A molecular signature for IL-10-producing Th1 cells in protozoan parasitic diseases.

Control of visceral leishmaniasis (VL) depends on proinflammatory Th1 cells that activate infected tissue macrophages to kill resident intracellular parasites. However, proinflammatory cytokines produced by Th1 cells can damage tissues and require tight regulation. Th1 cell IL-10 production is an important cell-autologous mechanism to prevent such damage. However, IL-10-producing Th1 (type 1 regulatory; Tr1) cells can also delay control of parasites and the generation of immunity following drug treatment or vaccination. To identify molecules to target in order to alter the balance between Th1 and Tr1 cells for improved antiparasitic immunity, we compared the molecular and phenotypic profiles of Th1 and Tr1 cells in experimental VL caused by Leishmania donovani infection of C57BL/6J mice. We also identified a shared Tr1 cell protozoan signature by comparing the transcriptional profiles of Tr1 cells from mice with experimental VL and malaria. We identified LAG3 as an important coinhibitory receptor in patients with VL and experimental VL, and we reveal tissue-specific heterogeneity of coinhibitory receptor expression by Tr1 cells. We also discovered a role for the transcription factor Pbx1 in suppressing CD4+ T cell cytokine production. This work provides insights into the development and function of CD4+ T cells during protozoan parasitic infections and identifies key immunoregulatory molecules.

COMMD3 loss drives invasive breast cancer growth by modulating copper homeostasis.

BACKGROUND: Despite overall improvement in breast cancer patient outcomes from earlier diagnosis and personalised treatment approaches, some patients continue to experience recurrence and incurable metastases. It is therefore imperative to understand the molecular changes that allow transition from a non-aggressive state to a more aggressive phenotype. This transition is governed by a number of factors. METHODS: As crosstalk with extracellular matrix (ECM) is critical for tumour cell growth and survival, we applied high throughput shRNA screening on a validated '3D on-top cellular assay' to identify novel growth suppressive mechanisms. RESULTS: A number of novel candidate genes were identified. We focused on COMMD3, a previously poorly characterised gene that suppressed invasive growth of ER + breast cancer cells in the cellular assay. Analysis of published expression data suggested that COMMD3 is normally expressed in the mammary ducts and lobules, that expression is lost in some tumours and that loss is associated with lower survival probability. We performed immunohistochemical analysis of an independent tumour cohort to investigate relationships between COMMD3 protein expression, phenotypic markers and disease-specific survival. This revealed an association between COMMD3 loss and shorter survival in hormone-dependent breast cancers and in particularly luminal-A-like tumours (ER+/Ki67-low; 10-year survival probability 0.83 vs. 0.73 for COMMD3-positive and -negative cases, respectively). Expression of COMMD3 in luminal-A-like tumours was directly associated with markers of luminal differentiation: c-KIT, ELF5, androgen receptor and tubule formation (the extent of normal glandular architecture; p < 0.05). Consistent with this, depletion of COMMD3 induced invasive spheroid growth in ER + breast cancer cell lines in vitro, while Commd3 depletion in the relatively indolent 4T07 TNBC mouse cell line promoted tumour expansion in syngeneic Balb/c hosts. Notably, RNA sequencing revealed a role for COMMD3 in copper signalling, via regulation of the Na+/K+-ATPase subunit, ATP1B1. Treatment of COMMD3-depleted cells with the copper chelator, tetrathiomolybdate, significantly reduced invasive spheroid growth via induction of apoptosis. CONCLUSION: Overall, we found that COMMD3 loss promoted aggressive behaviour in breast cancer cells.

IL-10-producing Th1 cells possess a distinct molecular signature in malaria

Control of intracellular parasites responsible for malaria requires host IFN-γ+T-bet+CD4+ T cells (Th1 cells) with IL-10 produced by Th1 cells to mitigate the pathology induced by this inflammatory response. However, these IL-10-producing Th1 (induced type I regulatory [Tr1]) cells can also promote parasite persistence or impair immunity to reinfection or vaccination. Here, we identified molecular and phenotypic signatures that distinguished IL-10-Th1 cells from IL-10+Tr1 cells in Plasmodium falciparum-infected people who participated in controlled human malaria infection studies, as well as C57BL/6 mice with experimental malaria caused by P. berghei ANKA. We also identified a conserved Tr1 cell molecular signature shared between patients with malaria, dengue, and graft-versus-host disease. Genetic manipulation of primary human CD4+ T cells showed that the transcription factor cMAF played an important role in the induction of IL-10, while BLIMP-1 promoted the development of human CD4+ T cells expressing multiple coinhibitory receptors. We also describe heterogeneity of Tr1 cell coinhibitory receptor expression that has implications for targeting these molecules for clinical advantage during infection. Overall, this work provides insights into CD4+ T cell development during malaria that offer opportunities for creation of strategies to modulate CD4+ T cell functions and improve antiparasitic immunity.

A novel negative pressure isolation device reduces aerosol exposure: A randomized controlled trial.

Background and aim: The COVID-19 pandemic has led to a proliferation of intubation barriers designed to protect healthcare workers from infection. We developed the Suction-Assisted Local Aerosol Containment Chamber (SLACC) and tested it in the operating room. The primary objectives were to determine the ease and safety of airway management with SLACC, and to measure its efficacy of aerosol containment to determine if it significantly reduces exposure to health care workers. Methods: In this randomized clinical trial, adult patients scheduled to undergo elective surgery with general endotracheal anesthesia were screened and informed consent obtained from those willing to participate. Patients were randomized to airway management either with or without the SLACC device. Patients inhaled nebulized saline before and during anesthesia induction to simulate the size and concentration of particles seen with severe symptomatic SARS-CoV-2 infection. Results: 79 patients were enrolled and randomized. Particle number concentration (PNC) at the patients' and healthcare workers' locations were measured and compared between the SLACC vs. control groups during airway management. Ease and success of tracheal intubation were recorded for each patient. All intubations were successful and time to intubation was similar between the two groups. Healthcare workers were exposed to significantly lower particle number concentrations (#/cm3) during airway management when SLACC was utilized vs. control. The particle count outside SLACC was reduced by 97% compared to that inside the device. Conclusions: The SLACC device does not interfere with airway management and significantly reduces healthcare worker exposure to aerosolized particles during airway management.

Ga+-Chabazite Zeolite: A Highly Selective Catalyst for Nonoxidative Propane Dehydrogenation.

Ga-chabazite zeolites (Ga-CHA) have been found to efficiently catalyze propane dehydrogenation with high propylene selectivity (96%). In situ Fourier transform infrared spectroscopy and pulse titrations are employed to determine that upon reduction, surface Ga2O3 is reduced and diffuses into the zeolite pores, displacing the Brønsted acid sites and forming extra-framework Ga+ sites. This isolated Ga+ site reacts reversibly with H2 to form GaHx (2034 cm-1) with an enthalpy of formation of ∼-51.2 kJ·mol-1, a result supported by density functional theory calculations. The initial C3H8 dehydrogenation rates decrease rapidly (40%) during the first 100 min and then decline slowly afterward, while the C3H6 selectivity is stable at ∼96%. The reduction in the reaction rate is correlated with the formation of polycyclic aromatics inside the zeolite (using UV-vis spectroscopy) indicating that the accumulation of polycyclic aromatics is the main cause of the deactivation. The carbon species formed can be easily oxidized at 600 °C with complete recovery of the PDH catalytic properties. The correlations between GaHx vs Ga/Al ratio and PDH rates vs Ga/Al ratio show that extra-framework Ga+ is the active center catalyzing propane dehydrogenation. The higher reaction rate on Ga+ than In+ in CHA zeolites, by a factor of 43, is the result of differences in the stabilization of the transition state due to the higher stability of Ga3+ vs In3+. The uniformity of the Ga+ sites in this material makes it an excellent model for the molecular understanding of metal cation-exchanged hydrocarbon interactions in zeolites.

Comparison of Preoperative Alpha-blockade for Resection of Paraganglioma and Pheochromocytoma.

OBJECTIVE: Phenoxybenzamine (nonselective, noncompetitive alpha-blocker) is the preferred drug for preoperative treatment of pheochromocytoma, but doxazosin (selective, competitive alpha-blocker) may be equally effective. We compared the efficacy of doxazosin vs phenoxybenzamine. METHODS: We conducted a prospective study of patients undergoing pheochromocytoma or paraganglioma resection by randomizing pretreatment with phenoxybenzamine or doxazosin at a single tertiary referral center. The high cost of phenoxybenzamine led to high crossover to doxazosin. Randomization was halted, and a consecutive historical cohort of phenoxybenzamine patients was included for a case-control study design. The efficacy of alpha-blockade was assessed with preinduction infusion of incremental doses of phenylephrine. The primary outcomes were mortality, cardiovascular complications, and intensive care unit admission. The secondary outcomes were hemodynamic instability index (proportion of operation outside of hemodynamic goals), adequacy of blockade by the phenylephrine titration test, and drug costs. RESULTS: Twenty-four patients were prospectively enrolled (doxazosin, n = 20; phenoxybenzamine, n = 4), and 15 historical patients treated with phenoxybenzamine were added (total phenoxybenzamine, n = 19). No major cardiovascular complications occurred in either group. The phenylephrine dose-response curves showed less blood pressure rise in the phenoxybenzamine than in the doxazosin group (linear regression coefficient = 0.008 vs 0.018, P = .01), suggesting better alpha-blockade in the phenoxybenzamine group. The median hemodynamic instability index was 14% vs 13% in the phenoxybenzamine and doxazosin groups, respectively (P = .56). The median highest daily cost of phenoxybenzamine was $442.20 compared to $5.06 for doxazosin. CONCLUSION: Phenoxybenzamine may blunt intraoperative hypertension better than doxazosin, but this difference did not translate to fewer cardiovascular complications and is offset by a considerably increased cost.

Combined Inhibition of G9a and EZH2 Suppresses Tumor Growth via Synergistic Induction of IL24-Mediated Apoptosis.

G9a and EZH2 are two histone methyltransferases commonly upregulated in several cancer types, yet the precise roles that these enzymes play cooperatively in cancer is unclear. We demonstrate here that frequent concurrent upregulation of both G9a and EZH2 occurs in several human tumors. These methyltransferases cooperatively repressed molecular pathways responsible for tumor cell death. In genetically distinct tumor subtypes, concomitant inhibition of G9a and EZH2 potently induced tumor cell death, highlighting the existence of tumor cell survival dependency at the epigenetic level. G9a and EZH2 synergistically repressed expression of genes involved in the induction of endoplasmic reticulum (ER) stress and the production of reactive oxygen species. IL24 was essential for the induction of tumor cell death and was identified as a common target of G9a and EZH2. Loss of function of G9a and EZH2 activated the IL24-ER stress axis and increased apoptosis in cancer cells while not affecting normal cells. These results indicate that G9a and EZH2 promotes the evasion of ER stress-mediated apoptosis by repressing IL24 transcription, therefore suggesting that their inhibition may represent a potential therapeutic strategy for solid cancers. SIGNIFICANCE: These findings demonstrate a novel role for G9a and EZH2 histone methyltransferases in suppressing apoptosis, which can be targeted with small molecule inhibitors as a potential approach to improve solid cancer treatment.

Single-Leg Vertical Hop Test Detects Greater Limb Asymmetries Than Horizontal Hop Tests After Anterior Cruciate Ligament Reconstruction in NCAA Division 1 Collegiate Athletes.

BACKGROUND: Knee function deficits may persist after anterior cruciate ligament reconstruction (ACLR). Return to sport (RTS) testing batteries assess recovery after ACLR and can guide RTS progression, but the ideal test components are debatable. The single leg vertical hop for height (SLVH) test using a commercially available jump mat may provide a valuable assessment of knee function. HYPOTHESIS/PURPOSE: The purpose of this study was to compare the limb symmetry index (LSI) of SLVH to horizontal hop testing in a cohort of National Collegiate Athletic Association (NCAA) Division 1 collegiate athletes after ACLR. The hypothesis was the SLVH would elicit significantly lower LSI than horizontal hop tests. STUDY DESIGN: Cross-Sectional Study. METHODS: Eighteen National Collegiate Athletic Association (NCAA) Division 1 collegiate athletes (7 males, 11 females) at 7.33 ± 2.05 months after ACLR were included in this retrospective study. LSI was calculated for single hop for distance (SHD), triple hop for distance (THD), cross-over hop for distance (CHD), timed 6-meter hop (T6H), and SLVH. A repeated measures ANOVA was performed to identify differences in LSI for each test. Spearman's Rho correlation coefficient was calculated to examine the relationship between LSIs for each test. RESULTS: The LSI for SLVH (84.48% ± 11.41%) was significantly lower than LSI for SHD (95.48 ± 8.02%, p = 0.003), THD (94.40 ± 3.70%, p = 0.002), CHD (95.85 ± 7.00, p = 0.007), and T6H (97.69 ± 6.60%, p = 0.001). The correlation of LSI between SLVH and the horizontal hop tests was weak and non-significant for SHD (rs = 0.166, p = 0.509), CHD (rs = 0.199, p = 0.428), and T6H (rs = 0.211, p = 0.401) and moderate and non-significant for THD (rs = 0.405, p = 0.096). CONCLUSIONS: Individuals after ACLR had lower LSI on the SLVH than on horizontal hop tests and weak to moderate correlations between the tests suggest SLVH detects performance deficits not identified by the horizontal hop tests. LEVEL OF EVIDENCE: 3.

Correction to 'Genome instability and pressure on non-homologous end joining drives chemotherapy resistance via a DNA repair crisis switch in triple negative breast cancer'.

[This corrects the article DOI: 10.1093/nar/zcab022.].

Genome instability and pressure on non-homologous end joining drives chemotherapy resistance via a DNA repair crisis switch in triple negative breast cancer.

Chemotherapy is used as a standard-of-care against cancers that display high levels of inherent genome instability. Chemotherapy induces DNA damage and intensifies pressure on the DNA repair pathways that can lead to deregulation. There is an urgent clinical need to be able to track the emergence of DNA repair driven chemotherapy resistance and tailor patient staging appropriately. There have been numerous studies into chemoresistance but to date no study has elucidated in detail the roles of the key DNA repair components in resistance associated with the frontline clinical combination of anthracyclines and taxanes together. In this study, we hypothesized that the emergence of chemotherapy resistance in triple negative breast cancer was driven by changes in functional signaling in the DNA repair pathways. We identified that consistent pressure on the non-homologous end joining pathway in the presence of genome instability causes failure of the key kinase DNA-PK, loss of p53 and compensation by p73. In-turn a switch to reliance on the homologous recombination pathway and RAD51 recombinase occurred to repair residual double strand DNA breaks. Further we demonstrate that RAD51 is an actionable target for resensitization to chemotherapy in resistant cells with a matched gene expression profile of resistance highlighted by homologous recombination in clinical samples.

Carbocation-Mediated Cyclization of Trienes in Acid Zeolites.

The mechanism by which acid zeolites catalyze the formation of aromatic species is not fully understood and is important in an array of industrial processes such as the methanol to gasoline reaction. The so-called "carbon pool" mechanism is generally agreed to be the main channel for the formation of hydrocarbons from methanol. There is, however, no agreed sequence of elementary steps that explains how linear intermediates transform to cyclic intermediates, let alone aromatic rings. Recent work suggests the formation of conjugated trienes during zeolite-catalyzed aromatization, but mechanisms involving triene-derived carbocations have never been investigated using modern computational tools. In this work, we propose a new mechanism for cyclization of hexatriene over the Brønsted acid site of faujasite zeolite. Microkinetic models (MKM) using the results of Density Functional Theory (DFT) calculations predict selectivity for neutral 5-membered-ring intermediates over 6-membered-ring intermediates, as suggested by infrared and UV-vis spectroscopic results reported by others. Given that the products of aromatization are 6-membered rings, this result suggests that triene cyclization can only explain how linear hydrocarbons become cyclic intermediates but not the mechanisms that ultimately lead to the aromatic rings seen in industrial zeolite-catalyzed hydrocarbon processes.

Histone Modifying Enzymes in Gynaecological Cancers.

Genetic and epigenetic factors contribute to the development of cancer. Epigenetic dysregulation is common in gynaecological cancers and includes altered methylation at CpG islands in gene promoter regions, global demethylation that leads to genome instability and histone modifications. Histones are a major determinant of chromosomal conformation and stability, and unlike DNA methylation, which is generally associated with gene silencing, are amenable to post-translational modifications that induce facultative chromatin regions, or condensed transcriptionally silent regions that decondense resulting in global alteration of gene expression. In comparison, other components, crucial to the manipulation of chromatin dynamics, such as histone modifying enzymes, are not as well-studied. Inhibitors targeting DNA modifying enzymes, particularly histone modifying enzymes represent a potential cancer treatment. Due to the ability of epigenetic therapies to target multiple pathways simultaneously, tumours with complex mutational landscapes affected by multiple driver mutations may be most amenable to this type of inhibitor. Interrogation of the actionable landscape of different gynaecological cancer types has revealed that some patients have biomarkers which indicate potential sensitivity to epigenetic inhibitors. In this review we describe the role of epigenetics in gynaecological cancers and highlight how it may exploited for treatment.

G9a Inhibition Enhances Checkpoint Inhibitor Blockade Response in Melanoma.

PURPOSE: G9a histone methyltransferase exerts oncogenic effects in several tumor types and its inhibition promotes anticancer effects. However, the impact on checkpoint inhibitor blockade response and the utility of G9a or its target genes as a biomarker is poorly studied. We aimed to examine whether G9a inhibition can augment the efficacy of checkpoint inhibitor blockade and whether LC3B, a G9a target gene, can predict treatment response. EXPERIMENTAL DESIGN: Clinical potential of LC3B as a biomarker of checkpoint inhibitor blockade was assessed using patient samples including tumor biopsies and circulating tumor cells from liquid biopsies. Efficacy of G9a inhibition to enhance checkpoint inhibitor blockade was examined using a mouse model. RESULTS: Patients with melanoma who responded to checkpoint inhibitor blockade were associated with not only a higher level of tumor LC3B but also a higher proportion of cells expressing LC3B. A higher expression of MAP1LC3B or LC3B protein was associated with longer survival and lower incidence of acquired resistance to checkpoint inhibitor blockade, suggesting LC3B as a potential predictive biomarker. We demonstrate that G9a histone methyltransferase inhibition is able to not only robustly induce LC3B level to augment the efficacy of checkpoint inhibitor blockade, but also induces melanoma cell death. CONCLUSIONS: Checkpoint inhibitor blockade response is limited to a subset of the patient population. These results have implications for the development of LC3B as a predictive biomarker of checkpoint inhibitor blockade to guide patient selection, as well as G9a inhibition as a strategy to extend the proportion of patients responding to immunotherapy.

ASC Modulates CTL Cytotoxicity and Transplant Outcome Independent of the Inflammasome.

The adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) is known to facilitate caspase-1 activation, which is essential for innate host immunity via the formation of the inflammasome complex, a multiprotein structure responsible for processing IL1ß and IL18 into their active moieties. Here, we demonstrated that ASC-deficient CD8+ T cells failed to induce severe graft-versus-host disease (GVHD) and had impaired capacity for graft rejection and graft-versus-leukemia (GVL) activity. These effects were inflammasome independent because GVHD lethality was not altered in recipients of caspase-1/11-deficient T cells. We also demonstrated that ASC deficiency resulted in a decrease in cytolytic function, with a reduction in granzyme B secretion and CD107a expression by CD8+ T cells. Altogether, our findings highlight that ASC represents an attractive therapeutic target for improving outcomes of clinical transplantation.

G9a-mediated repression of CDH10 in hypoxia enhances breast tumour cell motility and associates with poor survival outcome.

Rationale: Epigenetic mechanisms are fundamental processes that can modulate gene expression, allowing cellular adaptation to environmental conditions. Hypoxia is an important factor known to initiate the metastatic cascade in cancer, activating cell motility and invasion by silencing cell adhesion genes. G9a is a histone methyltransferase previously shown to accumulate in hypoxic conditions. While its oncogenic activity has been previously reported, not much is known about the role G9a plays in the hypoxia-mediated metastatic cascade. Methods: The role of G9a in cell motility in hypoxic condition was determined by inhibiting G9a either by short-hairpin mediated knock down or pharmacologically using a small molecule inhibitor. Through gene expression profiling, we identified CDH10 to be an important G9a target that regulates breast cancer cell motility. Lung metastasis assay in mice was used to determine the physiological significance of G9a. Results: We demonstrate that, while hypoxia enhances breast cancer migratory capacity, blocking G9a severely reduces cellular motility under both normoxic and hypoxic conditions and prevents the hypoxia-mediated induction of cellular movement. Moreover, inhibition of G9a histone methyltransferase activity in mice using a specific small molecule inhibitor significantly reduced growth and colonisation of breast cancer cells in the lung. We identify the type-II cadherin CDH10 as being a novel hypoxia-dependent gene, directly repressed by G9a through histone methylation. CDH10 overexpression significantly reduces cellular movements in breast cancer cell lines and prevents the hypoxia-mediated increase in cell motility. In addition, we show that CDH10 expression is prognostic in breast cancer and that it is inversely correlated to EHMT2 (G9a) transcript levels in many tumor-types, including breast cancer. Conclusion: We propose that G9a promotes cellular motility during hypoxic stress through the silencing of the cell adhesion molecule CDH10 and we describe CDH10 as a novel prognostic biomarker for breast cancer.

Chromatin interactome mapping at 139 independent breast cancer risk signals.

BACKGROUND: Genome-wide association studies have identified 196 high confidence independent signals associated with breast cancer susceptibility. Variants within these signals frequently fall in distal regulatory DNA elements that control gene expression. RESULTS: We designed a Capture Hi-C array to enrich for chromatin interactions between the credible causal variants and target genes in six human mammary epithelial and breast cancer cell lines. We show that interacting regions are enriched for open chromatin, histone marks for active enhancers, and transcription factors relevant to breast biology. We exploit this comprehensive resource to identify candidate target genes at 139 independent breast cancer risk signals and explore the functional mechanism underlying altered risk at the 12q24 risk region. CONCLUSIONS: Our results demonstrate the power of combining genetics, computational genomics, and molecular studies to rationalize the identification of key variants and candidate target genes at breast cancer GWAS signals.

Marine biofilm bacterial community response and carbon steel loss following Deepwater Horizon spill contaminant exposure.

Steel marine structures provide foci of biodiversity when they develop into artificial reefs. Development begins with deposition of a biofilm. The effects of contaminants from oil spills on biofilm microbiomes, microbially-induced corrosion (MIC) and metal loss may impact preservation of marine metal structures. A microcosm experiment exposed biofilms on carbon steel disks (CSDs) to crude oil, dispersant, and dispersed oil to address their impacts on bacterial composition and metal loss and pitting. Biofilm diversity increased over time in all exposures. Community composition in dispersant and dispersed oil treatments deviated from the controls for the duration of a 12-week experiment. As biofilms matured, Pseudomonadaceae increased while Rhodobacteraceae decreased in abundance in dispersed oil treatments compared to the controls and dispersant treatments. Greatest mass loss and deepest pitting on CSDs were observed in dispersed oil treatments, suggesting impacts manifest as a consequence of increased MIC potential on carbon steel.

Molecular basis of distinct oestrogen responses in endometrial and breast cancer.

Up to 80% of endometrial and breast cancers express oestrogen receptor alpha (ERα). Unlike breast cancer, anti-oestrogen therapy has had limited success in endometrial cancer, raising the possibility that oestrogen has different effects in both cancers. We investigated the role of oestrogen in endometrial and breast cancers using data from The Cancer Genome Atlas (TCGA) in conjunction with cell line studies. Using phosphorylation of ERα (ERα-pSer118) as a marker of transcriptional activation of ERα in TCGA datasets, we found that genes associated with ERα-pSer118 were predominantly unique between tumour types and have distinct regulators. We present data on the alternative and novel roles played by SMAD3, CREB-pSer133 and particularly XBP1 in oestrogen signalling in endometrial and breast cancer.