A heterotrimeric complex of Toxoplasma proteins promotes parasite survival in interferon gamma-stimulated human cells.

Toxoplasma gondii secretes protein effectors to subvert the human immune system sufficiently to establish a chronic infection. Relative to murine infections, little is known about which parasite effectors disarm human immune responses. Here, we used targeted CRISPR screening to identify secreted protein effectors required for parasite survival in IFNγ-activated human cells. Independent screens were carried out using 2 Toxoplasma strains that differ in virulence in mice, leading to the identification of effectors required for survival in IFNγ-activated human cells. We identify the secreted protein GRA57 and 2 other proteins, GRA70 and GRA71, that together form a complex which enhances the ability of parasites to persist in IFNγ-activated human foreskin fibroblasts (HFFs). Components of the protein machinery required for export of Toxoplasma proteins into the host cell were also found to be important for parasite resistance to IFNγ in human cells, but these export components function independently of the identified protein complex. Host-mediated ubiquitination of the parasite vacuole has previously been associated with increased parasite clearance from human cells, but we find that vacuoles from GRA57, GRA70, and GRA71 knockout strains are surprisingly less ubiquitinated by the host cell. We hypothesise that this is likely a secondary consequence of deletion of the complex, unlinked to the IFNγ resistance mediated by these effectors.

Author Correction: Metastatic-niche labelling reveals parenchymal cells with stem features.

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

Metastatic-niche labelling reveals parenchymal cells with stem features.

Direct investigation of the early cellular changes induced by metastatic cells within the surrounding tissue remains a challenge. Here we present a system in which metastatic cancer cells release a cell-penetrating fluorescent protein, which is taken up by neighbouring cells and enables spatial identification of the local metastatic cellular environment. Using this system, tissue cells with low representation in the metastatic niche can be identified and characterized within the bulk tissue. To highlight its potential, we applied this strategy to study the cellular environment of metastatic breast cancer cells in the lung. We report the presence of cancer-associated parenchymal cells, which exhibit stem-cell-like features, expression of lung progenitor markers, multi-lineage differentiation potential and self-renewal activity. In ex vivo assays, lung epithelial cells acquire a cancer-associated parenchymal-cell-like phenotype when co-cultured with cancer cells and support their growth. These results highlight the potential of this method as a platform for new discoveries.

RASSF8-mediated transport of Echinoid via the exocyst promotes Drosophila wing elongation and epithelial ordering.

Cell-cell junctions are dynamic structures that maintain cell cohesion and shape in epithelial tissues. During development, junctions undergo extensive rearrangements to drive the epithelial remodelling required for morphogenesis. This is particularly evident during axis elongation, where neighbour exchanges, cell-cell rearrangements and oriented cell divisions lead to large-scale alterations in tissue shape. Polarised vesicle trafficking of junctional components by the exocyst complex has been proposed to promote junctional rearrangements during epithelial remodelling, but the receptors that allow exocyst docking to the target membranes remain poorly understood. Here, we show that the adherens junction component Ras Association domain family 8 (RASSF8) is required for the epithelial re-ordering that occurs during Drosophila pupal wing proximo-distal elongation. We identify the exocyst component Sec15 as a RASSF8 interactor. Loss of RASSF8 elicits cytoplasmic accumulation of Sec15 and Rab11-containing vesicles. These vesicles also contain the nectin-like homophilic adhesion molecule Echinoid, the depletion of which phenocopies the wing elongation and epithelial packing defects observed in RASSF8 mutants. Thus, our results suggest that RASSF8 promotes exocyst-dependent docking of Echinoid-containing vesicles during morphogenesis.

Deep learning for automatic segmentation of the nuclear envelope in electron microscopy data, trained with volunteer segmentations.

Advancements in volume electron microscopy mean it is now possible to generate thousands of serial images at nanometre resolution overnight, yet the gold standard approach for data analysis remains manual segmentation by an expert microscopist, resulting in a critical research bottleneck. Although some machine learning approaches exist in this domain, we remain far from realizing the aspiration of a highly accurate, yet generic, automated analysis approach, with a major obstacle being lack of sufficient high-quality ground-truth data. To address this, we developed a novel citizen science project, Etch a Cell, to enable volunteers to manually segment the nuclear envelope (NE) of HeLa cells imaged with serial blockface scanning electron microscopy. We present our approach for aggregating multiple volunteer annotations to generate a high-quality consensus segmentation and demonstrate that data produced exclusively by volunteers can be used to train a highly accurate machine learning algorithm for automatic segmentation of the NE, which we share here, in addition to our archived benchmark data.

PTPN23 binds the dynein adaptor BICD1 and is required for endocytic sorting of neurotrophin receptors.

Signalling by target-derived neurotrophins is essential for the correct development of the nervous system and its maintenance throughout life. Several aspects concerning the lifecycle of neurotrophins and their receptors have been characterised over the years, including the formation, endocytosis and trafficking of signalling-competent ligand-receptor complexes. However, the molecular mechanisms directing the sorting of activated neurotrophin receptors are still elusive. Previously, our laboratory identified Bicaudal-D1 (BICD1), a dynein motor adaptor, as a key factor for lysosomal degradation of brain-derived neurotrophic factor (BDNF)-activated TrkB (also known as NTRK2) and p75NTR (also known as NGFR) in motor neurons. Here, using a proteomics approach, we identified protein tyrosine phosphatase, non-receptor type 23 (PTPN23), a member of the endosomal sorting complexes required for transport (ESCRT) machinery, in the BICD1 interactome. Molecular mapping revealed that PTPN23 is not a canonical BICD1 cargo; instead, PTPN23 binds the N-terminus of BICD1, which is also essential for the recruitment of cytoplasmic dynein. In line with the BICD1-knockdown phenotype, loss of PTPN23 leads to increased accumulation of BDNF-activated p75NTR and TrkB in swollen vacuole-like compartments, suggesting that neuronal PTPN23 is a novel regulator of the endocytic sorting of neurotrophin receptors.

Parent and patient perceptions of medical marijuana in the childhood cancer context.

BACKGROUND: Medical marijuana (MM) is legal in 34 US jurisdictions. Yet, little is known about patient and parent perceptions of MM in pediatric cancer care. We examined attitudes, beliefs, and experiences regarding MM among parents of children with cancer and adolescent and young adult (AYA) patients, to help frame future research initiatives. PROCEDURE: In this qualitative study, we conducted semi-structured, one-on-one interviews with parents and AYAs at a comprehensive cancer center. Interviews were audio-recorded, transcribed, and coded using both descriptive and inductive coding approaches. We used content and framework analysis to identify key themes. RESULTS: Fifteen parents and 15 AYAs enrolled. Participants were generally receptive to MM use, concurrently weighing benefits and risks. Participants most often endorsed MM use for relief of nausea, anorexia, and pain. Simultaneously, participants identified concerns about MM, including potential physiologic and psychological effects on children and lack of research. However, concerns were frequently minimized, relative to chemotherapy or supportive care medications with perceived greater side effect profiles. Many participants expressed uncertainty regarding legal access, citing complex processes to obtain MM. Few participants had discussed MM with their oncologist, instead seeking guidance from the internet, family, or peers. Importantly, we elicited several misconceptions regarding MM, including its utility as cancer-directed therapy. CONCLUSION: Patients and families are receptive to using MM, motivated by potential for symptom relief and cancer-directed effects. Yet, lack of empiric evidence is a barrier, underscoring the need for robust clinical trial data to support MM recommendations and use.

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy.

The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research.

Medical marijuana in pediatric oncology: A review of the evidence and implications for practice.

Medical marijuana (MM) has become increasingly legal at the state level and accessible to children with serious illness. Pediatric patients with cancer may be particularly receptive to MM, given purported benefits in managing cancer-related symptoms. In this review, we examine the evidence for MM as a supportive care agent in pediatric oncology. We describe the current legal status of MM, mechanism of action, common formulations, and potential benefits versus risks for pediatric oncology patients. We offer suggestions for how providers might approach MM requests. Throughout, we comment on avenues for future investigation on this growing trend in supportive care.

Generation of left ventricle-like cardiomyocytes with improved structural, functional, and metabolic maturity from human pluripotent stem cells.

Decreased left ventricle (LV) function caused by genetic mutations or injury often leads to debilitating and fatal cardiovascular disease. LV cardiomyocytes are, therefore, a potentially valuable therapeutical target. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are neither homogeneous nor functionally mature, which reduces their utility. Here, we exploit cardiac development knowledge to instruct differentiation of hPSCs specifically toward LV cardiomyocytes. Correct mesoderm patterning and retinoic acid pathway blocking are essential to generate near-homogenous LV-specific hPSC-CMs (hPSC-LV-CMs). These cells transit via first heart field progenitors and display typical ventricular action potentials. Importantly, hPSC-LV-CMs exhibit increased metabolism, reduced proliferation, and improved cytoarchitecture and functional maturity compared with age-matched cardiomyocytes generated using the standard WNT-ON/WNT-OFF protocol. Similarly, engineered heart tissues made from hPSC-LV-CMs are better organized, produce higher force, and beat more slowly but can be paced to physiological levels. Together, we show that functionally matured hPSC-LV-CMs can be obtained rapidly without exposure to current maturation regimes.

Expedited postpartum discharge during the COVID-19 pandemic and acute postpartum care utilization.

BACKGROUND: Early postpartum discharges increased organically during the COVID-19 pandemic. It is not known if this 'natural experiment' of shorter postpartum hospital stays resulted in increased risk for postpartum readmissions and other acute postpartum care utilization such as emergency room encounters. OBJECTIVE: The objectives of this study were to determine which clinical factors were associated with expedited postpartum discharge and whether the expedited postpartum discharge was associated with increased risk for acute postpartum care utilization. METHODS: This retrospective cohort study evaluated birth hospitalizations at affiliated hospitals during two periods: (i) the apex of the 'first wave' of the COVID-19 pandemic in New York City (3/22/20 to 4/30/20) and (ii) a historical control period of one year earlier (3/22/19 to 4/30/19). Routine postpartum discharge was defined as ≥2 d after vaginal birth and ≥3 d after cesarean birth. Expedited discharge was defined as <2 d after vaginal birth and <3 d after cesarean birth. Acute postpartum care utilization was defined as any emergency room visit, obstetric triage visit, or postpartum readmission ≤6 weeks after birth hospitalization discharge. Demographic and clinical variables were compared based on routine versus expedited postpartum discharge. Unadjusted and adjusted logistic regression models were performed to analyze factors associated with (i) expedited discharge and (ii) acute postpartum care utilization. Unadjusted (ORs) and adjusted odds ratios (aORs) with 95% CIs were used as measures of association. Stratified analysis was performed restricted to patients with chronic hypertension, preeclampsia, and gestational hypertension. RESULTS: A total of 1,358 birth hospitalizations were included in the analysis, 715 (52.7%) from 2019 and 643 (47.3%) from 2020. Expedited discharge was more common in 2020 than in 2019 (60.3% versus 5.0% of deliveries, p < .01). For 2020, clinical factors significantly associated with a decreased likelihood of expedited discharge included hypertensive disorders of pregnancy (OR 0.40, 95% CI 0.27-0.60), chronic hypertension (OR 0.14, 95% CI 0.06-0.29), and COVID-19 infection (OR 0.51, 95% CI 0.34-0.77). Cesarean (OR 3.00, 95% CI 2.14-4.19) and term birth (OR 3.34, 95% CI 2.03, 5.49) were associated with an increased likelihood of expedited discharge. Most of the associations retained significance in adjusted models. Expedited compared to routine discharge was not associated with significantly different odds of acute postpartum care utilization for 2020 deliveries (5.4% versus 5.9%; OR 0.92, 95% CI 0.47-1.82). Medicaid insurance (OR 2.30, 95% CI 1.06-4.98) and HDP (OR 5.16, 95% CI: 2.60-10.26) were associated with a higher risk of acute postpartum care utilization and retained significance in adjusted analyses. In the stratified analysis restricted to women with hypertensive diagnoses, expedited discharge was associated with significantly increased risk for postpartum readmission (OR 6.09, 95% CI 2.14, 17.33) but not overall acute postpartum care utilization (OR 2.17, 95% CI 1.00, 4.74). CONCLUSION: Expedited postpartum discharge was not associated with increased risk for acute postpartum care utilization. Among women with hypertensive diagnoses, expedited discharge was associated with a higher risk for readmission despite expedited discharge occurring less frequently.

CAR-associated vesicular transport of an adenovirus in motor neuron axons.

Axonal transport is responsible for the movement of signals and cargo between nerve termini and cell bodies. Pathogens also exploit this pathway to enter and exit the central nervous system. In this study, we characterised the binding, endocytosis and axonal transport of an adenovirus (CAV-2) that preferentially infects neurons. Using biochemical, cell biology, genetic, ultrastructural and live-cell imaging approaches, we show that interaction with the neuronal membrane correlates with coxsackievirus and adenovirus receptor (CAR) surface expression, followed by endocytosis involving clathrin. In axons, long-range CAV-2 motility was bidirectional with a bias for retrograde transport in nonacidic Rab7-positive organelles. Unexpectedly, we found that CAR was associated with CAV-2 vesicles that also transported cargo as functionally distinct as tetanus toxin, neurotrophins, and their receptors. These results suggest that a single axonal transport carrier is capable of transporting functionally distinct cargoes that target different membrane compartments in the soma. We propose that CAV-2 transport is dictated by an innate trafficking of CAR, suggesting an unsuspected function for this adhesion protein during neuronal homeostasis.

Semantic segmentation of HeLa cells: An objective comparison between one traditional algorithm and four deep-learning architectures.

The quantitative study of cell morphology is of great importance as the structure and condition of cells and their structures can be related to conditions of health or disease. The first step towards that, is the accurate segmentation of cell structures. In this work, we compare five approaches, one traditional and four deep-learning, for the semantic segmentation of the nuclear envelope of cervical cancer cells commonly known as HeLa cells. Images of a HeLa cancer cell were semantically segmented with one traditional image-processing algorithm and four three deep learning architectures: VGG16, ResNet18, Inception-ResNet-v2, and U-Net. Three hundred slices, each 2000 × 2000 pixels, of a HeLa Cell were acquired with Serial Block Face Scanning Electron Microscopy. The first three deep learning architectures were pre-trained with ImageNet and then fine-tuned with transfer learning. The U-Net architecture was trained from scratch with 36, 000 training images and labels of size 128 × 128. The image-processing algorithm followed a pipeline of several traditional steps like edge detection, dilation and morphological operators. The algorithms were compared by measuring pixel-based segmentation accuracy and Jaccard index against a labelled ground truth. The results indicated a superior performance of the traditional algorithm (Accuracy = 99%, Jaccard = 93%) over the deep learning architectures: VGG16 (93%, 90%), ResNet18 (94%, 88%), Inception-ResNet-v2 (94%, 89%), and U-Net (92%, 56%).

Choices, attitudes, and experiences of genetic screening in Latino/a and Ashkenazi Jewish individuals.

Genetic screening to inform personal risk has only recently become an option as the cost of sequencing decreases, and our ability to interpret sequence variants improves. Studies have demonstrated that people want to learn about their genetic information and do well after learning it, but minorities are underrepresented in these studies. We surveyed Ashkenazi Jewish (AJ) and Latino/a participants in a genetic screening study to solicit choices about genetic results to return, as well as their experience with learning these results and attitudes about genetic information secrecy and security. Participants had the option to proceed through the study self-guided, and few elected to have traditional pre-test genetic education and counseling. Despite this, the majority were satisfied with the process of selecting and receiving genetic results and felt that they understood their results. Concerns about privacy and confidentiality of genetic data were minimal, though some participants expressed modest concerns about keeping any potential results secret or the confidentiality of their genetic information. Our results support the feasibility of the option of self-guided genetic screening. Additional care will need to be taken when designing population-based screening studies to meet the needs of participants who come from communities with different experiences with genetics.

Engineering transplantable jejunal mucosal grafts using patient-derived organoids from children with intestinal failure.

Intestinal failure, following extensive anatomical or functional loss of small intestine, has debilitating long-term consequences for children1. The priority of patient care is to increase the length of functional intestine, particularly the jejunum, to promote nutritional independence2. Here we construct autologous jejunal mucosal grafts using biomaterials from pediatric patients and show that patient-derived organoids can be expanded efficiently in vitro. In parallel, we generate decellularized human intestinal matrix with intact nanotopography, which forms biological scaffolds. Proteomic and Raman spectroscopy analyses reveal highly analogous biochemical profiles of human small intestine and colon scaffolds, indicating that they can be used interchangeably as platforms for intestinal engineering. Indeed, seeding of jejunal organoids onto either type of scaffold reliably reconstructs grafts that exhibit several aspects of physiological jejunal function and that survive to form luminal structures after transplantation into the kidney capsule or subcutaneous pockets of mice for up to 2 weeks. Our findings provide proof-of-concept data for engineering patient-specific jejunal grafts for children with intestinal failure, ultimately aiding in the restoration of nutritional autonomy.

Segmentation and Modelling of the Nuclear Envelope of HeLa Cells Imaged with Serial Block Face Scanning Electron Microscopy.

This paper describes an unsupervised algorithm, which segments the nuclear envelope of HeLa cells imaged by Serial Block Face Scanning Electron Microscopy. The algorithm exploits the variations of pixel intensity in different cellular regions by calculating edges, which are then used to generate superpixels. The superpixels are morphologically processed and those that correspond to the nuclear region are selected through the analysis of size, position, and correspondence with regions detected in neighbouring slices. The nuclear envelope is segmented from the nuclear region. The three-dimensional segmented nuclear envelope is then modelled against a spheroid to create a two-dimensional (2D) surface. The 2D surface summarises the complex 3D shape of the nuclear envelope and allows the extraction of metrics that may be relevant to characterise the nature of cells. The algorithm was developed and validated on a single cell and tested in six separate cells, each with 300 slices of 2000 × 2000 pixels. Ground truth was available for two of these cells, i.e., 600 hand-segmented slices. The accuracy of the algorithm was evaluated with two similarity metrics: Jaccard Similarity Index and Mean Hausdorff distance. Jaccard values of the first/second segmentation were 93%/90% for the whole cell, and 98%/94% between slices 75 and 225, as the central slices of the nucleus are more regular than those on the extremes. Mean Hausdorff distances were 9/17 pixels for the whole cells and 4/13 pixels for central slices. One slice was processed in approximately 8 s and a whole cell in 40 min. The algorithm outperformed active contours in both accuracy and time.

Individual response variations in scaffold-guided bone regeneration are determined by independent strain- and injury-induced mechanisms.

This study explored the regenerative osteogenic response in the distal femur of sheep using scaffolds having stiffness values within, and above and below, the range of trabecular bone apparent modulus. Scaffolds 3D-printed from stiff titanium and compliant polyamide were implanted into a cylindrical metaphyseal defect 15 × 15 mm. After six weeks, bone ingrowth varied between 7 and 21% of the scaffold pore volume and this was generally inversely proportional to scaffold stiffness. The individual reparative response considerably varied among the animals, which could be divided into weak and strong responders. Notably, bone regeneration specifically within the interior of the scaffold was inversely proportional to scaffold stiffness and was strain-driven in strongly-responding animals. Conversely, bone regeneration at the periphery of the defect was injury-driven and equal in all scaffolds and in all strongly- and weakly-responding animals. The observation of the strain-driven response in some, but not all, animals highlights that scaffold compliance is desirable for triggering host bone regeneration, but scaffold permanence is important for the load-bearing, structural role of the bone-replacing device. Indeed, scaffolds may benefit from being nonresorbable and mechanically reliable for those unforeseeable cases of weakly responding recipients.

A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion.

Cancer-associated fibroblasts (CAFs) promote tumour invasion and metastasis. We show that CAFs exert a physical force on cancer cells that enables their collective invasion. Force transmission is mediated by a heterophilic adhesion involving N-cadherin at the CAF membrane and E-cadherin at the cancer cell membrane. This adhesion is mechanically active; when subjected to force it triggers ß-catenin recruitment and adhesion reinforcement dependent on α-catenin/vinculin interaction. Impairment of E-cadherin/N-cadherin adhesion abrogates the ability of CAFs to guide collective cell migration and blocks cancer cell invasion. N-cadherin also mediates repolarization of the CAFs away from the cancer cells. In parallel, nectins and afadin are recruited to the cancer cell/CAF interface and CAF repolarization is afadin dependent. Heterotypic junctions between CAFs and cancer cells are observed in patient-derived material. Together, our findings show that a mechanically active heterophilic adhesion between CAFs and cancer cells enables cooperative tumour invasion.

Targeting protein homeostasis in sporadic inclusion body myositis.

Sporadic inclusion body myositis (sIBM) is the commonest severe myopathy in patients more than 50 years of age. Previous therapeutic trials have targeted the inflammatory features of sIBM but all have failed. Because protein dyshomeostasis may also play a role in sIBM, we tested the effects of targeting this feature of the disease. Using rat myoblast cultures, we found that up-regulation of the heat shock response with arimoclomol reduced key pathological markers of sIBM in vitro. Furthermore, in mutant valosin-containing protein (VCP) mice, which develop an inclusion body myopathy, treatment with arimoclomol ameliorated disease pathology and improved muscle function. We therefore evaluated arimoclomol in an investigator-led, randomized, double-blind, placebo-controlled, proof-of-concept trial in sIBM patients and showed that arimoclomol was safe and well tolerated. Although arimoclomol improved some IBM-like pathology in the mutant VCP mouse, we did not see statistically significant evidence of efficacy in the proof-of-concept patient trial.

Modeling human neural functionality in vitro: three-dimensional culture for dopaminergic differentiation.

Advances in mechanistic knowledge of human neurological disorders have been hindered by the lack of adequate human in vitro models. Three-dimensional (3D) cellular models displaying higher biological relevance are gaining momentum; however, their lack of robustness and scarcity of analytical tools adapted to three dimensions hampers their widespread implementation. Herein we show that human midbrain-derived neural progenitor cells, cultured as 3D neurospheres in stirred culture systems, reproducibly differentiate into complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Moreover, an extensive toolbox of analytical methodologies has been adapted to 3D neural cell models, allowing molecular and phenotypic profiling and interrogation. The generated neurons underwent synaptogenesis and elicit spontaneous Ca(2+) transients. Synaptic vesicle trafficking and release of dopamine in response to depolarizing stimuli was also observed. Under whole-cell current-and-voltage clamp, recordings showed polarized neurons (Vm=-70 mV) and voltage-dependent potassium currents, which included A-type-like currents. Glutamate-induced currents sensitive to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate antagonists revealed the existence of functional glutamate receptors. Molecular and phenotypic profiling showed recapitulation of midbrain patterning events, and remodeling toward increased similarity to human brain features, such as extracellular matrix composition and metabolic signature. We have developed a robust and reproducible human 3D neural cell model, which may be extended to patient-derived induced pluripotent stem cells, broadening the applicability of this model.