involved in all steps of the research process. Results Barriers experienced during the transition process were classified into four categories: (1) different treatment cultures that describe differences in how parents are included in CAMHS and AMHS; (2) mistrust between CAMHS and AMHS that can create a lack of collaboration and predictability for the patients’ transition; (3) Clinicians` factors such as lack of professional self-confidence can influence continuity of care for patients; and (4) lack of trust between services and not enough focus on building a new alliance in AMHS negatively influences the transition. Conclusions The present study revealed four important categories that professionals needs to consider when participating in the transition for patients with AN from CAMHS to AMHS. Awareness of these challenges might improve the transition process for patients with AN.

Abstract Background The transition between the Child and Adolescent Mental Health Services (CAMHS) and the Adult Mental Health Services (AMHS) is identified as an especially critical time for the continuity of care for patients with anorexia nervosa (AN). However, research on this topic is scarce. In the present study, we explore the patients’ experiences of the transition between CAMHS and AMHS. Methods A qualitative explorative study was carried out based on recorded interviews from one multi-step focus group and six individual interviews with patients who experienced the transition from CAMHS to AHMS in Norway. This study is service user-initiated, meaning service users were involved in all steps of the research process. Results The adolescents’ experiences are characterized by four overall themes regarding the transition process between CAMHS and AMHS: (1) “Being unprepared and alone in the transition process” describes how a lack of preparation for the transition between CAMHS and AMHS makes them feel alone and increases stress. (2) “It takes time to create a trusting relationship” describes how time influences patients’ trust in therapists and motivation for treatment. (3) “We are not all the same” describes how adolescents develop differently but are not treated differently despite their diverse ability to be self-sufficient. (4) “How they see me and treat me affects my hope for the future” describes the interaction between adolescents and therapists. Conclusions Acknowledging the patients’ needs during the transition period and considering their readiness for the transition is important. Taking into account the four dimensions described in the present study might improve the transition process and enhance the patients’ self-sufficiency and maturity.

Abstract Background In recent years there has been a growth in economic evaluations that consider indirect health benefits to populations due to advances in mathematical modeling. In addition, economic evaluations guidelines have suggested the inclusion of impact inventories to include non-health direct and indirect consequences. We aim to bring together this literature, together with the broader literature on internalities and externalities to propose a comprehensive approach for analysts to identify and characterize all unintended consequences in economic evaluations. Methods We present a framework to assist analysts identify and characterize additional costs and effects beyond that of direct health impact primarily intended to be influenced by the intervention/technology. We build on previous checklists to provide analysts with a comprehensive framework to justify the inclusion or exclusion of effects, supporting the use of current guidelines, to ensure any unintended effects are considered. We illustrate this framework with examples from immunization. These were identified from a previous systematic review, PhD thesis work, and general search scoping in PubMed databases. Results We present a comprehensive framework to consider additional consequences, exemplified by types and categories. We bring this and other guidance together to assist analysts identify possible unintended consequences whether taking a provider or societal perspective. Conclusions Although there are many challenges ahead to standardize the inclusion of additional consequences in economic evaluation, we hope by moving beyond generic statements to reporting against a comprehensive framework of additional effects we can support further consistency in this aspect of cost-effectiveness analysis going forward.

Introduction: We assessed the usefulness of SARS-CoV-2 RT-PCR cycle thresholds (Ct) values trends produced by the LHUB-ULB (a consolidated microbiology laboratory located in Brussels, Belgium) for monitoring the epidemic's dynamics at local and national levels and for improving forecasting models.Methods: SARS-CoV-2 RT-PCR Ct values produced from April 1, 2020, to May 15, 2021, were compared with national COVID-19 confirmed cases notifications according to their geographical and time distribution. These Ct values were evaluated against both a phase diagram predicting the number of COVID-19 patients requiring intensive care and an age-structured model estimating COVID-19 prevalence in Belgium.Results: Over 155,811 RT-PCR performed, 12,799 were positive and 7,910 Ct values were available for analysis. The 14-day median Ct values were negatively correlated with the 14-day mean daily positive tests with a lag of 17 days. In addition, the 14-day mean daily positive tests in LHUB-ULB were strongly correlated with the 14-day mean confirmed cases in the Brussels-Capital and in Belgium with coinciding start, peak, and end of the different waves of the epidemic. Ct values decreased concurrently with the forecasted phase-shifts of the diagram. Similarly, the evolution of 14-day median Ct values was negatively correlated with daily estimated prevalence for all age-classes.Conclusion: We provide preliminary evidence that trends of Ct values can help to both follow and predict the epidemic's trajectory at local and national levels, underlining that consolidated microbiology laboratories can act as epidemic sensors as they gather data that are representative of the geographical area they serve.

Individuals with familial hypercholesterolemia (FH) have an increased risk of cardiovascular disease. Treatment is mainly low-density lipoprotein cholesterol (LDL-C) reduction. How omega-3 polyunsaturated fatty acids (n-3 PUFAs) supplements affect lipoproteins in FH subjects is unknown. We hypothesized that a high-dose n-3 PUFA supplement would reduce atherogenic lipoproteins and influence the high-density lipoprotein cholesterol (HDL-C) function. We performed a randomized, double-blinded crossover study with 34 genetically verified FH individuals (18–75 years, clinically stable, statin treatment > 12 months). Treatment was 4 g n-3 PUFAs (1840 mg eicosapentaenoic acid and 1520 mg docosahexaenoic acid daily) or four capsules of olive oil for three months in a crossover design with a washout period of three months. The defined outcomes were changes in triglycerides, lipoproteins, lipoprotein subfractions, apolipoproteins, and HDL-C function. After treatment with n-3 PUFAs, total cholesterol, LDL-C, and triglycerides were reduced compared to placebo (p ≤ 0.01 for all). Total HDL-C levels were unchanged, but the subfraction of large HDL-C was higher (p ≤ 0.0001) after n-3 PUFAs than after placebo, and intermediate HDL-C and small HDL-C were reduced after n-3 PUFAs compared to placebo (p = 0.02 and p ≤ 0.001, respectively). No changes were found in apolipoproteins and HDL-C function. N-3 PUFAs supplements reduced atherogenic lipoproteins in FH subjects, leaving HDL-C function unaffected.

Abstract Background There is emerging evidence for the effectiveness of psychodynamic psychotherapy for depressive disorders. However, we know less of how this relation-focused therapy mode is experienced and what the patients themselves identify as helpful. Hence, the purpose of this study is to explore adolescents’ experiences of factors promoting improvement in psychodynamic therapy. Methods Eight female patients participating in a Norwegian study on psychodynamic therapy, the First Experimental Study of Transference Work – In Teenagers (FEST-IT), were included. The participants were offered a total number of 28 sessions. Semi-structured qualitative interviews about experiences with therapy were then conducted and analysed with systematic text condensation and hermeneutic interpretation. Results The analysis revealed four main themes. ‘Exploring oneself’ comprises autonomy and acknowledgment, openness, insight and acceptance of oneself. ‘Therapist relation and characteristics’ includes confidence and trust in and support from the therapist as well as having a trustworthy and experienced therapist. ‘Focusing on everyday life’ includes learning and practical orientation. ‘Time factors’ refers to duration and frequency. Conclusions Together with a supportive and listening therapist, the adolescents improve by exploring themselves within the frames of a time-limited treatment period. Improvement seems to be experienced through better relations to oneself and to others and by finding one’s place in the family, or at school. Adolescents value problem solving and help with concrete challenges. Hence, therapy should be tailored to the needs of adolescents with depression and incorporate the challenges they face in their everyday life. Trial registration ClinicalTrials.gov. Id: NCT01531101. Date of registry: 8 February 2012, retrospectively registered.

Spoil heaps of surplus rock from hydropower tunnel construction negatively impact alpine landscapes unless restored. Such spoil heaps have been created for more than 100 years, but we still lack knowledge about the relative importance of compensatory mitigation (seeding and fer-tilization), spoil-heap construction method, local environ-mental factors, and regional climatic factors for restoration success. We studied the species composition of 19 alpine spoil heaps in Western Norway and their undisturbed surroundings using ordination and statistical modeling. Substrate grain size was the principal factor explaining differences in species composition between spoil heaps and their surroundings. Soil characteristics, that is, organic matter content and pH, and reutilization of topsoil were also important. Seeding and fertilization had negligible effects on restoration success. Slow recovery was observed for total vegetation cover and species richness of vascu-lar plants and lichens while bryophyte cover recovered rapidly. Lower bryophyte cover and bryophyte and vas-cular plant species richness on older than on younger spoil heaps indicated recent changes in spoil-heap construction practices that favor plant colonization. Our results indi-cate that spoil-heap design is more important for restora-tion success than compensatory mitigation. We therefore suggest spoil heaps designed with a fine-grained top sub-strate preferably from stockpiled local topsoil, with uneven surface topography that mimics natural topographic vari-ation, and recommend discontinuation of seeding and fertilization. [ABSTRACT FROM AUTHOR]

This paper discusses how archaeologists can approach ways in which the ritual treatment of the dead body was a means of reproducing a sense of identity and community in the past. The approach combines a theoretical framework grounded in practice and body theory with a methodological approach based on taphonomic analysis. This framework is introduced to analyze the mortuary practices at the Mesolithic cemeteries of Skateholm I and II, Vedbæk, Bøgebakken and Zvejnieki. Beyond the immediate context, the study seeks to reflect on how similarities and differences noticeable over time and space may provide an insight into changing identity processes.

The debatc concerning repatriation and reburial is attracting increasing attention in Sweden. While most archaeologists today understand the importancc of repatriation and the arguments underlying the claim, the process is not completely unproblematic and certainly not in all cases. This article explores some tendencies within the international debate about repatriation, and frames them within a more general discussion about human rights, the right to culture, and the role of cultural heritage within this debatc. Through a critical approach to the debate, it is argucd that archaeology needs to be a more active party in the negotiations.

Commander is a multiprotein complex that orchestrates endosomal recycling of integral cargo proteins and is essential for normal development. While the structure of this complex has recently been described, how cargo proteins are selected for Commander-mediated recycling remains unclear. Here we identify the mechanism through which the unstructured carboxy-terminal tail of the cargo adaptor sorting nexin-17 (SNX17) directly binds to the Retriever sub-complex of Commander. SNX17 adopts an autoinhibited conformation where its carboxy-terminal tail occupies the cargo binding groove. Competitive cargo binding overcomes this autoinhibition, promoting SNX17 endosomal residency and the release of the tail for Retriever association. Furthermore, our study establishes the central importance of SNX17-Retriever association in the handover of integrin and lipoprotein receptor cargoes into pre-existing endosomal retrieval sub-domains. In describing the principal mechanism of cargo entry into the Commander recycling pathway we provide key insight into the function and regulation of this evolutionary conserved sorting pathway., (© 2024. The Author(s).)

Elucidating the 3D nanoscale structure of tissues and cells is essential for understanding the complexity of biological processes. Electron microscopy (EM) offers the resolution needed for reliable interpretation, but the limited throughput of electron microscopes has hindered its ability to effectively image large volumes. We report a workflow for volume EM with FAST-EM, a novel multibeam scanning transmission electron microscope that speeds up acquisition by scanning the sample in parallel with 64 electron beams. FAST-EM makes use of optical detection to separate the signals of the individual beams. The acquisition and 3D reconstruction of ultrastructural data from multiple biological samples is demonstrated. The results show that the workflow is capable of producing large reconstructed volumes with high resolution and contrast to address biological research questions within feasible acquisition time frames., Competing Interests: Competing interests: A.J.K., B.H.P.D., R.L, C.H., D.B.B.H., T.H., A.H.G.W., N.L., and B.N.G.G. declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. FAST-EM is a product of Delmic BV, J.P.H. is co-founder of and shareholder in Delmic BV., (© 2024 the author(s), published by De Gruyter on behalf of Thoss Media.)

Tissue repair is disturbed in fibrotic diseases like systemic sclerosis (SSc), where the deposition of large amounts of extracellular matrix components such as collagen interferes with organ function. LAIR-1 is an inhibitory collagen receptor highly expressed on tissue immune cells. We questioned whether in SSc, impaired LAIR-1-collagen interaction is contributing to the ongoing inflammation and fibrosis. We found that SSc patients do not have an intrinsic defect in LAIR-1 expression or function. Instead, fibroblasts from healthy controls and SSc patients stimulated by soluble factors that drive inflammation and fibrosis in SSc deposit disorganized collagen products in vitro, which are dysfunctional LAIR-1 ligands. This is dependent of matrix metalloproteinases and platelet-derived growth factor receptor signaling. In support of a non-redundant role of LAIR-1 in the control of fibrosis, we found that LAIR-1-deficient mice have increased skin fibrosis in response to repeated injury and in the bleomycin mouse model for SSc. Thus, LAIR-1 represents an essential control mechanism for tissue repair. In fibrotic disease, excessive collagen degradation may lead to a disturbed feedback loop. The presence of functional LAIR-1 in patients provides a therapeutic opportunity to reactivate this intrinsic negative feedback mechanism in fibrotic diseases., Competing Interests: Declaration of competing interest DF, MR and MEMG were full time employees of Boehringer Ingelheim. TRDJR was a principal investigator in the immune catalyst program of GlaxoSmith-Kline, which was an independent research program. He did not receive any financial support. Currently, TRDJR is an employee of Abbvie where he holds stock. TRDJR had no part in the design and interpretation of the study results after he started at Abbvie. LM's research lab has received financial support for investigator-initiated studies from Boehringer Ingelheim, NextCure and NGM biopharmaceuticals. The remaining 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 © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The multisubunit HOPS tethering complex is a well-established regulator of lysosome fusion with late endosomes and autophagosomes. However, the role of the HOPS complex in other stages of endo-lysosomal trafficking is not well understood. To address this, we made HeLa cells knocked out for the HOPS-specific subunits Vps39 or Vps41, or the HOPS-CORVET-core subunits Vps18 or Vps11. In all four knockout cells, we found that endocytosed cargos were trapped in enlarged endosomes that clustered in the perinuclear area. By correlative light-electron microscopy, these endosomes showed a complex ultrastructure and hybrid molecular composition, displaying markers for early (Rab5, PtdIns3P, EEA1) as well as late (Rab7, CD63, LAMP1) endosomes. These "HOPS bodies" were not acidified, contained enzymatically inactive cathepsins and accumulated endocytosed cargo and cation-independent mannose-6-phosphate receptor (CI-MPR). Consequently, CI-MPR was depleted from the TGN, and secretion of lysosomal enzymes to the extracellular space was enhanced. Strikingly, HOPS bodies also contained the autophagy proteins p62 and LC3, defining them as amphisomes. Together, these findings show that depletion of the lysosomal HOPS complex has a profound impact on the functional organization of the entire endosomal system and suggest the existence of a HOPS-independent mechanism for amphisome formation.

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal details that are important for understanding the autophagic process. However, EM methods often lack molecular information, obstructing the correlation of ultrastructural information obtained by EM to fluorescence microscopy-based localization of specific autophagy proteins. Furthermore, the rarity of autophagosomes in unaltered cellular conditions hampers investigation by EM, which requires high magnification, and hence provides a limited field of view. In answer to both challenges, an on-section correlative light-electron microscopy (CLEM) method based on fluorescent labeling was applied to correlate a common autophagosomal marker, LC3, to EM ultrastructure. The method was used to rapidly screen cells in fluorescence microscopy for LC3 labeling in combination with other relevant markers. Subsequently, the underlying ultrastructural features of selected LC3-labeled spots were identified by CLEM. The method was applied to starved cells without adding inhibitors of lysosomal acidification. In these conditions, LC3 was found predominantly on autophagosomes and rarely in autolysosomes, in which LC3 is rapidly degraded. These data show both the feasibility and sensitivity of this approach, demonstrating that CLEM can be used to provide ultrastructural insights on LC3-mediated autophagy in native conditions-without drug treatments or genetic alterations. Overall, this method presents a valuable tool for ultrastructural localization studies of autophagy proteins and other scarce antigens by bridging light microscopy to EM data.

Arl8b, an Arf-like GTP-binding protein, regulates cargo trafficking and positioning of lysosomes. However, it is unknown whether Arl8b regulates lysosomal cargo sorting. Here, we report that Arl8b binds to the Rab4 and Rab14 interaction partner, RUN and FYVE domain-containing protein (RUFY) 1, a known regulator of cargo sorting from recycling endosomes. Arl8b determines RUFY1 endosomal localization through regulating its interaction with Rab14. RUFY1 depletion led to a delay in CI-M6PR retrieval from endosomes to the TGN, resulting in impaired delivery of newly synthesized hydrolases to lysosomes. We identified the dynein-dynactin complex as an RUFY1 interaction partner, and similar to a subset of activating dynein adaptors, the coiled-coil region of RUFY1 was required for interaction with dynein and the ability to mediate dynein-dependent organelle clustering. Our findings suggest that Arl8b and RUFY1 play a novel role on recycling endosomes, from where this machinery regulates endosomes to TGN retrieval of CI-M6PR and, consequently, lysosomal cargo sorting., (© 2022 Rawat et al.)

Fluorescent biosensors are valuable tools to monitor protein activities and the functional state of organelles in live cells. However, the information provided by fluorescent microscopy (FM) is mostly limited in resolution and lacks ultrastructural context information. Protein activities are confined to organelle zones with a distinct membrane morphology, which can only be seen by electron microscopy (EM). EM, however, intrinsically lacks information on protein activities. The lack of methods to integrate these two imaging modalities has hampered understanding the functional organization of cellular organelles. Here we introduce "functional correlative microscopy" (functional CLEM) to directly infer functional information from live cells to EM with nanometer resolution. We label and visualize live cells with fluorescent biosensors after which they are processed for EM and imaged using a volume electron microscopy technique. Within a single dataset we correlate hundreds of fluorescent spots enabling quantitative analysis of the functional-ultrastructural data. We employ our method to monitor essential functional parameters of late endo-lysosomal compartments, i.e., pH, calcium, enzyme activities and cholesterol content. Our data reveal a steep functional difference in enzyme activity between late endosomes and lysosomes and unexpectedly high calcium levels in late endosomes. The presented CLEM workflow is compatible with a large repertoire of probes and paves the way for large scale functional studies of all types of cellular structures., (Copyright © 2023 Elsevier Inc. All rights reserved.)

We present a bimodal endocytic tracer, fluorescent BSA-gold (fBSA-Au), as a fiducial marker for 2D and 3D correlative light and electron microscopy (CLEM) applications. fBSA-Au consists of colloidal gold (Au) particles stabilized with fluorescent BSA. The conjugate is efficiently endocytosed and distributed throughout the 3D endolysosomal network of cells and has an excellent visibility in both fluorescence microscopy (FM) and electron microscopy (EM). We demonstrate that fBSA-Au facilitates rapid registration in several 2D and 3D CLEM applications using Tokuyasu cryosections, resin-embedded material, and cryoelectron microscopy (cryo-EM). Endocytosed fBSA-Au benefits from a homogeneous 3D distribution throughout the endosomal system within the cell, does not obscure any cellular ultrastructure, and enables accurate (50-150 nm) correlation of fluorescence to EM data. The broad applicability and visibility in both modalities makes fBSA-Au an excellent endocytic fiducial marker for 2D and 3D (cryo)CLEM applications., Competing Interests: The endocytic fBSA-Au5 and fBSA-Au10 fiducials reported here are available in the product listing of Cell Microscopy Core, UMC Utrecht., (© 2022 The Author(s).)

Intracellular processes depend on a strict spatial and temporal organization of proteins and organelles. Therefore, directly linking molecular to nanoscale ultrastructural information is crucial in understanding cellular physiology. Volume or three-dimensional (3D) correlative light and electron microscopy (volume-CLEM) holds unique potential to explore cellular physiology at high-resolution ultrastructural detail across cell volumes. However, the application of volume-CLEM is hampered by limitations in throughput and 3D correlation efficiency. In order to address these limitations, we describe a novel pipeline for volume-CLEM that provides high-precision (<100 nm) registration between 3D fluorescence microscopy (FM) and 3D electron microscopy (EM) datasets with significantly increased throughput. Using multi-modal fiducial nanoparticles that remain fluorescent in epoxy resins and a 3D confocal fluorescence microscope integrated into a Focused Ion Beam Scanning Electron Microscope (FIB.SEM), our approach uses FM to target extremely small volumes of even single organelles for imaging in volume EM and obviates the need for post-correlation of big 3D datasets. We extend our targeted volume-CLEM approach to include live-cell imaging, adding information on the motility of intracellular membranes selected for volume-CLEM. We demonstrate the power of our approach by targeted imaging of rare and transient contact sites between the endoplasmic reticulum (ER) and lysosomes within hours rather than days. Our data suggest that extensive ER-lysosome and mitochondria-lysosome interactions restrict lysosome motility, highlighting the unique capabilities of our integrated CLEM pipeline for linking molecular dynamic data to high-resolution ultrastructural detail in 3D., 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 © 2022 Loginov, Fermie, Fokkema, Agronskaia, De Heus, Blab, Klumperman, Gerritsen and Liv.)

Elevated expression of non-receptor tyrosine kinase FER is an independent prognosticator that correlates with poor survival of high-grade and basal/triple-negative breast cancer (TNBC) patients. Here, we show that high FER levels are also associated with improved outcomes after adjuvant taxane-based combination chemotherapy in high-risk, HER2-negative patients. In TNBC cells, we observe a causal relation between high FER levels and sensitivity to taxanes. Proteomics and mechanistic studies demonstrate that FER regulates endosomal recycling, a microtubule-dependent process that underpins breast cancer cell invasion. Using chemical genetics, we identify DCTN2 as a FER substrate. Our work indicates that the DCTN2 tyrosine 6 is essential for the development of tubular recycling domains in early endosomes and subsequent propagation of TNBC cell invasion in 3D. In conclusion, we show that high FER expression promotes endosomal recycling and represents a candidate predictive marker for the benefit of adjuvant taxane-containing chemotherapy in high-risk patients, including TNBC patients., Competing Interests: Declaration of interests S.C.L. and H.M.O. received funding from Amgen, Sanofi, and the Dutch Cancer Society during the conduct of the study. S.C.L. reports grants and nonfinancial support from AstraZeneca, Genentech/Roche, Tesaro, and Immunomedics. S.C.L. received funding from Eurocept Pharmaceuticals, Novartis, and Pfizer and other support from Cergentis, IBM, Daiichi Sankyo, and Bayer outside the submitted work. S.C.L. is an advisory board member for Cergentis, IBM, Novartis, Pfizer, Roche, and Sanofi. H.M.O. is an advisory board member for Roche, Novartis, Pfizer, and MSD. J.K. received funding from Genentech/Roche. All other authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Inborn errors of metabolism (IEMs) comprise a diverse group of individually rare monogenic disorders that affect metabolic pathways. Mutations lead to enzymatic deficiency or dysfunction, which results in intermediate metabolite accumulation or deficit leading to disease phenotypes. Currently, treatment options for many IEMs are insufficient. Rarity of individual IEMs hampers therapy development and phenotypic and genetic heterogeneity suggest beneficial effects of personalized approaches. Recently, cultures of patient-own liver-derived intrahepatic cholangiocyte organoids (ICOs) have been established. Since most metabolic genes are expressed in the liver, patient-derived ICOs represent exciting possibilities for in vitro modeling and personalized drug testing for IEMs. However, the exact application range of ICOs remains unclear. To address this, we examined which metabolic pathways can be studied with ICOs and what the potential and limitations of patient-derived ICOs are to model metabolic functions. We present functional assays in patient ICOs with defects in branched-chain amino acid metabolism (methylmalonic acidemia), copper metabolism (Wilson disease), and transporter defects (cystic fibrosis). We discuss the broad range of functional assays that can be applied to ICOs, but also address the limitations of these patient-specific cell models. In doing so, we aim to guide the selection of the appropriate cell model for studies of a specific disease or metabolic process., (© 2021 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Microglia are the major innate immune cells in the brain and are essential for maintaining homeostasis in a neuronal microenvironment. Currently, a genetic tool to modify microglial gene expression in specific brain regions is not available. In this report, we introduce a tailor-designed method that uses lipid and polymer hybridized nanoparticles (LPNPs) for the local delivery of small interfering RNAs (siRNAs), allowing the silencing of specific microglial genes in the hypothalamus. Our physical characterization proved that this LPNP-siRNA was uniform and stable. We demonstrated that, due to their natural phagocytic behavior, microglial cells are the dominant cell type taking up these LPNPs in the hypothalamus of rats. We then tested the silencing efficiency of LPNPs carrying a cluster of differentiation molecule 11b (CD11b) or Toll-like receptor 4 (TLR4) siRNA using different in vivo and in vitro approaches. In cultured microglial cells treated with LPNP-CD11b siRNA or LPNP-TLR4 siRNA, we found a silencing efficiency at protein expression levels of 65 or 77%, respectively. In line with this finding, immunohistochemistry and western blotting results from in vivo experiments showed that LPNP-CD11b siRNA significantly inhibited microglial CD11b protein expression in the hypothalamus. Furthermore, following lipopolysaccharide (LPS) stimulation of cultured microglial cells, gene expression of the TLR4 downstream signaling component myeloid differentiation factor 88 and its associated cytokines was significantly inhibited in LPNP-TLR4 siRNA-treated microglial cells compared with cells treated with LPNP-scrambled siRNA. Finally, after LPNP-TLR4 siRNA injection into the rat hypothalamus, we observed a significant reduction in microglial activation in response to LPS compared with the control rats injected with LPNP-scrambled siRNA. Our results indicate that LPNP-siRNA is a promising tool to manipulate microglial activity locally in the brain and may serve as a prophylactic approach to prevent microglial dysfunction-associated diseases.

The key endosomal regulators Rab5, EEA1, and APPL1 are frequently applied in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as a marker for late endosomes and lysosomes. However, endogenous levels of these proteins localize poorly in immuno-EM, and systematic studies on their native ultrastructural distributions are lacking. To address this gap, we here present a quantitative, on-section correlative light and electron microscopy (CLEM) approach. Using the sensitivity of fluorescence microscopy, we label hundreds of organelles that are subsequently visualized by EM and classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 colocalizes with Rab5 on early endosomes, but unexpectedly also labels Rab5-negative late endosomes, which are positive for PI(3)P but lack Rab7. APPL1 is restricted to small Rab5-positive, tubulo-vesicular profiles. Rab7 primarily labels late endosomes and lysosomes. These data increase our understanding of the structural-functional organization of the endosomal system and introduce quantitative CLEM as a sensitive alternative for immuno-EM., (© 2021 van der Beek et al.)

Malignant rhabdoid tumors (MRTs) represent one of the most aggressive childhood malignancies. No effective treatment options are available, and prognosis is, therefore, dismal. Previous studies have demonstrated that tumor organoids capture the heterogeneity of patient tumors and can be used to predict patient response to therapy. Here, we perform drug screening on patient-derived normal and tumor organoids to identify MRT-specific therapeutic vulnerabilities. We identify neddylation inhibitor MLN4924 as a potential therapeutic agent. Mechanistically, we find increased neddylation in MRT organoids and tissues and show that MLN4924 induces a cytotoxic response via upregulation of the unfolded protein response. Lastly, we demonstrate in vivo efficacy in an MRT PDX mouse model, in which single-agent MLN4924 treatment significantly extends survival. Our study demonstrates that organoids can be used to find drugs selectively targeting tumor cells while leaving healthy cells unharmed and proposes neddylation inhibition as a therapeutic strategy in MRT., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neuronal function relies on careful coordination of organelle organization and transport. Kinesin-1 mediates transport of the endoplasmic reticulum (ER) and lysosomes into the axon and it is increasingly recognized that contacts between the ER and lysosomes influence organelle organization. However, it is unclear how organelle organization, inter-organelle communication and transport are linked and how this contributes to local organelle availability in neurons. Here, we show that somatic ER tubules are required for proper lysosome transport into the axon. Somatic ER tubule disruption causes accumulation of enlarged and less motile lysosomes at the soma. ER tubules regulate lysosome size and axonal translocation by promoting lysosome homo-fission. ER tubule - lysosome contacts often occur at a somatic pre-axonal region, where the kinesin-1-binding ER-protein P180 binds microtubules to promote kinesin-1-powered lysosome fission and subsequent axonal translocation. We propose that ER tubule - lysosome contacts at a pre-axonal region finely orchestrate axonal lysosome availability for proper neuronal function., (© 2021. The Author(s).)