Discussions at a recent conference on microscopy technology dissemination spotlighted the importance of setting technology adoption capable of producing scientific outcome as the end goal. This Comment examines current global efforts in microscopy dissemination and summarizes the challenges and paths forward.

In the dynamic landscape of scientific research, imaging core facilities are vital hubs propelling collaboration and innovation at the technology development and dissemination frontier. Here, we present a collaborative effort led by Global BioImaging (GBI), introducing international recommendations geared towards elevating the careers of Imaging Scientists in core facilities. Despite the critical role of Imaging Scientists in modern research ecosystems, challenges persist in recognising their value, aligning performance metrics and providing avenues for career progression and job security. The challenges encompass a mismatch between classic academic career paths and service-oriented roles, resulting in a lack of understanding regarding the value and impact of Imaging Scientists and core facilities and how to evaluate them properly. They further include challenges around sustainability, dedicated training opportunities and the recruitment and retention of talent. Structured across these interrelated sections, the recommendations within this publication aim to propose globally applicable solutions to navigate these challenges. These recommendations apply equally to colleagues working in other core facilities and research institutions through which access to technologies is facilitated and supported. This publication emphasises the pivotal role of Imaging Scientists in advancing research programs and presents a blueprint for fostering their career progression within institutions all around the world.

Conventional optical microscopy imaging of obligate intracellular bacteria is hampered by the small size of bacterial cells, tight clustering exhibited by some bacterial species and challenges relating to labelling such as background from host cells, a lack of validated reagents, and a lack of tools for genetic manipulation. In this study, we imaged intracellular bacteria from the species Orientia tsutsugamushi (Ot) using five different fluorescence microscopy techniques: standard confocal, Airyscan confocal, instant Structured Illumination Microscopy (iSIM), three-dimensional Structured Illumination Microscopy (3D-SIM) and Stimulated Emission Depletion Microscopy (STED). We compared the ability of each to resolve bacterial cells in intracellular clumps in the lateral (xy) axis, using full width half-maximum (FWHM) measurements of a labelled outer membrane protein (ScaA) and the ability to detect small, outer membrane vesicles external to the cells. Comparing the techniques readily available to us (above), 3D-SIM microscopy, in combination with the shortest-wavelength dyes, was found overall to give the best lateral resolution. We next compared the ability of each technique to sufficiently resolve bacteria in the axial (z) direction and found 3D-STED to be the most successful method for this. We then combined this 3D-STED approach with a custom 3D cell segmentation and analysis pipeline using the open-source, deep learning software, Cellpose to segment the cells and subsequently the commercial software Imaris to analyse their 3D shape and size. Using this combination, we demonstrated differences in bacterial shape, but not their size, when grown in different mammalian cell lines. Overall, we compare the advantages and disadvantages of different super-resolution microscopy techniques for imaging this cytoplasmic obligate intracellular bacterium based on the specific research question being addressed.

The use of polychromatic immunofluorescent staining on whole-mount skin enables cell type characterization and aids in the delineation of the physiological and immunological strategies used by the skin to combat pathogens. Using whole-mount skin for polychromatic immunofluorescent staining removes the need for histological sectioning and enables the visualization of anatomical structures and immune cell types in three dimensions. Here we present a detailed protocol for immunostaining with fluorescence-conjugated primary antibodies in whole-mount skin to reveal structural landmarks and specific immune cell types using confocal laser scanning microscopy (CLSM) (Basic Protocol 1). The optimized staining panel reveals structural features such as blood vessels (CD31 antibody) and the lymphatic network (LYVE-1 antibody), in combination with MHCII antibodies for antigen-presenting cells (APCs), CD64 for macrophages and monocytes, CD103 for dendritic epidermal T cells (DETC), and CD326 for Langerhans cells (LC). Basic Protocol 2 describes image visualization pipelines using open-source software (ImageJ/FIJI), enabling four visualization options (z-projections, orthogonal views, 3D visualization, and animation). Basic Protocol 3 describes a quantitative analysis pipeline using CellProfiler to characterize the spatial relationship between cell types using mathematical indices such as Spatial Distribution Index (SDI), Neighborhood Frequency (NF), and Normalized Median Evenness (NME). These protocols will enable researchers to stain, record, analyze, and interpret data from whole-mount skin using commercially available reagents in a CLSM-equipped laboratory and freely available analysis software. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescent staining and imaging for whole-mount mouse skin Basic Protocol 2: File rendering and visualization using FIJI Basic Protocol 3: Spatial image analysis using CellProfiler.

Human ageing is accompanied by poor responses to infection and decreased vaccine efficacy. While the causes of this can be attributed to defects in the immune system that increase with age, it is unknown whether mitochondrial dysfunction may also contribute to these phenomena. This study aims to assess mitochondrial dysfunction in CD4+ terminal effector memory T cells re-expressing CD45RA (TEMRA) cells and other CD4+ memory T cell subtypes, which are increased in number in the elderly population, with respect to how their metabolic responses to stimulation are altered compared to CD4+ naïve T cells. In this study, we show that CD4+ TEMRA cells exhibit altered mitochondrial dynamics compared to CD4+ naïve cells and CD4+ central and effector memory cells, with a 25% reduction in OPA1 expression. CD4+ TEMRA and memory cells show increased upregulation of Glucose transporter 1 following stimulation and higher levels of mitochondrial mass compared to CD4+ naïve T cells. Additionally, TEMRA cells exhibit a decrease in mitochondrial membrane potential compared to other CD4+ memory cell subsets by up to 50%. By comparing young to aged individuals, more significant mitochondria mass and lower membrane potential were observed in CD4+ TEMRA of young individuals. In conclusion, we suggest that CD4+ TEMRA cells may be impaired with respect to their metabolic response to stimulation, possibly contributing to impaired responses to infection and vaccination.

Background Lamins, key nuclear lamina components, have been proposed as candidate risk biomarkers in different types of cancer but their accuracy is still debated. AKTIP is a telomeric protein with the property of being enriched at the nuclear lamina. AKTIP has similarity with the tumor susceptibility gene TSG101. AKTIP deficiency generates genome instability and, in p53 −/− mice, the reduction of the mouse counterpart of AKTIP induces the exacerbation of lymphomas. Here, we asked whether the distribution of AKTIP is altered in cancer cells and whether this is associated with alterations of lamins. Methods We performed super-resolution imaging, quantification of lamin expression and nuclear morphology on HeLa, MCF7, and A549 tumor cells, and on non-transformed fibroblasts from healthy donor and HGPS (LMNA c.1824C > T p.Gly608Gly) and EDMD2 (LMNA c.775 T > G) patients. As proof of principle model combining a defined lamin alteration with a tumor cell setting, we produced HeLa cells exogenously expressing the HGPS lamin mutant progerin that alters nuclear morphology. Results In HeLa cells, AKTIP locates at less than 0.5 µm from the nuclear rim and co-localizes with lamin A/C. As compared to HeLa, there is a reduced co-localization of AKTIP with lamin A/C in both MCF7 and A549. Additionally, MCF7 display lower amounts of AKTIP at the rim. The analyses in non-transformed fibroblasts show that AKTIP mislocalizes in HGPS cells but not in EDMD2. The integrated analysis of lamin expression, nuclear morphology, and AKTIP topology shows that positioning of AKTIP is influenced not only by lamin expression, but also by nuclear morphology. This conclusion is validated by progerin-expressing HeLa cells in which nuclei are morphologically altered and AKTIP is mislocalized. Conclusions Our data show that the combined alteration of lamin and nuclear morphology influences the localization of the tumor-associated factor AKTIP. The results also point to the fact that lamin alterations per se are not predictive of AKTIP mislocalization, in both non-transformed and tumor cells. In more general terms, this study supports the thesis that a combined analytical approach should be preferred to predict lamin-associated changes in tumor cells. This paves the way of next translational evaluation to validate the use of this combined analytical approach as risk biomarker.

BACKGROUND AND OBJECTIVESAgeing is associated with an impaired cellular function that can affect tissue architecture and wound healing in gingival and periodontal tissues. However, the impact of oral fibroblast ageing on the structural organization of the extracellular matrix (ECM) proteins is poorly understood. Hence, in this study, we investigated the impact of cellular ageing of oral fibroblasts on the production and structural organization of collagen and other ECM proteins.METHODSOral fibroblasts were serially subcultured, and replicative cellular senescence was assessed using population doubling time, Ki67 counts and expression of P21WAFI . The production and structural organization of ECM proteins were assessed at early (young-oFB) and late (aged-oFB) passages. The thickness and pattern of collagen produced by live cultures of young- and aged-oFB were assessed using a label-free and non-invasive second harmonic generation (SHG)-based multiphoton imaging. Expression of other ECM proteins (fibronectin, fibrillin, collagen-IV and laminins) was evaluated using immunocytochemistry and confocal microscopy-based depth profile analysis.RESULTSAged-oFB displayed a higher population doubling time, lower Ki67+ cells and higher expression of P21WAFI indicative of slower proliferation rate and senescence phenotype. SHG imaging demonstrated that young-oFB produced a thick, interwoven network of collagen fibres, while the aged-oFB produced thin and linearly organized collagen fibres. Similarly, analysis of immunostained cultures showed that young-oFB produced a rich, interwoven mesh of fibronectin, fibrillin and collagen-IV fibres. In contrast, the aged-oFB produced linearly organized fibronectin, fibrillin and collagen-IV fibres. Lastly, there was no observable difference in production and organization of laminins among the young- and aged-oFB.CONCLUSIONOur results suggest that oral fibroblast ageing impairs ECM production and more importantly the organization of ECM fibres, which could potentially impair wound healing in the elderly.

High prevalence of non-healing chronic wounds contributes to a huge healthcare burden across the world. Early treatment interventions for non-healing wounds are vital. It was previously shown that accumulation of 15% or more of senescent cells in a chronic wound edge is an indicator that the wound is unlikely to heal. However, determining the presence of senescent cells would require invasive procedures such as tissue biopsies to be taken. In this study, we found a strong correlation between decreased collagen area and presence of senescent cells in human chronic wounds i.e. venous leg ulcer (VLU), diabetic foot ulcer (DFU) and pressure ulcer (PRU). We also report that the lowest collagen levels were found in VLU patients less than 60 years of age, with a persistent wound of > 24 months. Elevated levels of senescent cells were also found in VLU of males. Second harmonic imaging of collagen at the edge of chronic wounds with a handheld multiphoton device could be used to predict the number of senescent cells, indicating if the wound is on a healing trajectory or not. Our data support the use of collagen imaging in cutaneous wound assessment for a faster and non-invasive method to predict cellular senescence and determining wound trajectory of healing.

Desmosomes, strong cell-cell junctions of epithelia and cardiac muscle, link intermediate filaments to cell membranes and mechanically integrate cells across tissues, dissipating mechanical stress. They comprise 5 major protein classes - desmocollins and desmogleins (the desmosomal cadherins), plakoglobin, plakophilins and desmoplakin - whose individual contribution to the structure and turnover of desmosomes is poorly understood. Using live-cell imaging together with FRAP and FLAP we show that desmosomes consist of two contrasting protein fractions or modules: a very stable desmosomal core of desmosomal cadherins and plakoglobin, and a highly mobile plakophilin. As desmosomes mature from calcium-dependence to calciumindependent hyper-adhesion, core stability increases, but Pkp2a remains highly mobile. Desmoplakin is initially mobile but stabilises with hyper-adhesion. We show that desmosome down-regulation during growth factor-induced cell scattering proceeds by internalisation of whole desmosomes, which still retain a stable core and highly mobile Pkp2a. This molecular mobility of Pkp2a suggests a transient and probably regulatory role for Pkp2a in the desmosome.

Vinculin, a mechanotransducer associated with both adherens junctions (AJ) and focal adhesions (FA) plays a central role in force transmission through these cell-cell and cell-substratum contacts. Here we describe the conditional knock out (KO) of vinculin in murine skin. Remarkably, we find that the loss of vinculin function results in the loss of bulge stem cell (BuSC) quiescence. We demonstrate that vinculin KO cells are impaired in force generation resulting in mechanically weak AJs. Mechanistically, vinculin functions by keeping α-catenin in a stretched conformation, which in turn regulates the retention of YAP1, another potent mechanotransducer and regulator of cell proliferation, to the junctions. Conditional KO of α-catenin specifically in the BuSCs further corroborates the importance of stable AJs in the maintenance of quiescence and stemness. Altogether, our data provides definitive mechanistic insights into the hitherto unexplored regulatory link between the mechanical stability of cell-junctions and the maintenance of BuSC quiescence.

Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related sensor proteins NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, their mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIIND UPA -CARD) of NLRP1 and CARD8 self-oligomerize to assemble distinct inflammasome complexes. Recombinant FIIND UPA -CARD of NLRP1 forms a two-layered filament, with an inner core of oligomerized CARD surrounded by an outer ring of FIIND UPA . Biochemically, self-assembled NLRP1-CARD filaments are sufficient to drive ASC speck formation in cultured human cells—a process that is greatly enhanced by NLRP1-FIIND UPA which forms oligomers in vitro. The cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments, solved here at 3.7 Å, uncover unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide structural insight into the mechanisms of activation for human NLRP1 and CARD8 and reveal how highly specific signaling can be achieved by heterotypic CARD interactions within the inflammasome complexes.

A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated , quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist's experiments, while more than half have even failed to reproduce their own experiments. One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique. Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g. DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common standards and guidelines for quality assessment and reproducibility. In April 2020, the QUality Assessment and REProducibility for instruments and images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper (1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; (2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers and observers of such; (3) outlines the current actions of the QUAREP-LiMi initiative and (4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics.

A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated, quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist’s experiments, while more than half have even failed to reproduce their own experiments 1 . One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique 2 . Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g., DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE 3 ), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common standards and guidelines for quality assessment and reproducibility 4 . In April 2020, the QUality Assessment and REProducibility for Instruments and Images in Light Microscopy (QUAREP-LiMi) initiative 5 was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models, and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research.ThisWhite Paper 1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; 2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists 6 , bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers, and observers of such; 3) outlines the current actions of the QUAREP-LiMi initiative, and 4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC.

ABSTRACT Desmosomes, strong cell–cell junctions of epithelia and cardiac muscle, link intermediate filaments to cell membranes and mechanically integrate cells across tissues, dissipating mechanical stress. They comprise five major protein classes – desmocollins and desmogleins (the desmosomal cadherins), plakoglobin, plakophilins and desmoplakin – whose individual contribution to the structure and turnover of desmosomes is poorly understood. Using live-cell imaging together with fluorescence recovery after photobleaching (FRAP) and fluorescence loss and localisation after photobleaching (FLAP), we show that desmosomes consist of two contrasting protein moieties or modules: a very stable moiety of desmosomal cadherins, desmoplakin and plakoglobin, and a highly mobile plakophilin (Pkp2a). As desmosomes mature from Ca2+ dependence to Ca2+-independent hyper-adhesion, their stability increases, but Pkp2a remains highly mobile. We show that desmosome downregulation during growth-factor-induced cell scattering proceeds by internalisation of whole desmosomes, which still retain a stable moiety and highly mobile Pkp2a. This molecular mobility of Pkp2a suggests a transient and probably regulatory role for Pkp2a in desmosomes. This article has an associated First Person interview with the first author of the paper.

Actinic keratosis (AK), also known as solar keratosis, are thick, scaly or crusty skin commonly found on older, fair-skinned individuals due to chronic exposure to ultraviolet radiation (UVR) from sunlight. Premalignant AK is an incipient form of squamous cell carcinoma (SCC) [ [1] Carpenter P.M. Linden K.G. McLaren C.E. Li K.T. Arain S. Barr R.J. et al. Nuclear morphometry and molecular biomarkers of actinic keratosis, sun-damaged, and nonexposed skin. Cancer Epidemiol. Biomark. Prev. 2004; 13: 1996-2002 PubMed Google Scholar ]. UVR is also a potent inducer of cellular senescence, a state of irreversible growth arrest that is essential during embryonic development and tissue repair [ 2 Wang A.S. Dreesen O. Biomarkers of cellular senescence and skin aging. Front. Genet. 2018; Crossref Scopus (134) Google Scholar , 3 Fitsiou E. Pulido T. Campisi J. Alimirah F. Demaria M. Cellular senescence and the senescence-associated secretory phenotype as drivers of skin photoaging. J. Invest. Dermatol. 2021; 141: 1119-1126 Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar ]. Senescent cells accumulate in UV-induced skin pathologies and premalignant lesions [ 4 Collado M. Gil J. Efeyan A. Guerra C. Schuhmacher A.J. Barradas M. Benguría A. Zaballos A. Flores J.M. Barbacid M. Beach D. Serrano M. Tumour biology: senescence in premalignant tumours. Nature. 2005; 436: 642 Crossref PubMed Scopus (1128) Google Scholar , 5 Ivanov A. Pawlikowski J. Manoharan I. Van Tuyn J. Nelson D.M. Rai T.S. Shah P.P. Hewitt G. Korolchuk V.I. Passos J.F. Wu H. Berger S.L. Adams P.D. Lysosome-mediated processing of chromatin in senescence. J. Cell Biol. 2013; 202: 129-143 Crossref PubMed Scopus (283) Google Scholar , 6 Wang A.S. Ong P.F. Chojnowski A. Clavel C. Dreesen O. Loss of lamin B1 is a biomarker to quantify cellular senescence in photoaged skin. Sci. Rep. 2017; 7 Google Scholar ] and express a number of biomarkers, including senescence associated-β-galactosidase (SA-β-gal) activity, up-regulation of cell cycle regulators such as p53, p21WAF1 and p16INK4a, and increased secretion of cytokines, chemokines and growth factors, collectively termed as senescence-associated secretory phenotype (SASP) [ 2 Wang A.S. Dreesen O. Biomarkers of cellular senescence and skin aging. Front. Genet. 2018; Crossref Scopus (134) Google Scholar , 3 Fitsiou E. Pulido T. Campisi J. Alimirah F. Demaria M. Cellular senescence and the senescence-associated secretory phenotype as drivers of skin photoaging. J. Invest. Dermatol. 2021; 141: 1119-1126 Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar ]. Due to technical limitations and cell type specific differences, it remains challenging to adequately quantify senescent cells within complex tissues such as human skin. To circumvent these limitations, we and others identified lamin B1 and high mobility group box 1 (HMGB1) as novel senescence markers in vitro and in vivo. Lamin B1 levels decrease in various skin cell types in vitro during replicative, UV- and oncogene-induced senescence [ 6 Wang A.S. Ong P.F. Chojnowski A. Clavel C. Dreesen O. Loss of lamin B1 is a biomarker to quantify cellular senescence in photoaged skin. Sci. Rep. 2017; 7 Google Scholar , 7 Dreesen O. Chojnowski A. Ong P.F. Zhao T.Y. Common J.E. Lunny D. Lane E.B. Lee S.J. Vardy L.A. Stewart C.L. Colman A. Lamin B1 fluctuations have differential effects on cellular proliferation and senescence. J. Cell Biol. 2013; 200: 605-617 Crossref PubMed Scopus (142) Google Scholar , 8 Freund A. Laberge R.-M. Demaria M. Campisi J. Lamin B1 loss is a senescence-associated biomarker. Mol. Biol. Cell. 2012; 23: 2066-2075 Crossref PubMed Scopus (440) Google Scholar ], and in pre-neoplastic naevi [ [5] Ivanov A. Pawlikowski J. Manoharan I. Van Tuyn J. Nelson D.M. Rai T.S. Shah P.P. Hewitt G. Korolchuk V.I. Passos J.F. Wu H. Berger S.L. Adams P.D. Lysosome-mediated processing of chromatin in senescence. J. Cell Biol. 2013; 202: 129-143 Crossref PubMed Scopus (283) Google Scholar ] and chronologically-aged human skin in vivo [ [7] Dreesen O. Chojnowski A. Ong P.F. Zhao T.Y. Common J.E. Lunny D. Lane E.B. Lee S.J. Vardy L.A. Stewart C.L. Colman A. Lamin B1 fluctuations have differential effects on cellular proliferation and senescence. J. Cell Biol. 2013; 200: 605-617 Crossref PubMed Scopus (142) Google Scholar ], whilst HMGB1 was diminished in senescent fibroblasts in vitro [ [9] Davalos A.R. Kawahara M. Malhotra G.K. Schaum N. Huang J. Ved U. Beausejour C.M. Coppe J.P. Rodier F. Campisi J. p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes. J. Cell Biol. 2013; 201: 613-629 Crossref PubMed Scopus (231) Google Scholar ] and in aged skin in vivo [ [10] Victorelli S. Lagnado A. Halim J. Moore W. Talbot D. Barrett K. Chapman J. Birch J. Ogrodnik M. Meves A. Pawlikowski J.S. Jurk D. Adams P.D. Heemst D. Beekman M. Slagboom P.E. Gunn D.A. Passos J.F. Senescent human melanocytes drive skin ageing via paracrine telomere dysfunction. EMBO J. 2019; 38e101982 Crossref PubMed Scopus (58) Google Scholar ]. In this study, we aim to use lamin B1 and HMGB1 as biomarkers to rapidly detect and quantify senescent cell types in AK lesions in vivo.

BACKGROUND & AIMS: There are few in vitro models for studying the 3-dimensional interactions among different liver cell types during organogenesis or disease development. We aimed to generate hepatic organoids that comprise different parenchymal liver cell types and have structural features of the liver, using human pluripotent stem cells. METHODS: We cultured H1 human embryonic stem cells (WA-01, passage 27-40) and induced pluripotent stem cells (GM23338) with a series of chemically defined and serum-free media to induce formation of posterior foregut cells, which were differentiated in 3 dimensions into hepatic endoderm spheroids and stepwise into hepatoblast spheroids. Hepatoblast spheroids were reseeded in a high-throughput format and induced to form hepatic organoids; development of functional bile canaliculi was imaged live. Levels of albumin and apolipoprotein B were measured in cell culture supernatants using an enzyme-linked immunosorbent assay. Levels of gamma glutamyl transferase and alkaline phosphatase were measured in cholangiocytes. Organoids were incubated with troglitazone for varying periods and bile transport and accumulation were visualized by live-imaging microscopy. Organoids were incubated with oleic and palmitic acid, and formation of lipid droplets was visualized by staining. We compared gene expression profiles of organoids incubated with free fatty acids or without. We also compared gene expression profiles between liver tissue samples from patients with nonalcoholic steatohepatitis (NASH) versus without. We quantified hepatocyte and cholangiocyte populations in organoids using immunostaining and flow cytometry; cholangiocyte proliferation of cholangiocytes was measured. We compared the bile canaliculi network in the organoids incubated with versus without free fatty acids by live imaging. RESULTS: Cells in organoids differentiated into hepatocytes and cholangiocytes, based on the expression of albumin and cytokeratin 7. Hepatocytes were functional, based on secretion of albumin and apolipoprotein B and cytochrome P450 activity; cholangiocytes were functional, based on gamma glutamyl transferase and alkaline phosphatase activity and proliferative responses to secretin. The organoids organized a functional bile canaliculi system, which was disrupted by cholestasis-inducing drugs such as troglitazone. Organoids incubated with free fatty acids had gene expression signatures similar to those of liver tissues from patients with NASH. Incubation of organoids with free fatty acid-enriched media resulted in structural changes associated with nonalcoholic fatty liver disease, such as decay of bile canaliculi network and ductular reactions. CONCLUSIONS: We developed a hepatic organoid platform with human cells that can be used to model complex liver diseases, including NASH.

Advances in high-content high-throughput fluorescence microscopy have emerged as a powerful tool for several stages of drug discovery process, leading to the identification of a drug candidate with the potential for becoming a marketed drug. This high-content screening (HCS) technology has recently involved the application of machine learning methods for automated analysis of large amount of data generated from screening of large compound libraries to identify drug induced perturbations. However, high costs associated with large-scale HCS drug assays and the limitations of producing abundant high-quality data required to train machine learning models, pose major challenges. In this work, we have developed a computational framework based on deep convolutional generative adversarial network (DCGAN), for the generation of synthetic high-content imaging data to augment the limited real data. The proposed framework was applied on cell-based drug screening image data to derive phenotypic profiles of drug induced effects on the cells and to compute phenotypic similarities between different drugs. Such analysis can provide important insights into repurposing of previously approved drugs for different conditions. Moreover, a generative modeling-based approach of creating augmented datasets can allow to screen more drug compounds within the same imaging assay, thus reducing experimental costs.

The study of skin pigmentation requires determining the rate of melanin production in melanocytes and quantifying the rate of melanosome transfer to keratinocytes. Here, we describe a method to quantify melanosome transfer using immunofluorescence microscopy coupled with automated image analysis of in vitro human melanocytes and keratinocytes in co-culture. In this method, the number of melanin capped keratinocyte nuclei is quantified.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Alzheimer’s disease (AD), characterized by memory loss and cognitive decline, is one of the diseases with the highest attrition rate in drug development. As with other neurodegenerative diseases, AD manifests on several scales (molecular, cellular and organismal), resulting in an organismal phenotype that is difficult to replicate and exploit for screening in vitro. Development of high-throughput drug screening platforms using invertebrate organisms may therefore facilitate drug discovery in AD, at least for compounds that target mechanisms that can be modelled in invertebrates, such as metabolic defects and mitochondrial toxicity. Here we present a potentially high-throughput screening platform against AD in Caenorhabditis elegans (C. elegans). The system is based on our transgenic AD model strain (GRU102) that expresses a pathogenic human amyloid-beta peptide (Aβ1-42) specifically in neurons and we use this approach to identify Metformin, Lithium and Curcumin as potential “hits”. We find that the ability to rescue the swim-exhaustion phenotype correlates well with lifespan and healthspan improvements in GRU102. Importantly, several other drugs, such as Thioflavin T that have been reported previously to extend lifespan, modify aspects of ageing or for which protective effects in AD model might be suspected did not rescue the swim performance nor lifespan of GRU102. This illustrates the predictive value of the screening assay and confirms that not all compounds targeting ageing are capable of compensating for the toxic effects of Aβ1-42 in GRU102. Together, these findings demonstrate the utility of our screen towards the Aβ-induced defects in GRU102.