NOTCH1 signaling during CD4+ T-cell activation alters transcription factor networks and enhances antigen responsiveness.

Adoptive transfer of T cells expressing chimeric antigen receptors (CAR-T) effectively treats refractory hematologic malignancies in a subset of patients but can be limited by poor T-cell expansion and persistence in vivo. Less differentiated T-cell states correlate with the capacity of CAR-T to proliferate and mediate antitumor responses, and interventions that limit tumor-specific T-cell differentiation during ex vivo manufacturing enhance efficacy. NOTCH signaling is involved in fate decisions across diverse cell lineages and in memory CD8+ T cells was reported to upregulate the transcription factor FOXM1, attenuate differentiation, and enhance proliferation and antitumor efficacy in vivo. Here, we used a cell-free culture system to provide an agonistic NOTCH1 signal during naïve CD4+ T-cell activation and CAR-T production and studied the effects on differentiation, transcription factor expression, cytokine production, and responses to tumor. NOTCH1 agonism efficiently induced a stem cell memory phenotype in CAR-T derived from naïve but not memory CD4+ T cells and upregulated expression of AhR and c-MAF, driving heightened production of interleukin-22, interleukin-10, and granzyme B. NOTCH1-agonized CD4+ CAR-T demonstrated enhanced antigen responsiveness and proliferated to strikingly higher frequencies in mice bearing human lymphoma xenografts. NOTCH1-agonized CD4+ CAR-T also provided superior help to cotransferred CD8+ CAR-T, driving improved expansion and curative antitumor responses in vivo at low CAR-T doses. Our data expand the mechanisms by which NOTCH can shape CD4+ T-cell behavior and demonstrate that activating NOTCH1 signaling during genetic modification ex vivo is a potential strategy for enhancing the function of T cells engineered with tumor-targeting receptors.

Separation of coherent and incoherent light using image plane vortex phase masks.

We consider the application of a modified optical vortex coronagraph as a transmissometer. We find, through theory and simulation, that the rejection of scattered light benefits from increasing the charge number of the vortex masks in the image plane, and that a combination of a vortex mask and binary pinhole can outperform the pinhole alone.

Hierarchical Self-Assembly of Nanowires on the Surface by Metallo-Supramolecular Truncated Cuboctahedra.

Parastichy, the spiral arrangement of plant organs, is an example of the long-range apparent order seen in biological systems. These ordered arrangements provide scientists with both an aesthetic challenge and a mathematical inspiration. Synthetic efforts to replicate the regularity of parastichy may allow for molecular-scale control over particle arrangement processes. Here we report the packing of a supramolecular truncated cuboctahedron (TCO) into double-helical (DH) nanowires on a graphite surface with a non-natural parastichy pattern ascribed to the symmetry of the TCOs and interactions between TCOs. Such a study is expected to advance our understanding of the design inputs needed to create complex, but precisely controlled, hierarchical materials. It is also one of the few reported helical packing structures based on Platonic or Archimedean solids since the discovery of the Boerdijk-Coxeter helix. As such, it may provide experimental support for studies of packing theory at the molecular level.

Role of zinc transporter ZIP12 in susceptibility-weighted brain magnetic resonance imaging (MRI) phenotypes and mitochondrial function.

Brain zinc dysregulation is linked to many neurological disorders. However, the mechanisms regulating brain zinc homeostasis are poorly understood. We performed secondary analyses of brain MRI GWAS and exome sequencing data from adults in the UK Biobank. Coding ZIP12 polymorphisms in zinc transporter ZIP12 (SLC39A12) were associated with altered brain susceptibility weighted MRI (swMRI). Conditional and joint association analyses revealed independent GWAS signals in linkage disequilibrium with 2 missense ZIP12 polymorphisms, rs10764176 and rs72778328, with reduced zinc transport activity. ZIP12 rare coding variants predicted to be deleterious were associated with similar impacts on brain swMRI. In Neuro-2a cells, ZIP12 deficiency by short hairpin RNA (shRNA) depletion or CRISPR/Cas9 genome editing resulted in impaired mitochondrial function, increased superoxide presence, and detectable protein carbonylation. Inhibition of Complexes I and IV of the electron transport chain reduced neurite outgrowth in ZIP12 deficient cells. Transcriptional coactivator PGC-1α, mitochondrial superoxide dismutase (SOD2), and chemical antioxidants α-tocopherol, MitoTEMPO, and MitoQ restored neurite extension impaired by ZIP12 deficiency. Mutant forms of α-synuclein and tau linked to familial Parkinson's disease and frontotemporal dementia, respectively, reduced neurite outgrowth in cells deficient in ZIP12. Zinc and ZIP12 may confer resilience against neurological diseases or premature aging of the brain.

Activating PAX gene family paralogs to complement PAX5 leukemia driver mutations.

PAX5, one of nine members of the mammalian paired box (PAX) family of transcription factors, plays an important role in B cell development. Approximately one-third of individuals with pre-B acute lymphoblastic leukemia (ALL) acquire heterozygous inactivating mutations of PAX5 in malignant cells, and heterozygous germline loss-of-function PAX5 mutations cause autosomal dominant predisposition to ALL. At least in mice, Pax5 is required for pre-B cell maturation, and leukemic remission occurs when Pax5 expression is restored in a Pax5-deficient mouse model of ALL. Together, these observations indicate that PAX5 deficiency reversibly drives leukemogenesis. PAX5 and its two most closely related paralogs, PAX2 and PAX8, which are not mutated in ALL, exhibit overlapping expression and function redundantly during embryonic development. However, PAX5 alone is expressed in lymphocytes, while PAX2 and PAX8 are predominantly specific to kidney and thyroid, respectively. We show that forced expression of PAX2 or PAX8 complements PAX5 loss-of-function mutation in ALL cells as determined by modulation of PAX5 target genes, restoration of immunophenotypic and morphological differentiation, and, ultimately, reduction of replicative potential. Activation of PAX5 paralogs, PAX2 or PAX8, ordinarily silenced in lymphocytes, may therefore represent a novel approach for treating PAX5-deficient ALL. In pursuit of this strategy, we took advantage of the fact that, in kidney, PAX2 is upregulated by extracellular hyperosmolarity. We found that hyperosmolarity, at potentially clinically achievable levels, transcriptionally activates endogenous PAX2 in ALL cells via a mechanism dependent on NFAT5, a transcription factor coordinating response to hyperosmolarity. We also found that hyperosmolarity upregulates residual wild type PAX5 expression in ALL cells and modulates gene expression, including in PAX5-mutant primary ALL cells. These findings specifically demonstrate that osmosensing pathways may represent a new therapeutic target for ALL and more broadly point toward the possibility of using gene paralogs to rescue mutations driving cancer and other diseases.

Characterization of the Uniformity of High-Flux CdZnTe Material.

Since the late 2000s, the availability of high-quality cadmium zinc telluride (CdZnTe) has greatly increased. The excellent spectroscopic performance of this material has enabled the development of detectors with volumes exceeding 1 cm3 for use in the detection of nuclear materials. CdZnTe is also of great interest to the photon science community for applications in X-ray imaging cameras at synchrotron light sources and free electron lasers. Historically, spatial variations in the crystal properties and temporal instabilities under high-intensity irradiation has limited the use of CdZnTe detectors in these applications. Recently, Redlen Technologies have developed high-flux-capable CdZnTe material (HF-CdZnTe), which promises improved spatial and temporal stability. In this paper, the results of the characterization of 10 HF-CdZnTe detectors with dimensions of 20.35 mm × 20.45 mm × 2.00 mm are presented. Each sensor has 80 × 80 pixels on a 250-m pitch and were flip-chip-bonded to the STFC HEXITEC ASIC. These devices show excellent spectroscopic performance at room temperature, with an average Full Width at Half Maximum (FWHM) of 0.83 keV measured at 59.54 keV. The effect of tellurium inclusions in these devices was found to be negligible; however, some detectors did show significant concentrations of scratches and dislocation walls. An investigation of the detector stability over 12 h of continuous operation showed negligible changes in performance.

Synthesis and SAR of novel capsazepine analogs with significant anti-cancer effects in multiple cancer types.

We previously demonstrated that capsazepine (CPZ), a synthetic transient receptor potential Vanilloid subtype 1 (TRPV1) antagonist, has significant anti-cancer effects in vivo. The purpose of this study was to develop more potent analogs based upon CPZ pharmacophore and structure-activity relationships (SAR) across analogs. We generated 30 novel compounds and screened for their anti-proliferative effects in cultured HeLa cervical cancer cells. Cell viability assays identified multiple compounds with IC50s < 15 µM and one compound, 29 with an IC50 < 5 µM; six fold more potent than CPZ. We validated the anti-proliferative efficacy of two lead compounds, 17 and 29, in vivo using HeLa-derived xenografts in athymic nude mice. Both analogs significantly reduced tumor volumes by day 8 compared to control treated animals (p < 0.001) with no observable adverse effects. Calcium imaging determined that compound 17 activates TRPV1 whereas 29 neither activates nor inhibits TRPV1; indicating a unique mechanism-of-action that does not involve TRPV1 signaling. Cell viability assays using a panel of additional tumor types including oral squamous cell carcinoma, non-small cell lung cancer (NSCLC), breast cancer, and prostate cancer cell lines (HSC-3, H460, MDA-231, and PC-3 respectively) demonstrated that both lead compounds were efficacious against every cancer type tested. Compounds 29 displayed IC50s of 1-2.5 µM in HSC-3and PC-3cells. Thus, we propose that these novel CPZ analogs may serve as efficacious therapeutic agents against multiple tumor types that warrant further development for clinical application.

The novel capsazepine analog, CIDD-99, significantly inhibits oral squamous cell carcinoma in vivo through a TRPV1-independent induction of ER stress, mitochondrial dysfunction, and apoptosis.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a deadly disease with a mere 40% five-year survival rate for patients with advanced disease. Previously, we discovered that capsazepine (CPZ), a transient receptor potential channel, Vanilloid subtype 1 (TRPV1) antagonist, has significant anti-tumor effects against OSCC via a unique mechanism-of-action that is independent of TRPV1. Thus, we developed novel CPZ analogs with more potent anti-proliferative effects (CIDD-24, CIDD-99, and CIDD-111). METHODS: Using OSCC xenograft models, we determined the efficacy of these analogs in vivo. TRPV1 interactions were evaluated using calcium imaging and a rat model of orofacial pain. Anti-cancer mechanism(s)-of-action were assessed by cell cycle analysis and mitochondrial depolarization assays. RESULTS: CIDD-99 was the most potent analog demonstrating significant anti-tumor effects in vivo (P < 0.001). CIDD-24 was equipotent to the parent compound CPZ, but less potent than CIDD-99. CIDD-111 was the least efficacious analog. Calcium imaging studies confirmed that CIDD-99 neither activates nor inhibits TRPV1 confirming that TRPV1 activity is not involved in its anti-cancer effects. All analogs induced an S-phase block, dose-dependent mitochondrial depolarization, and apoptosis. Histological analyses revealed increased apoptosis and reduced cell proliferation in tumors treated with these analogs. Importantly, CIDD-99 had the most dramatic anti-tumor effects with 85% of tumors resolving leaving only minute traces of viable tissue. Additionally, CIDD-99 was non-noxious and demonstrated no observable adverse reactions CONCLUSION: This study describes a novel, highly efficacious, CPZ analog, CIDD-99, with dramatic anti-tumor effects against OSCC that may be efficacious as a lone therapy or in combination with standard therapies.

An engineered transforming growth factor ß (TGF-ß) monomer that functions as a dominant negative to block TGF-ß signaling.

The transforming growth factor ß isoforms, TGF-ß1, -ß2, and -ß3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-ß pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-ßs in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-ß monomer, lacking the heel helix, a structural motif essential for binding the TGF-ß type I receptor (TßRI) but dispensable for binding the other receptor required for TGF-ß signaling, the TGF-ß type II receptor (TßRII), as an alternative therapeutic modality for blocking TGF-ß signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-ß monomers and bound TßRII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-ß signaling with a Ki of 20-70 nm Investigation of the mechanism showed that the high affinity of the engineered monomer for TßRII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit TßRI, enabled it to bind endogenous TßRII but prevented it from binding and recruiting TßRI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-ß signaling and may inform similar modifications of other TGF-ß family members.

Luteolin inhibits Musashi1 binding to RNA and disrupts cancer phenotypes in glioblastoma cells.

RNA binding proteins have emerged as critical oncogenic factors and potential targets in cancer therapy. In this study, we evaluated Musashi1 (Msi1) targeting as a strategy to treat glioblastoma (GBM); the most aggressive brain tumor type. Msi1 expression levels are often high in GBMs and other tumor types and correlate with poor clinical outcome. Moreover, Msi1 has been implicated in chemo- and radio-resistance. Msi1 modulates a range of cancer relevant processes and pathways and regulates the expression of stem cell markers and oncogenic factors via mRNA translation/stability. To identify Msi1 inhibitors capable of blocking its RNA binding function, we performed a ~ 25,000 compound fluorescence polarization screen. NMR and LSPR were used to confirm direct interaction between Msi1 and luteolin, the leading compound. Luteolin displayed strong interaction with Msi1 RNA binding domain 1 (RBD1). As a likely consequence of this interaction, we observed via western and luciferase assays that luteolin treatment diminished Msi1 positive impact on the expression of pro-oncogenic target genes. We tested the effect of luteolin treatment on GBM cells and showed that it reduced proliferation, cell viability, colony formation, migration and invasion of U251 and U343 GBM cells. Luteolin also decreased the proliferation of patient-derived glioma initiating cells (GICs) and tumor-organoids but did not affect normal astrocytes. Finally, we demonstrated the value of combined treatments with luteolin and olaparib (PARP inhibitor) or ionizing radiation (IR). Our results show that luteolin functions as an inhibitor of Msi1 and demonstrates its potential use in GBM therapy.

Germinal Center B Cell and T Follicular Helper Cell Responses to Viral Vector and Protein-in-Adjuvant Vaccines.

There is great interest in the development of Ab-inducing subunit vaccines targeting infections, including HIV, malaria, and Ebola. We previously reported that adenovirus vectored vaccines are potent in priming Ab responses, but uncertainty remains regarding the optimal approach for induction of humoral immune responses. In this study, using OVA as a model Ag, we assessed the magnitude of the primary and anamnestic Ag-specific IgG responses of mice to four clinically relevant vaccine formulations: replication-deficient adenovirus; modified vaccinia Ankara (a poxvirus); protein with alum; and protein in the squalene oil-in-water adjuvant Addavax. We then used flow cytometric assays capable of measuring total and Ag-specific germinal center (GC) B cell and follicular Th cell responses to compare the induction of these responses by the different formulations. We report that adenovirus vectored vaccines induce Ag insert-specific GC B cell and Ab responses of a magnitude comparable to those induced by a potent protein/squalene oil-in-water formulation whereas-despite a robust overall GC response-the insert-specific GC B cell and Ab responses induced by modified vaccinia Ankara were extremely weak. Ag-specific follicular Th cell responses to adenovirus vectored vaccines exceeded those induced by other platforms at day 7 after immunization. We found little evidence that innate immune activation by adenovirus may act as an adjuvant in such a manner that the humoral response to a recombinant protein may be enhanced by coadministering with an adenovirus lacking a transgene of interest. Overall, these studies provide further support for the use of replication-deficient adenoviruses to induce humoral responses.

Vascular mTOR-dependent mechanisms linking the control of aging to Alzheimer's disease.

Aging is the strongest known risk factor for Alzheimer's disease (AD). With the discovery of the mechanistic target of rapamycin (mTOR) as a critical pathway controlling the rate of aging in mice, molecules at the interface between the regulation of aging and the mechanisms of specific age-associated diseases can be identified. We will review emerging evidence that mTOR-dependent brain vascular dysfunction, a universal feature of aging, may be one of the mechanisms linking the regulation of the rate of aging to the pathogenesis of Alzheimer's disease. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock.

Neuroprotective Sirtuin ratio reversed by ApoE4.

The canonical pathogenesis of Alzheimer's disease links the expression of apolipoprotein E ε4 allele (ApoE) to amyloid precursor protein (APP) processing and Aß peptide accumulation by a set of mechanisms that is incompletely defined. The development of a simple system that focuses not on a single variable but on multiple factors and pathways would be valuable both for dissecting the underlying mechanisms and for identifying candidate therapeutics. Here we show that, although both ApoE3 and ApoE4 associate with APP with nanomolar affinities, only ApoE4 significantly (i) reduces the ratio of soluble amyloid precursor protein alpha (sAPPα) to Aß; (ii) reduces Sirtuin T1 (SirT1) expression, resulting in markedly differing ratios of neuroprotective SirT1 to neurotoxic SirT2; (iii) triggers Tau phosphorylation and APP phosphorylation; and (iv) induces programmed cell death. We describe a subset of drug candidates that interferes with the APP-ApoE interaction and returns the parameters noted above to normal. Our data support the hypothesis that neuronal connectivity, as reflected in the ratios of critical mediators such as sAPPα:Aß, SirT1:SirT2, APP:phosphorylated (p)-APP, and Tau:p-Tau, is programmatically altered by ApoE4 and offer a simple system for the identification of program mediators and therapeutic candidates.

Optical measurements from single levitated particles using a linear electrodynamic quadrupole trap.

We have recently made advancements in a linear electrodynamic quadrupole (LEQ) device for capturing and levitating either single or multiple micro-particles that provides significant improvements in capture efficiency, reliability, and optical measurement access. We have used our LEQ to trap particles ranging from 30 to less than 0.5 µm in size and provide a controlled environment to study particle physical/chemical dependencies on temperature, relative humidity, and gas constituents. To demonstrate this approach, we present data and analysis of liquid-droplet evaporation rates for two materials: glycerol and dibutyl sebacate. Droplet size was monitored as a function of time by two independent optical methods: direct imaging and fixed-angle light scattering. This new approach provides a means to rapidly characterize a wide range of aerosol particle properties and a platform for development of new aerosol optical-diagnostic measurements.

Metallic-nanoparticles-enhanced fluorescence from individual micron-sized aerosol particles on-the-fly.

Fluorescence spectra from individual aerosol particles that were either coated or embedded with metallic nanoparticles (MNPs) was acquired on-the-fly using 266 nm and 355 nm excitation. Using aqueous suspensions of MNPs with either polystyrene latex (PSL) spheres or dissolved proteins (tryptophan or ovalbumin), we generated PSL spheres coated with MNPs, or protein clusters embedded with MNPs as aerosols. Both enhanced and quenched fluorescence intensities were observed as a function of MNP concentration. Optimizing MNP material, size and spacing should yield enhanced sensitivity for specific aerosol materials that could be exploited to improve detection limits of single-particle, on-the-fly fluorescence or Raman based spectroscopic sensors.

Inactive ERBB receptors cooperate with reactive oxygen species to suppress cancer progression.

The ERBB receptors are a family of heterodimerization partners capable of driving transformation and metastasis. While the therapeutic targeting of single receptors has proven efficacious, optimal targeting of this receptor family should target all oncogenic members simultaneously. The juxtamembrane domains of ERBB1, ERBB2, and ERBB3 are highly conserved and control various aspects of ERBB-dependent biology. In an effort to block those functions, we have targeted this domain with decoy peptides synthesized in tandem with a cell-penetrating peptide, termed EJ1. Treatment with EJ1 induces cell death, promotes the formation of inactive ERBB multimers, and results in simultaneous reduction of ERBB1, ERBB2, and ERBB3 activation. Treatment also results in the activation of myosin light chain-dependent cell blebbing while inactivating CaMKII signaling, coincident with the induction of cell death. EJ1 also directly translocates to mitochondria, correlating with a loss of mitochondrial membrane potential and production of reactive oxygen species. Finally, treatment of a mouse model of breast cancer with EJ1 results in the inhibition of tumor growth and metastasis without associated toxicities in normal cells. Overall, these data demonstrate that a portion of the ERBB jxm domain, when used as an intracellular decoy, can inhibit tumor growth and metastasis, representing a novel anticancer therapeutic.

Lipid-lowering optimisation for secondary prevention vascular and diabetic foot patients in a pharmacist-led clinic.

Patients attending vascular or diabetic foot clinics commonly have atherosclerotic disease, are at increased risk of cardiovascular disease (CVD), merit high-intensity lipid-modifying therapy to maintain secondary prevention targets and are often sub optimally treated in primary care. We set out to assess the impact of a pharmacist led lipid optimisation clinic in these patients in an area with high levels of social deprivation. METHODS: We performed a clinical cohort study to assess the effectiveness of a pharmacist led clinic to optimise lipid lowering therapy by optimising of statin therapy and commencing additional lipid lowering therapy if applicable with monitoring of blood lipid profiles. RESULTS: Of the 216 patients (166 (77%) on statins) triaged by the pharmacist, 175 (81%) had non-HDL cholesterol levels above the target value of 97 mg/dL (2.5 mmol/L) with a mean non-HDL cholesterol level of 135.73 mg/dL (3.51 mmol/L). Pre optimisation by the prescribing clinical pharmacist 41/216 (19%) patients were at target with a mean non-HDL cholesterol of 135.5 mg/dL improving to 92/137 (67%) patients achieving the target non-HDL cholesterol level with a mean post optimisation non-HDL cholesterol of 94.35 mg/dL (2.44 mmol/L), odds ratio for being at target 8.67 [95% CI 5.30-14.20]. The calculated LDL cholesterol levels (Friedewald) demonstrated a mean reduction of 35.19 [95% CI 29.23-41.38] mg/dL (0.91 [95% CI 0.76-1.07] mmol/l). Proportion on high intensity statin increased from 65 out of 166 (39%) to 129 of 170 (76%) at follow up O.R. 4.89 [3.06-7.82], equivalent to an NNT = 3. CONCLUSIONS: A pharmacist led service in undertreated and clinically challenging vascular and diabetic foot patients in an area of high social deprivation produced significant improvements in utilization of high intensity statin and other lipid lowering therapies and attainment of lipid goals.

High throughput application of ASTM D8332: Detailed prototype design and operating conditions for microplastic sampling of riverine systems.

Microplastic sampling strategies for aquatic systems commonly employ small mesh nets to collect suspended microparticles. These methods work well for marine sampling campaigns; however, complex water systems such as freshwater rivers, effluent discharges, and stormwater ponds characterized by high total suspended solids and fast-moving water can cause the nets to clog, rip, or tear. Published in 2020, ASTM D8332 is an alternative approach to sampling complex water systems for microplastics involving pumping large volumes of water across a cascading stack of sieves to collect suspended particles. Here we show that ASTM D8332 can be applied to sample freshwater rivers for microplastic collection. A high throughput sampling prototype developed in this work is capable of pumping 1500 L of river water in 45 min to collect particles as small as 45 µm. The system is lightweight, modular, and easily transportable. It has a discrete power supply, allowing for the collection of microplastics anywhere along the river, including municipal discharges. The design minimizes the amount of plastic in the flow path and provides a practical way to measure field contamination. Finally, we outline lessons learned through extensive field trials and testing using this system sampling the North Saskatchewan River in Edmonton, Alberta. •Existing small mesh nets face limitations in freshwater rivers, encountering clogging and tearing issues from high suspended solids and fast moving water.•Using a standardized method, ASTM D8332 - a pumping-based approach is efficient for microplastic collection in freshwater rivers.•Lightweight, modular, plastic free prototype system pumps 1500 L of river water in 45 min, collecting particles as small as 45 µm. Successfully tested in the North Saskatchewan River.