Gremlin 1+ fibroblastic niche maintains dendritic cell homeostasis in lymphoid tissues.

Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.

LRRC15+ myofibroblasts dictate the stromal setpoint to suppress tumour immunity.

Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15)1-3. However, the molecular signals that underlie the development of LRRC15+ cancer-associated fibroblasts (CAFs) and their direct impact on anti-tumour immunity are uncharacterized. Here in mouse models of pancreatic cancer, we provide in vivo genetic evidence that TGFß receptor type 2 signalling in healthy dermatopontin+ universal fibroblasts is essential for the development of cancer-associated LRRC15+ myofibroblasts. This axis also predominantly drives fibroblast lineage diversity in human cancers. Using newly developed Lrrc15-diphtheria toxin receptor knock-in mice to selectively deplete LRRC15+ CAFs, we show that depletion of this population markedly reduces the total tumour fibroblast content. Moreover, the CAF composition is recalibrated towards universal fibroblasts. This relieves direct suppression of tumour-infiltrating CD8+ T cells to enhance their effector function and augments tumour regression in response to anti-PDL1 immune checkpoint blockade. Collectively, these findings demonstrate that TGFß-dependent LRRC15+ CAFs dictate the tumour-fibroblast setpoint to promote tumour growth. These cells also directly suppress CD8+ T cell function and limit responsiveness to checkpoint blockade. Development of treatments that restore the homeostatic fibroblast setpoint by reducing the population of pro-disease LRRC15+ myofibroblasts may improve patient survival and response to immunotherapy.

Collapse of the hepatic gene regulatory network in the absence of FoxA factors.

The FoxA transcription factors are critical for liver development through their pioneering activity, which initiates a highly complex regulatory network thought to become progressively resistant to the loss of any individual hepatic transcription factor via mutual redundancy. To investigate the dispensability of FoxA factors for maintaining this regulatory network, we ablated all FoxA genes in the adult mouse liver. Remarkably, loss of FoxA caused rapid and massive reduction in the expression of critical liver genes. Activity of these genes was reduced back to the low levels of the fetal prehepatic endoderm stage, leading to necrosis and lethality within days. Mechanistically, we found FoxA proteins to be required for maintaining enhancer activity, chromatin accessibility, nucleosome positioning, and binding of HNF4α. Thus, the FoxA factors act continuously, guarding hepatic enhancer activity throughout adult life.

Cross-tissue organization of the fibroblast lineage.

Fibroblasts are non-haematopoietic structural cells that define the architecture of organs, support the homeostasis of tissue-resident cells and have key roles in fibrosis, cancer, autoimmunity and wound healing1. Recent studies have described fibroblast heterogeneity within individual tissues1. However, the field lacks a characterization of fibroblasts at single-cell resolution across tissues in healthy and diseased organs. Here we constructed fibroblast atlases by integrating single-cell transcriptomic data from about 230,000 fibroblasts across 17 tissues, 50 datasets, 11 disease states and 2 species. Mouse fibroblast atlases and a DptIRESCreERT2 knock-in mouse identified two universal fibroblast transcriptional subtypes across tissues. Our analysis suggests that these cells can serve as a reservoir that can yield specialized fibroblasts across a broad range of steady-state tissues and activated fibroblasts in disease. Comparison to an atlas of human fibroblasts from perturbed states showed that fibroblast transcriptional states are conserved between mice and humans, including universal fibroblasts and activated phenotypes associated with pathogenicity in human cancer, fibrosis, arthritis and inflammation. In summary, a cross-species and pan-tissue approach to transcriptomics at single-cell resolution has identified key organizing principles of the fibroblast lineage in health and disease.

Considerations for dual barrel electrode fabrication and experimentation.

New electrochemical probes offer the opportunity to investigate new systems. A dual barrel electrode can be laser pulled to produce micron-sized platinum disk electrodes. Here, we detail several important considerations for both the fabrication process and for experimental implimentation of the probe. We provide parameters for a Sutter P-2000 laser puller, methods for optical and electrochemical characterization, tips for how to successfully bevel the microelectrodes, and how salt concentrations and electrostatic discharge affect the voltammetry. This paper serves as a guide for how to successfully implement dual barrel electrodes from fabrication to experimentation.

OpenChemIE: An Information Extraction Toolkit for Chemistry Literature.

Information extraction from chemistry literature is vital for constructing up-to-date reaction databases for data-driven chemistry. Complete extraction requires combining information across text, tables, and figures, whereas prior work has mainly investigated extracting reactions from single modalities. In this paper, we present OpenChemIE to address this complex challenge and enable the extraction of reaction data at the document level. OpenChemIE approaches the problem in two steps: extracting relevant information from individual modalities and then integrating the results to obtain a final list of reactions. For the first step, we employ specialized neural models that each address a specific task for chemistry information extraction, such as parsing molecules or reactions from text or figures. We then integrate the information from these modules using chemistry-informed algorithms, allowing for the extraction of fine-grained reaction data from reaction condition and substrate scope investigations. Our machine learning models attain state-of-the-art performance when evaluated individually, and we meticulously annotate a challenging dataset of reaction schemes with R-groups to evaluate our pipeline as a whole, achieving an F1 score of 69.5%. Additionally, the reaction extraction results of OpenChemIE attain an accuracy score of 64.3% when directly compared against the Reaxys chemical database. OpenChemIE is most suited for information extraction on organic chemistry literature, where molecules are generally depicted as planar graphs or written in text and can be consolidated into a SMILES format. We provide OpenChemIE freely to the public as an open-source package, as well as through a web interface.

Relapsing Polychondritis as a Cause of Sudden and Unexpected Death With Central Nervous System Involvement.

ABSTRACT: Relapsing polychondritis (RP) is a rare inflammatory disease process that affects cartilaginous tissues throughout the body. Although the pathogenesis remains unknown, RP is thought to be an autoimmune disorder in which host immune cells are conditioned to attack the body's cartilage, such as the ears, nose, eyes, joints, and airways, resulting in inflammation and destruction of otherwise healthy tissues. In rare and unusual cases, neurological involvement has been described.We report a case of a 36-year-old man with a medical history of asthma and suspected seronegative rheumatoid arthritis/RP and panuveitis who was found deceased in his residence. Postmortem examination revealed cartilaginous destruction of the external ear and large airways and meningoencephalitis involving the left medial temporal lobe without an underlying infectious cause.Progressive destruction of airway tissue and increased susceptibility to pulmonary infection is the most common cause of death in RP. Central nervous system involvement is exceedingly rare, presenting with highly variable clinical and pathological manifestations. A review of RP and systemic manifestations will follow. Accurate recognition of this multisystem autoimmune disease as a cause of sudden and unexpected death is critical for proper death certification and to broaden our understanding of this disease.

Understanding levels of technology integration: A TPACK scale for EFL teachers to promote 21st-century learning.

Education worldwide has emphasized 21st-century competencies, including language competence, computer competence, and thinking skills. Research on Technological Pedagogical Content Knowledge (TPACK), essential teacher knowledge, has attempted to address the need for technology integration to support thinking skills. However, existing TPACK assessments have not intended to help teachers understand the levels of technology integration in teaching English as a foreign language (EFL). Therefore, this study proposed a two-dimensional TPACK scale, allowing EFL teachers to assess their TPACK in integrating technology and thinking skills. In total, 525 EFL teachers responded to this survey online. Scores of this scale were collected to test and establish validity and reliability. The statistical evidence showed that this instrument has high reliability and validity and is helpful for understanding levels of technology integration. The results showed that the EFL teachers were less confident in their TPACK teaching higher-order thinking skills. The EFL teachers in different cultures reported different confidence levels in TPACK and thinking skills (F(6, 518) = 7.83, p < .001). The high-achieving EFL teachers reported high TPACK self-efficacy (r = .210, p < .05). This TPACK survey would be helpful for EFL teachers to understand their development of TPACK in integrating technology and thinking skills in teaching English.

A High-Content Screen Identifies MicroRNAs That Regulate Liver Repopulation After Injury in Mice.

BACKGROUND & AIMS: Liver regeneration is impaired in mice with hepatocyte-specific deficiencies in microRNA (miRNA) processing, but it is not clear which miRNAs regulate this process. We developed a high-throughput screen to identify miRNAs that regulate hepatocyte repopulation after toxic liver injury using fumarylacetoacetate hydrolase-deficient mice. METHODS: We constructed plasmid pools encoding more than 30,000 tough decoy miRNA inhibitors (hairpin nucleic acids designed to specifically inhibit interactions between miRNAs and their targets) to target hepatocyte miRNAs in a pairwise manner. The plasmid libraries were delivered to hepatocytes in fumarylacetoacetate hydrolase-deficient mice at the time of liver injury via hydrodynamic tail-vein injection. Integrated transgene-containing transposons were quantified after liver repopulation via high-throughput sequencing. Changes in polysome-bound transcripts after miRNA inhibition were determined using translating ribosome affinity purification followed by high-throughput sequencing. RESULTS: Analyses of tough decoy abundance in hepatocyte genomic DNA and input plasmid pools identified several thousand miRNA inhibitors that were significantly depleted or increased after repopulation. We classified a subset of miRNA binding sites as those that have strong effects on liver repopulation, implicating the targeted hepatocyte miRNAs as regulators of this process. We then generated a high-content map of pairwise interactions between 171 miRNA-binding sites and identified synergistic and redundant effects. CONCLUSIONS: We developed a screen to identify miRNAs that regulate liver repopulation after injury in live mice.

Combinatorial genetics in liver repopulation and carcinogenesis with a in vivo CRISPR activation platform.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 activation (CRISPRa) systems have enabled genetic screens in cultured cell lines to discover and characterize drivers and inhibitors of cancer cell growth. We adapted this system for use in vivo to assess whether modulating endogenous gene expression levels can result in functional outcomes in the native environment of the liver. We engineered the catalytically dead CRISPR-associated 9 (dCas9)-positive mouse, cyclization recombination-inducible (Cre) CRISPRa system for cell type-specific gene activation in vivo. We tested the capacity for genetic screening in live animals by applying CRISPRa in a clinically relevant model of liver injury and repopulation. We targeted promoters of interest in regenerating hepatocytes using multiple single guide RNAs (gRNAs), and employed high-throughput sequencing to assess enrichment of gRNA sequences during liver repopulation and to link specific gRNAs to the initiation of carcinogenesis. All components of the CRISPRa system were expressed in a cell type-specific manner and activated endogenous gene expression in vivo. Multiple gRNA cassettes targeting a proto-oncogene were significantly enriched following liver repopulation, indicative of enhanced division of cells expressing the proto-oncogene. Furthermore, hepatocellular carcinomas developed containing gRNAs that activated this oncogene, indicative of cancer initiation events. Also, we employed our system for combinatorial cancer genetics in vivo as we found that while clonal hepatocellular carcinomas were dependent on the presence of the oncogene-inducing gRNAs, they were depleted for multiple gRNAs activating tumor suppressors. CONCLUSION: The in vivo CRISPRa platform developed here allows for parallel and combinatorial genetic screens in live animals; this approach enables screening for drivers and suppressors of cell replication and tumor initiation. (Hepatology 2017).

Normonatremic osmotic demyelination in the setting of acquired immune deficiency syndrome and malnutrition: case report and literature review.

In this report, we present the case of a 43-year-old woman with AIDS, disseminated aspergillosis, and malnutrition who developed osmotic demyelination syndrome. AIDS-related osmotic demyelination has only been documented in a handful of cases to date, and it appears independent of the classic mechanism of rapid correction of hyponatremia. In this manuscript, we review the six prior cases of osmotic demyelination in AIDS patients and compare their circumstances to that of our own patient. It appears that complications of malnutrition, possibly related to depletion of organic osmolytes in the central nervous system, may place AIDS patients at greater risk of osmotic demyelination. These, and other proposed mechanisms, deserve further inquiry.

Feasibility of the internal mammary lymph node flap as a vascularized lymph node transfer: A cadaveric dissection study.

BACKGROUND: We performed cadaveric dissections to examine the feasibility of an internal mammary-based lymph node flap as a donor site for vascularized lymph node transfer. METHODS: Internal mammary vessels and adjacent nodes were dissected in ten fresh cadaver specimens. Surgeon inspection and palpation identified the number of nodes in the specimen. Specimens were examined macro- and microscopically by a pathologist for correlation of lymph node counts. Kappa statistic correlated surgeon- and pathologist-reported node counts. RESULTS: Surgeon- and pathologist-reported node counts were moderately correlated (kappa 0.57). Inspection and palpation correctly predicted node presence or absence in 80% of specimens. Sixty percent of flaps contained between 1 and 3 nodes, with a mean of 2.0 nodes when nodes were present. CONCLUSIONS: Inspection and palpation predicts the presence or absence of nodes in 80% of flaps. Nodes were present in 60% of internal mammary-based flaps, and one to three nodes can be transferred. © 2015 Wiley Periodicals, Inc. Microsurgery 36:485-490, 2016.

Cognitive and Linguistic Predictors of Mathematical Word Problems With and Without Irrelevant Information.

The purpose of this study was to identify cognitive and linguistic predictors of word problems with versus without irrelevant information. The sample was 701 2nd-grade students who received no specialized intervention on word problems. In the fall, they were assessed on initial arithmetic and word-problem skill as well as language ability, working memory capacity, and processing speed; in the spring, they were tested on a word-problem measure that included items with versus without irrelevant information. Significant predictors common to both forms of word problems were initial arithmetic and word problem-solving skill as well as language and working memory. Nonverbal reasoning predicted word problems with irrelevant information, but not word problems without irrelevant information. Findings are discussed in terms of implications for intervention and future research.

Is Word-Problem Solving a Form of Text Comprehension?

This study's hypotheses were that (a) word-problem (WP) solving is a form of text comprehension that involves language comprehension processes, working memory, and reasoning, but (b) WP solving differs from other forms of text comprehension by requiring WP-specific language comprehension as well as general language comprehension. At the start of the 2nd grade, children (n = 206; on average, 7 years, 6 months) were assessed on general language comprehension, working memory, nonlinguistic reasoning, processing speed (a control variable), and foundational skill (arithmetic for WPs; word reading for text comprehension). In spring, they were assessed on WP-specific language comprehension, WPs, and text comprehension. Path analytic mediation analysis indicated that effects of general language comprehension on text comprehension were entirely direct, whereas effects of general language comprehension on WPs were partially mediated by WP-specific language. By contrast, effects of working memory and reasoning operated in parallel ways for both outcomes.

Phase I Study of ORIC-101, a Glucocorticoid Receptor Antagonist, in Combination with Enzalutamide in Patients with Metastatic Castration-resistant Prostate Cancer Progressing on Enzalutamide.

PURPOSE: Increased glucocorticoid receptor (GR) signaling is a proposed compensatory mechanism of resistance to androgen receptor (AR) inhibition in metastatic castration-resistant prostate cancer (mCRPC). ORIC-101 is a potent and selective orally-bioavailable GR antagonist. PATIENTS AND METHODS: Safety, pharmacokinetic/pharmacodynamic, and antitumor activity of ORIC-101 in combination with enzalutamide were studied in patients with mCRPC progressing on enzalutamide. ORIC-101 doses ranging from 80 to 240 mg once daily were tested in combination with enzalutamide 160 mg once daily. Pharmacokinetics/pharmacodynamics was assessed after a single dose and at steady state. Disease control rate (DCR) at 12 weeks was evaluated at the recommended phase 2 dose (RP2D). RESULTS: A total of 41 patients were enrolled. There were no dose-limiting toxicities and the RP2D was selected as 240 mg of ORIC-101 and 160 mg of enzalutamide daily. At the RP2D, the most common treatment-related adverse events were fatigue (38.7%), nausea (29.0%), decreased appetite (19.4%), and constipation (12.9%). Pharmacokinetic/pharmacodynamic data confirmed ORIC-101 achieved exposures necessary for GR target engagement. Overall, for 31 patients treated at the RP2D, there was insufficient clinical benefit based on DCR (25.8%; 80% confidence interval: 15.65-38.52) which did not meet the prespecified target rate, leading to termination of the study. Exploratory subgroup analyses based on baseline GR expression, presence of AR resistance variants, and molecular features of aggressive variant prostate cancer suggested possible benefit in patients with high GR expression and no other resistance markers, although this would require confirmation. CONCLUSIONS: Although the combination of ORIC-101 and enzalutamide demonstrated an acceptable tolerability profile, GR target inhibition with ORIC-101 did not produce clinical benefit in men with metastatic prostate cancer resistant to enzalutamide.

Combined PD-L1/TGFß blockade allows expansion and differentiation of stem cell-like CD8 T cells in immune excluded tumors.

TGFß signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFß signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFß and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFß/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFß therapy efficacy. Our data suggest that TGFß works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.

In vivo tumor immune microenvironment phenotypes correlate with inflammation and vasculature to predict immunotherapy response.

Response to immunotherapies can be variable and unpredictable. Pathology-based phenotyping of tumors into 'hot' and 'cold' is static, relying solely on T-cell infiltration in single-time single-site biopsies, resulting in suboptimal treatment response prediction. Dynamic vascular events (tumor angiogenesis, leukocyte trafficking) within tumor immune microenvironment (TiME) also influence anti-tumor immunity and treatment response. Here, we report dynamic cellular-level TiME phenotyping in vivo that combines inflammation profiles with vascular features through non-invasive reflectance confocal microscopic imaging. In skin cancer patients, we demonstrate three main TiME phenotypes that correlate with gene and protein expression, and response to toll-like receptor agonist immune-therapy. Notably, phenotypes with high inflammation associate with immunostimulatory signatures and those with high vasculature with angiogenic and endothelial anergy signatures. Moreover, phenotypes with high inflammation and low vasculature demonstrate the best treatment response. This non-invasive in vivo phenotyping approach integrating dynamic vasculature with inflammation serves as a reliable predictor of response to topical immune-therapy in patients.

The Role of T-box Transcription Factor in a Pituitary Adenoma Diagnostic Algorithm.

CONTEXT.­: We previously examined pituitary adenomas with immunohistochemical (IHC) stains for steroidogenic factor 1, Pit-1, anterior pituitary hormones, cytokeratin CAM 5.2, and the α-subunit of human chorionic gonadotropin and found that a screening panel comprising stains for steroidogenic factor 1, Pit-1, and adrenocorticotropic hormone successfully classified most cases and reduced the overall number of stains required. OBJECTIVES.­: To examine the potential role of IHC stain for T-box transcription factor (Tpit) in the classification of our series of pituitary adenomas and to update our screening panel as necessary. DESIGN.­: We collected 157 pituitary adenomas from 2 institutions and included these in tissue microarrays. Immunostains for Tpit were scored in a blinded fashion using the Allred system. Adenomas were assigned to a gold standard class based on IHC pattern followed by application of available clinical and serologic information. Test characteristics were calculated. Correlation analyses, cluster analyses, and classification tree analyses were used to see whether IHC staining patterns reliably reflected adenoma class. RESULTS.­: Of the cases collected, 147 (93.6%) had sufficient material for Tpit analysis. IHC stain for Tpit identified 8 null cell adenomas (all nonfunctioning clinically) as silent corticotrophs; Tpit stains showed better sensitivity, specificity, positive predictive value, and negative predictive value than IHC for adrenocorticotropic hormone and cytokeratin CAM 5.2. Correlation analyses continued to show the expected relationships among IHC stains. Cluster analyses showed grouping of adenomas into clinically consistent groups. Classification tree analysis underscored the central role of transcription factor IHC stains, including Tpit, in adenoma classification. CONCLUSIONS.­: Substitution of Tpit stain for the adrenocorticotropic hormone stain improves our prior algorithm by reducing the number of false-negatives and false-positives. As a result, fewer adenomas are classified as null cell adenoma, and more adenomas are classified as silent corticotroph adenoma.

Oxycodone prescribing in the emergency department during the opioid crisis.

OBJECTIVE: Misuse of prescription opioids is a significant public health issue in Australia. There has been a rapid rise in prescription opioid use, with an associated increase in overdose and death. The over-prescribing of oral opioids, especially oxycodone, in the ED has been identified as a contributor to this problem overseas. It is unclear if similar practice occurs in the Australian ED. The primary aim of our study was to identify the incidence of oral oxycodone administration to patients within the ED. The secondary outcome was to identify the incidence of oxycodone prescribed to patients on discharge from the ED into the community. METHODS: Our study was designed as an observational, retrospective data analysis of the incidence of oxycodone prescribed within the three EDs of a large Australian public health service. All immediate-release (IR) and slow-release (SR) oral oxycodone prescribed over a 4-year period (2015-2018) was included. RESULTS: There were 890 557 presentations to the three EDs during the period, which resulted in 288 242 episodes of oxycodone administration within department, equivalent to 324 administrations per 1000 presentations. There were 39 381 prescriptions for oxycodone provided on discharge, resulting in an incidence of 44 prescriptions per 1000 discharged. The most frequently prescribed opioid medication in the ED was oxycodone IR 5 mg, 78.6% of discharge prescriptions generated provided a maximum quantity (20 for IR formulation or 28 for SR) of tablets allowable under the pharmaceutical benefits scheme. CONCLUSIONS: There is a higher incidence of oxycodone prescribing in the Australian ED than previously recognised. An overuse of oxycodone may be contributing to adverse patient outcomes and a public health crisis. Hospitals should consider appropriate steps to reduce the incidence of opioid prescribing and the supply of these medications into the community.