IL-1ß Inhibition Partially Negates the Beneficial Effects of Diet-Induced Atherosclerosis Regression in Mice.

BACKGROUND: Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions is the global leading cause of death. The most common and effective means to reduce these major adverse cardiovascular events, including myocardial infarction and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, we know little regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. METHODS: Smooth muscle cell lineage-tracing Apoe-/- mice were fed a high-cholesterol Western diet for 18 weeks and then a zero-cholesterol standard laboratory diet for 12 weeks before treating them with an IL (interleukin)-1ß or control antibody for 8 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of smooth muscle cell and other lesion cells by smooth muscle cell lineage tracing combined with single-cell RNA sequencing, cytometry by time-of-flight, and immunostaining plus high-resolution confocal microscopic z-stack analysis. RESULTS: Lipid lowering by switching Apoe-/- mice from a Western diet to a standard laboratory diet reduced LDL cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden, as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1ß antibody treatment after diet-induced reductions in lipids resulted in multiple detrimental changes including increased plaque burden and brachiocephalic artery lesion size, as well as increasedintraplaque hemorrhage, necrotic core area, and senescence as compared with IgG control antibody-treated mice. Furthermore, IL-1ß antibody treatment upregulated neutrophil degranulation pathways but downregulated smooth muscle cell extracellular matrix pathways likely important for the protective fibrous cap. CONCLUSIONS: Taken together, IL-1ß appears to be required for the maintenance of standard laboratory diet-induced reductions in plaque burden and increases in multiple indices of plaque stability.

IL-1ß inhibition partially negates the beneficial effects of diet-induced lipid lowering.

Background: Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions are the leading cause of death in the world. The most common and effective means to reduce these major adverse cardiovascular events (MACE), including myocardial infarction (MI) and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, little is known regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. Methods: Smooth muscle cell (SMC)-lineage tracing Apoe-/- mice were fed a Western diet (WD) for 18 weeks and then switched to a low-fat chow diet for 12 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery (BCA) lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of SMC, and other lesion cells by SMC-lineage tracing combined with scRNA-seq, CyTOF, and immunostaining plus high resolution confocal microscopic z-stack analysis. In addition, to determine if treatment with a potent inhibitor of inflammation could augment the benefits of chow diet-induced reductions in LDL-cholesterol, SMC-lineage tracing Apoe-/- mice were fed a WD for 18 weeks and then chow diet for 12 weeks prior to treating them with an IL-1ß or control antibody (Ab) for 8-weeks. Results: Lipid-lowering by switching Apoe-/- mice from a WD to a chow diet reduced LDL-cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1ß Ab treatment resulted in multiple detrimental changes including increased plaque burden, BCA lesion size, as well as increased cholesterol crystal accumulation, intra-plaque hemorrhage, necrotic core area, and senescence as compared to IgG control Ab treated mice. Furthermore, IL-1ß Ab treatment upregulated neutrophil degranulation pathways but down-regulated SMC extracellular matrix pathways likely important for the protective fibrous cap. Conclusions: Taken together, IL-1ß appears to be required for chow diet-induced reductions in plaque burden and increases in multiple indices of plaque stability.

Operationalizing inclusion: moving from an elusive goal to strategic action.

To mitigate the structural and institutional biases that contribute to inequities in health, we need a diverse cadre of individuals to feel included and advance within our field in order to bring a multicultural set of perspectives to the studies we conduct, the science we generate, the health and academic systems we design, and the medical and scientific knowledge we impart. There has been increasing focus on diversity, inclusion, and equity in recent years; however, often these terms are presented without adequate precision and, therefore, the inability to effectively operationalize inclusion and achieve diversity within organizations. This narrative review details several key studies, with the primary objective of presenting a roadmap to guide defining, measuring, and operationalizing inclusion within work and learning environments.

A developmental atlas of somatosensory diversification and maturation in the dorsal root ganglia by single-cell mass cytometry.

Precisely controlled development of the somatosensory system is essential for detecting pain, itch, temperature, mechanical touch and body position. To investigate the protein-level changes that occur during somatosensory development, we performed single-cell mass cytometry on dorsal root ganglia from C57/BL6 mice of both sexes, with litter replicates collected daily from embryonic day 11.5 to postnatal day 4. Measuring nearly 3 million cells, we quantified 30 molecularly distinct somatosensory glial and 41 distinct neuronal states across all timepoints. Analysis of differentiation trajectories revealed rare cells that co-express two or more Trk receptors and over-express stem cell markers, suggesting that these neurotrophic factor receptors play a role in cell fate specification. Comparison to previous RNA-based studies identified substantial differences between many protein-mRNA pairs, demonstrating the importance of protein-level measurements to identify functional cell states. Overall, this study demonstrates that mass cytometry is a high-throughput, scalable platform to rapidly phenotype somatosensory tissues.

Intramolecular, Interrupted Homo-Nazarov Cascade Biscyclizations to Angular (Hetero)Aryl-Fused Polycycles.

Herein, a SnCl4 -catalyzed intramolecular, interrupted homo-Nazarov cascade biscyclization to access angular (hetero)aryl-fused polycycles is reported. Subsequent decarboxylation of the readily enolizable products afforded the angular products in up to 71 % yield over two steps, with the trans-diastereomers as the major products. The cyclopropyl homo-Nazarov cyclization precursors were formed using a scalable and modular synthetic route that, ultimately, offers access to 6,6,6-, 6,6,5-, 6,5,6-, 6,6,5,6-, and 6,6,6,5-fused angular polycyclic products. To showcase the rigor and utility of the method, an 8-step total synthesis of (±)-1-oxoferruginol, an antibacterial aromatic abietane diterpenoid, was disclosed.

Evidence-Based Determination of Cut-Off Points for Increased Cardiac-Surgery Mortality Risk With EuroSCORE II and STS: The Best-Performing Risk Scoring Models in a Single-Centre Australian Population.

OBJECTIVES: Risk scoring models (RSMs) are commonly used for estimation of postoperative-mortality risk in patients undergoing cardiac surgery, but their prediction accuracy may vary in different populations and clinical situations. The prognostic accuracies of some RSMs have not yet been fully evaluated in the Australian population. In this retrospective observational study, our aims were to assess the performance of four contemporary RSMs, to identify the best RSMs for prediction of postoperative-mortality in the single-centre cohort, and to determine a statistical threshold for classification of patients with increased or "higher" mortality risk. METHODS: The study population included patients who underwent cardiac surgery at Liverpool Hospital between January 2013 and December 2014. Demographic information was collected, and mortality risks were estimated with the ES2 (EuroSCORE II), STS (Society of Thoracic Surgeons Score), AS (AusSCORE total) and ASMR (AusSCORE multi-risk) RSMs. (Additive EuroSCORE) (AES) and LES (logistic EuroSCORE) were included for historical interest. Discrimination, the ability to stratify patients between mortality and no mortality outcomes, and calibration, the comparison of risk score estimated and observed outcome in the population, were evaluated for each RSM, to determine their predictive accuracy in the study population. Discrimination was assessed by the AUC (area under the receiver operating characteristic curve), and acceptable calibration by the p-value greater than 0.05 for the Hosmer-Lemeshow (H-L) test. The best AUCs in contempory models were compared using the DeLong test. For ES2 and STS risk scores, cut-off points, or thresholds, for patients at increased risk of mortality were derived using Youden's J-statistics, calculated from sensitivity and specificity of models in predicting mortality. RESULTS: From a total study population of 898 patients, 738 had scores for all six RSMs. The three EuroSCORE risk models and Youden's J-statistics analysis included the total population. Of the models in contemporary use, ES2 had higher discrimination (AUC=0.850) in this population than ASMR (AUC=0.767, p=0.024) and AS (AUC=0.739) and non-significantly higher discrimination than STS (AUC=0.806, p=0.19). All contemporary models had acceptable calibration but the older LES (H-L p=0.024) did not. Estimated mortality was closest to observed mortality with the ES2 model. Both AES and LES over predicted mortality. The RSM with the highest discrimination in isolated coronary artery bypass graft surgery (CAGs) (AUC=0.847), isolated valves (AUC=0.830), and females (AUC=0.784) was the ES2 model. STS discrimination was highest in CAGs plus valve procedures (AUC 0.891), and males (STS AUC=0.891). Cut-off points for risk scores to define increased risk populations were 3.0% for ES2 and 1.7% for STS. Similar proportions of patients in each RSM (ES2-26% to STS-32%) were defined as higher risk by the model threshold score depending on type of procedure. CONCLUSION: Among RSMs in contemporary use, ES2 and STS showed the best discrimination and acceptable calibration. Caution is recommended in specific subgroups. Increased mortality risk score cut-off points could be identified for these two RSMs in this single-centre cohort.

A silent burden-prolapse, incontinence, and infertility in Australian Aboriginal and Torres Strait Islander women: A systematic search and narrative review.

BACKGROUND: Pelvic organ prolapse (POP), urinary incontinence, and infertility are all prevalent conditions associated with considerable reduction in quality of life. As a group, Aboriginal and Torres Strait Islander women may be at higher risk of these conditions, but studies are scarce. OBJECTIVE: To review the literature pertaining to the epidemiology, diagnosis, and management of these conditions in Indigenous Australian women. SEARCH STRATEGY: Medline, Embase, and Scopus were searched for articles published between 1980 and 2021 pertaining to these conditions in Indigenous Australian women. SELECTION CRITERIA: Studies that did not directly address the epidemiology, diagnosis, and management of these conditions were excluded. MAIN RESULTS: It was possible to identify only 11 papers dealing with these conditions in Indigenous Australian women. Only one dealt with POP and was a retrospective audit of a health outreach program in the Northern Territory concluding that there was significant underreporting of the condition. Five papers dealt with urinary incontinence and, again, described significant underreporting and poor referral pathways. Five papers reported small studies about infertility, one reporting poor engagement from clinical directors. CONCLUSION: It was concluded that despite the importance of these conditions, there is almost no body of research and this is an urgent national problem.

Identification of human immune cell subtypes most responsive to IL-1ß-induced inflammatory signaling using mass cytometry.

IL-1ß is a key mediator of the cytokine storm linked to high morbidity and mortality from COVID-19, and IL-1ß blockade with anakinra and canakinumab during COVID-19 infection has entered clinical trials. Using mass cytometry of human peripheral blood mononuclear cells, we identified effector memory CD4+ T cells and CD4-CD8low/-CD161+ T cells, specifically those positive for the chemokine receptor CCR6, as the circulating immune subtypes with the greatest response to IL-1ß. This response manifested as increased phosphorylation and, thus, activation of the proinflammatory transcription factor NF-κB and was also seen in other subsets, including CD11c+ myeloid dendritic cells, classical monocytes, two subsets of natural killer cells (CD16-CD56brightCD161- and CD16-CD56dimCD161+), and lineage- (Lin-) cells expressing CD161 and CD25. IL-1ß also induced a rapid but less robust increase in the phosphorylation of the kinase p38 as compared to that of NF-κB in most of these immune cell subsets. Prolonged IL-1ß stimulation increased the phosphorylation of the transcription factor STAT3 and to a lesser extent that of STAT1 and STAT5 across various immune cell types. IL-1ß-induced production of IL-6 likely led to the activation of STAT1 and STAT3 at later time points. Interindividual heterogeneity and inhibition of STAT activation by anakinra raise the possibility that assays measuring NF-κB phosphorylation in response to IL-1ß in CCR6+ T cell subtypes could identify those patients at higher risk of cytokine storm and most likely to benefit from IL-1ß-neutralizing therapies.

Generation of Functional Gene Knockout Melanoma Cell Lines by CRISPR-Cas9 Gene Editing.

Recent advances in the treatment of metastatic melanoma have emerged only from advances in our understanding of melanoma development and progression at the cellular and molecular levels. Despite the impact that such advances have made on the clinical management of this cancer over the last decade, additional insights into factors that promote melanoma progression and therapeutic resistance are needed to combat this disease. CRISPR-Cas9 gene editing technology is a powerful tool for studying gene function in a timely and cost-effective manner, enabling the manipulation of specific DNA sequences via a targeted approach. Herein, we describe a protocol for generating functional gene knockouts in melanoma cell lines by CRISPR-Cas9 gene editing, and we present an example application of this protocol for the successful knockout of the Foxc2 transcription factor-encoding gene in the B16-F1 murine melanoma cell line.

Day 1 to day 4 serum hCG change in predicting single-dose methotrexate treatment failure for tubal ectopic pregnancies.

OBJECTIVE: To determine if changes in Day 1 to Day 4 serum human chorionic gonadotropin (hCG) levels can predict treatment failure of single-dose methotrexate (MTX) in medical management of tubal ectopic pregnancies (EP). STUDY DESIGN: This retrospective cohort study was conducted at a tertiary level hospital. Files were reviewed for all women who received at least one dose of 50 mg/m2 intramuscular MTX for treatment of ultrasound-confirmed tubal EPs between 2013 and 2018. "Treatment failure" is defined as needing additional MTX or surgery to manage the EP. The primary purpose is to establish a threshold percentage change in Day 1 to Day 4 (Day 1/4) hCG that best predicts treatment failure, with clinically and statistically significant sensitivity and specificity, based on receiver-operator characteristic (ROC) analysis. RESULTS: 252 files were reviewed, with 108 included for final analysis. 17% of cases required a second dose of MTX and 12% required surgery to manage the EP. Women in the treatment failure group had significantly higher median hCG levels on Day 1, 4 and 7, but were otherwise similar to women who were successful in age, parity, history of previous EP, and EP size. ROC curve analysis of Day 1/4 hCG demonstrates that ≥5% rise best predicts treatment failure with sensitivity 68% (95% confidence interval [CI] 49-83%), specificity 69% (95%CI 56-78%), and AUC 0.77 (95%CI 0.68-0.86, p < 0.001). The positive predictive value is 46% (95%CI 36-56%) and negative predictive value is 84% (95%CI 75-90%). In comparison, ROC analysis of Day 4 to Day 7 hCG demonstrates that a drop of ≤17% best predicted failure, with sensitivity 83% (95%CI 64-94%), specificity 82% (95%CI 71-90%), and AUC 0.90 (95%CI 0.84-0.96), p < 0.001. CONCLUSION: This study suggests that ≥5% rise in Day 1/4 serum hCG levels could potentially predict treatment failure of single-dose MTX for tubal EPs, and that conversely, <5% rise or any drop in Day 1/4 hCG levels can reliably predict treatment success. Clinicians could consider factoring-in Day 1/4 hCG changes during the course of medically managing patients. They must bear in mind, however, that acting on the Day 1/4 hCG change would lead to increased interventions.

Stem Cell Pluripotency Genes Klf4 and Oct4 Regulate Complex SMC Phenotypic Changes Critical in Late-Stage Atherosclerotic Lesion Pathogenesis.

BACKGROUND: Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability. METHODS: We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis. RESULTS: We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11-, Lgals3+ population with a chondrocyte-like gene signature that was markedly reduced with SMC-Klf4 knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene-positive, extracellular matrix-remodeling, "pioneer" cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization. CONCLUSIONS: Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3+ osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.

Smad3 Regulates Neuropilin 2 Transcription by Binding to its 5' Untranslated Region.

Background Vascular smooth muscle cell phenotypic change and consequential intimal hyperplasia (IH) cause arterial stenosis and posttreatment restenosis. Smad3 is a master transcription factor, yet its underlying functional mechanisms in this disease context are not well defined. Methods and Results In cultured smooth muscle cells, Smad3 silencing and overexpression respectively reduced and increased the mRNA and protein of NRP2 (neuropilin 2), a recently reported pro-IH signaling factor. Smad3 silencing attenuated pro-IH smooth muscle cell phenotypes including proliferation, migration, and dedifferentiation (reduced smooth muscle α-actin). While increased Smad3 enhanced these phenotypes, NRP2 silencing abolished this enhancement. Interestingly, the 5' untranslated region but not the promoter of NRP2 was indispensable for Smad3-enhanced transcriptional activity (luciferase assay); both chromatin immunoprecipitation and electrophoretic mobility shift assay showed predominant Smad3 binding in the +51 to +78 bp region of NRP2's 5' untranslated region. In vivo, Smad3 haploinsufficiency reduced NRP2 (immunostaining) and IH (by 47%) in wire-injured mouse femoral arteries. Conclusions Smad3 controls NRP2 expression by occupying its 5' untranslated region in promoting smooth muscle cell phenotypic change in vitro. This and in vivo results shed new light on the long-debated role of Smad3 in IH.

Genital Shedding of Human Immunodeficiency Virus Type-1 (HIV) When Antiretroviral Therapy Suppresses HIV Replication in the Plasma.

BACKGROUND: During antiretroviral treatment (ART) with plasma HIV RNA below the limit of quantification, HIV RNA can be detected in genital or rectal secretions, termed discordant shedding (DS). We hypothesized that proliferating cells produce virions without HIV replication. METHODS: ART-naive Peruvians initiating ART were observed for DS over 2 years. HIV env and pol genomes were amplified from DS. Antiretrovirals and cytokines/chemokines concentrations were compared at DS and control time points. RESULTS: Eighty-two participants had ART suppression. DS was detected in 24/82 (29%) participants: 13/253 (5%) cervicovaginal lavages, 20/322 (6%) seminal plasmas, and 6/85 (7%) rectal secretions. HIV RNA in DS specimens was near the limit of quantification and not reproducible. HIV DNA was detected in 6/13 (46%) DS cervicovaginal lavages at low levels. Following DNase treatment, 5/39 DS specimens yielded HIV sequences, all without increased genetic distances. Women with and without DS had similar plasma antiretroviral levels and DS in 1 woman was associated with inflammation. CONCLUSIONS: HIV RNA and DNA sequences and therapeutic antiretroviral plasma levels did not support HIV replication as the cause of DS from the genital tract. Rather, our findings infer that HIV RNA is shed due to proliferation of infected cells with virion production.

FLOW-MAP: a graph-based, force-directed layout algorithm for trajectory mapping in single-cell time course datasets.

High-dimensional single-cell technologies present new opportunities for biological discovery, but the complex nature of the resulting datasets makes it challenging to perform comprehensive analysis. One particular challenge is the analysis of single-cell time course datasets: how to identify unique cell populations and track how they change across time points. To facilitate this analysis, we developed FLOW-MAP, a graphical user interface (GUI)-based software tool that uses graph layout analysis with sequential time ordering to visualize cellular trajectories in high-dimensional single-cell datasets obtained from flow cytometry, mass cytometry or single-cell RNA sequencing (scRNAseq) experiments. Here we provide a detailed description of the FLOW-MAP algorithm and how to use the open-source R package FLOWMAPR via its GUI or with text-based commands. This approach can be applied to many dynamic processes, including in vitro stem cell differentiation, in vivo development, oncogenesis, the emergence of drug resistance and cell signaling dynamics. To demonstrate our approach, we perform a step-by-step analysis of a single-cell mass cytometry time course dataset from mouse embryonic stem cells differentiating into the three germ layers: endoderm, mesoderm and ectoderm. In addition, we demonstrate FLOW-MAP analysis of a previously published scRNAseq dataset. Using both synthetic and experimental datasets for comparison, we perform FLOW-MAP analysis side by side with other single-cell analysis methods, to illustrate when it is advantageous to use the FLOW-MAP approach. The protocol takes between 30 min and 1.5 h to complete.

The FOXC2 Transcription Factor Promotes Melanoma Outgrowth and Regulates Expression of Genes Associated With Drug Resistance and Interferon Responsiveness.

BACKGROUND/AIM: The FOXC2 transcription factor promotes the progression of several cancer types, but has not been investigated in the context of melanoma cells. To study FOXC2's influence on melanoma progression, we generated a FOXC2-deficient murine melanoma cell line and evaluated The Cancer Genome Atlas (TCGA) patient datasets. MATERIALS AND METHODS: We compared tumor growth kinetics and RNA-seq/qRT-PCR gene expression profiles from wild-type versus FOXC2-deficient murine melanomas. We also performed Kaplan-Meier survival analysis of TCGA data to assess the influence of FOXC2 gene expression on melanoma patients' response to chemotherapy and immunotherapy. RESULTS: FOXC2 promotes melanoma progression and regulates the expression of genes associated with multiple oncogenic pathways, including the oxidative stress response, xenobiotic metabolism, and interferon responsiveness. FOXC2 expression in melanoma correlates negatively with patient response to chemotherapy and immunotherapy. CONCLUSION: FOXC2 drives a tumor-promoting gene expression program in melanoma and is a prognostic indicator of patient response to multiple cancer therapies.

BBS4 is required for intraflagellar transport coordination and basal body number in mammalian olfactory cilia.

Bardet-Beidl syndrome (BBS) manifests from genetic mutations encoding for one or more BBS proteins. BBS4 loss impacts olfactory ciliation and odor detection, yet the cellular mechanisms remain unclear. Here, we report that Bbs4-/- mice exhibit shorter and fewer olfactory sensory neuron (OSN) cilia despite retaining odorant receptor localization. Within Bbs4-/- OSN cilia, we observed asynchronous rates of IFT-A/B particle movements, indicating miscoordination in IFT complex trafficking. Within the OSN dendritic knob, the basal bodies are dynamic, with incorporation of ectopically expressed centrin-2 and γ-tubulin occurring after nascent ciliogenesis. Importantly, BBS4 loss results in the reduction of basal body numbers separate from cilia loss. Adenoviral expression of BBS4 restored OSN cilia lengths and was sufficient to re-establish odor detection, but failed to rescue ciliary and basal body numbers. Our results yield a model for the plurality of BBS4 functions in OSNs that includes intraciliary and periciliary roles that can explain the loss of cilia and penetrance of ciliopathy phenotypes in olfactory neurons.

Role for intraflagellar transport in building a functional transition zone.

Genetic disorders caused by cilia dysfunction, termed ciliopathies, frequently involve the intraflagellar transport (IFT) system. Mutations in IFT subunits-including IFT-dynein motor DYNC2H1-impair ciliary structures and Hedgehog signalling, typically leading to "skeletal" ciliopathies such as Jeune asphyxiating thoracic dystrophy. Intriguingly, IFT gene mutations also cause eye, kidney and brain ciliopathies often linked to defects in the transition zone (TZ), a ciliary gate implicated in Hedgehog signalling. Here, we identify a C. elegans temperature-sensitive (ts) IFT-dynein mutant (che-3; human DYNC2H1) and use it to show a role for retrograde IFT in anterograde transport and ciliary maintenance. Unexpectedly, correct TZ assembly and gating function for periciliary proteins also require IFT-dynein. Using the reversibility of the novel ts-IFT-dynein, we show that restoring IFT in adults (post-developmentally) reverses defects in ciliary structure, TZ protein localisation and ciliary gating. Notably, this ability to reverse TZ defects declines as animals age. Together, our findings reveal a previously unknown role for IFT in TZ assembly in metazoans, providing new insights into the pathomechanism and potential phenotypic overlap between IFT- and TZ-associated ciliopathies.

Immune checkpoint blockade therapy for cancer: An overview of FDA-approved immune checkpoint inhibitors.

Although T lymphocytes have long been appreciated for their role in the immunosurveillance of cancer, it has been the realization that cancer cells may ultimately escape a response from tumor-reactive T cells that has ignited efforts to enhance the efficacy of anti-tumor immune responses. Recent advances in our understanding of T cell immunobiology have been particularly instrumental in informing therapeutic strategies to overcome mechanisms of tumor immune escape, and immune checkpoint blockade has emerged as one of the most promising therapeutic options for patients in the history of cancer treatment. Designed to interfere with inhibitory pathways that naturally constrain T cell reactivity, immune checkpoint blockade releases inherent limits on the activation and maintenance of T cell effector function. In the context of cancer, where negative T cell regulatory pathways are often overactive, immune checkpoint blockade has proven to be an effective strategy for enhancing the effector activity and clinical impact of anti-tumor T cells. Checkpoint inhibitors targeting CTLA-4, PD-1, and PD-L1 have yielded unprecedented and durable responses in a significant percentage of cancer patients in recent years, leading to U.S. FDA approval of six checkpoint inhibitors for numerous cancer indications since 2011. In this review, we highlight the clinical success of these FDA-approved immune checkpoint inhibitors and discuss current challenges and future strategies that must be considered going forward to maximize the efficacy of immune checkpoint blockade therapy for cancer.

α-Alkylidene-γ-butyrolactone Formation via Bi(OTf)3-Catalyzed, Dehydrative, Ring-Opening Cyclizations of Cyclopropyl Carbinols: Understanding Substituent Effects and Predicting E/Z Selectivity.

A Bi(OTf)3-catalyzed ring-opening cyclization of (hetero)aryl cyclopropyl carbinols to form α-alkylidene-γ-butyrolactones (ABLs) is reported. This transformation represents different chemoselectivity from previous reports that demonstrated formation of (hetero)aryl-fused cyclohexa-1,3-dienes upon acid-promoted cyclopropyl carbinol ring opening. ABLs are obtained in up to 89% yield with a general preference for the E-isomers. Mechanistically, Bi(OTf)3 serves as a stable and easy to handle precursor to TfOH. TfOH then catalyzes the formation of cyclopropyl carbinyl cations, which undergo ring opening, intramolecular trapping by the neighboring ester group, subsequent hydrolysis, and loss of methanol resulting in the formation of the ABLs. The nature and relative positioning of the substituents on both the carbinol and the cyclopropane determine both chemo- and stereoselective outcomes. Carbinol substituents determine the extent of cyclopropyl carbinyl cation formation. The cyclopropane donor substituents determine the overall reaction chemoselectivity. Weakly stabilizing or electron-poor donor groups provide better yields of the ABL products. In contrast, copious amounts of competing products are observed with highly stabilizing cyclopropane donor substituents. Finally, a predictive model for E/Z selectivity was developed using DFT calculations.