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Inhalational general anesthesia results from the poorly understood interactions of haloethers with multiple protein targets, which prominently includes ion channels in the nervous system. Previously, we reported that the commonly used inhaled anesthetic sevoflurane potentiates the activity of voltage-gated K+ (Kv) channels, specifically, several mammalian Kv1 channels and the Drosophila K-Shaw2 channel. Also, previous work suggested that the S4-S5 linker of K-Shaw2 plays a role in the inhibition of this Kv channel by n-alcohols and inhaled anesthetics. Here, we hypothesized that the S4-S5 linker is also a determinant of the potentiation of Kv1.2 and K-Shaw2 by sevoflurane. Following functional expression of these Kv channels in Xenopus oocytes, we found that converse mutations in Kv1.2 (G329T) and K-Shaw2 (T330G) dramatically enhance and inhibit the potentiation of the corresponding conductances by sevoflurane, respectively. Additionally, Kv1.2-G329T impairs voltage-dependent gating, which suggests that Kv1.2 modulation by sevoflurane is tied to gating in a state-dependent manner. Toward creating a minimal Kv1.2 structural model displaying the putative sevoflurane binding sites, we also found that the positive modulations of Kv1.2 and Kv1.2-G329T by sevoflurane and other general anesthetics are T1-independent. In contrast, the positive sevoflurane modulation of K-Shaw2 is T1-dependent. In silico docking and molecular dynamics-based free-energy calculations suggest that sevoflurane occupies distinct sites near the S4-S5 linker, the pore domain and around the external selectivity filter. We conclude that the positive allosteric modulation of the Kv channels by sevoflurane involves separable processes and multiple sites within regions intimately involved in channel gating.

Multiple physiological systems interact throughout the development of a complex disease. Knowledge of the dynamics and connectivity of interactions across physiological systems could facilitate the prevention or mitigation of organ damage underlying complex diseases, many of which are currently refractory to available therapeutics (e.g., hypertension). We studied the regulatory interactions operating within and across organs throughout disease development by integrating in vivo analysis of gene expression dynamics with a reverse engineering approach to infer data-driven dynamic network models of multi-organ gene regulatory influences. We obtained experimental data on the expression of 22 genes across five organs, over a time span that encompassed the development of autonomic nervous system dysfunction and hypertension. We pursued a unique approach for identification of continuous-time models that jointly described the dynamics and structure of multi-organ networks by estimating a sparse subset of ∼12,000 possible gene regulatory interactions. Our analyses revealed that an autonomic dysfunction-specific multi-organ sequence of gene expression activation patterns was associated with a distinct gene regulatory network. We analyzed the model structures for adaptation motifs, and identified disease-specific network motifs involving genes that exhibited aberrant temporal dynamics. Bioinformatic analyses identified disease-specific single nucleotide variants within or near transcription factor binding sites upstream of key genes implicated in maintaining physiological homeostasis. Our approach illustrates a novel framework for investigating the pathogenesis through model-based analysis of multi-organ system dynamics and network properties. Our results yielded novel candidate molecular targets driving the development of cardiovascular disease, metabolic syndrome, and immune dysfunction.

We applied a set of in silico and in vitro assays, compliant with the Comprehensive In Vitro Proarrhythmia Assay (CiPA) paradigm, to assess the risk of chloroquine (CLQ) or hydroxychloroquine (OH‐CLQ)‐mediated QT prolongation and Torsades de Pointes (TdP), alone and combined with erythromycin (ERT) and azithromycin (AZI), drugs repurposed during the first wave of coronavirus disease 2019 (COVID‐19). Each drug or drug combination was tested in patch clamp assays on seven cardiac ion channels, in in silico models of human ventricular electrophysiology (Virtual Assay) using control (healthy) or high‐risk cell populations, and in human‐induced pluripotent stem cell (hiPSC)‐derived cardiomyocytes. In each assay, concentration‐response curves encompassing and exceeding therapeutic free plasma levels were generated. Both CLQ and OH‐CLQ showed blocking activity against some potassium, sodium, and calcium currents. CLQ and OH‐CLQ inhibited IKr (half‐maximal inhibitory concentration [IC50]: 1 µM and 3–7 µM, respectively) and IK1 currents (IC50: 5 and 44 µM, respectively). When combining OH‐CLQ with AZI, no synergistic effects were observed. The two macrolides had no or very weak effects on the ion currents (IC50 > 300–1000 µM). Using Virtual Assay, both antimalarials affected several TdP indicators, CLQ being more potent than OH‐CLQ. Effects were more pronounced in the high‐risk cell population. In hiPSC‐derived cardiomyocytes, all drugs showed early after‐depolarizations, except AZI. Combining CLQ or OH‐CLQ with a macrolide did not aggravate their effects. In conclusion, our integrated nonclinical CiPA dataset confirmed that, at therapeutic plasma concentrations relevant for malaria or off‐label use in COVID‐19, CLQ and OH‐CLQ use is associated with a proarrhythmia risk, which is higher in populations carrying predisposing factors but not worsened with macrolide combination.

A genetic variant in the gene PTPN22 (R620W, rs2476601) is strongly associated with increased risk for multiple autoimmune diseases and linked to altered TCR regulation and T cell activation. Here, we utilize Crispr/Cas9 gene editing with donor DNA repair templates in human cord blood-derived, naive T cells to generate PTPN22 risk edited (620W), non-risk edited (620R), or knockout T cells from the same donor. PTPN22 risk edited cells exhibited increased activation marker expression following non-specific TCR engagement, findings that mimicked PTPN22 KO cells. Next, using lentiviral delivery of T1D patient-derived TCRs against the pancreatic autoantigen, islet-specific glucose-6 phosphatase catalytic subunit-related protein (IGRP), we demonstrate that loss of PTPN22 function led to enhanced signaling in T cells expressing a lower avidity self-reactive TCR, but not a high-avidity TCR. In this setting, loss of PTPN22 mediated enhanced proliferation and Th1 skewing. Importantly, expression of the risk variant in association with a lower avidity TCR also increased proliferation relative to PTPN22 non-risk T cells. Together, these findings suggest that, in primary human T cells, PTPN22 rs2476601 contributes to autoimmunity risk by permitting increased TCR signaling and activation in mildly self-reactive T cells, thereby potentially expanding the self-reactive T cell pool and skewing this population toward an inflammatory phenotype.

This most up-to-date, one-stop reference combines coverage of both theory and observational techniques, with introductory sections to bring all readers up to the same level. Written by outstanding researchers directly involved with the scientific program of the Laser Interferometer Gravitational-Wave Observatory (LIGO), the book begins with a brief review of general relativity before going on to describe the physics of gravitational waves and the astrophysical sources of gravitational radiation. Further sections cover gravitational wave detectors, data analysis, and the outlook of gravitational wave astronomy and astrophysics.

The popular refrain for Detroit’s economic downfall is the collapse of the auto industry. This is unsatisfactory as the manufacturing decline affected the entire Detroit metro region but all of Detroit suburbs have not suffered the same fate as the city. This article offers a different yet more complete argument for Detroit’s poor performance by analyzing the politics of the city along various dimensions. Detroit differs from its neighbors by having more corrupt and destructive politics accompanied with high tax rates. Tiebout competition allows for Detroit residents to leave its poor yet expensive governance. While the decrease of auto-manufacturing jobs clearly negatively impacted the city, poor governance set Detroit apart from nearby areas and better describes its long run condition.

Coordinated interactions between cytokine signaling and morphological dynamics of microglial cells regulate neuroinflammation in CNS injury and disease. We found that pro-inflammatory cytokine gene expression showed a pronounced recovery following systemic LPS. We performed a novel multivariate analysis of microglial morphology and identified changes in specific morphological properties of microglia that matched the expression dynamics of pro-inflammatory cytokine TNFα. The adaptive recovery kinetics of TNFα expression and microglial soma size showed comparable profiles and dependence on anti-inflammatory cytokine IL-10 expression. The recovery of cytokine variations and microglial morphology responses to inflammation were negatively regulated by IL-10. Our novel morphological analysis of microglia is able to detect subtle changes and can be used widely. We implemented simulations of cytokine network dynamics which showed-counter-intuitively, but in line with our experimental observations-that negative feedback from IL-10 was sufficient to impede the adaptive recovery of TNFα-mediated inflammation. Our integrative approach is a powerful tool to study changes in specific components of microglial morphology for insights into their functional states, in relation to cytokine network dynamics, during CNS injury and disease.

BackgroundThe success of CAR-T cell therapy in treating solid tumors has faced several limitations. Key factors limiting CAR-T efficacy in the hostile tumor microenvironment (TME) are lack of T cell persistence and poor durability due to the immunosuppressive and hypoxic milieu. To equip CAR-T cells with attributes that improve their function against solid tumors, we have developed a novel method to condition CAR-T cells during ex vivo expansion. Without the need for complex engineering, our conditioning regimen is a radically simple and effective way to epigenetically program CAR-T cells for long-term persistence and durable effector function, proven in in vivo models. Our proprietary conditioning regimen generates a unique brand of OUTLAST™ CAR-T cells (OTX), that exhibit superior effector function against solid tumors.MethodsOTX CAR-T cells were engineered and expanded ex vivo in our proprietary OUTLAST conditioning media. This ex vivo conditioning epigenetically skews T cells to exhibit favorable attributes. Briefly, T cells are isolated from peripheral blood, activated via CD3, tranduced with lentivirus vector to express the CD70 CAR and expanded for 10 days in the OUTLAST conditioning media.ResultsOur first OTX CAR-T product is an autologous anti-CD70 CAR-T to treat clear cell renal cell carcinoma (ccRCC). When compared to conventional T cell (Tconv), we have demonstrated that OTX CD70 CAR-T cells have 1) superior metabolic fitness, 2) superior activation state (by CD25 MFI) in the presence of target cells, 3) lower exhaustion state after repeated stimulation (by PD-1, LAG-3 and KLRG1 MFI), 4) superior resistance to TGFβ suppression and 5) superior control of tumor cells in vivo in an aggressive repeat challenge model.ConclusionsOverall, we show that OTX CD70 CAR-T cells exhibit multiple attributes desired of CAR-T cells for effective and superior function in the solid tumor microenvironment, and that they ‘outlast’ conventional CAR-T cells leading to more durable control in vivo.

A popular theory explains that marijuana’s criminalization resulted from DuPont’s lobbying as DuPont viewed hemp as competition for their products and was politically connected. To test the effects of this rent seeking story I analyze DuPont’s stock price. Initially, Congress taxed marijuana at a prohibitive level instead of an outright ban on consumption. Such legislative authority of prohibitive taxation remained in question until a Supreme Court decision in March 1937. DuPont’s Sharpe Ratio saw a break in trend immediately upon this ruling. However, in a panel dataset the effects of the ruling are found to be highly insignificant. While DuPont may have gained from marijuana’s criminalization, ultimately it was not reflected in its stock price.

BackgroundMyeloid cells, unlike other immune cells such as T cells or NK cells, are known residents in the solid tumor microenvironment (TME). In the absence of checkpoints and in proinflammatory conditions, M1 macrophages are known to be capable of direct phagocytosis of tumor cells and can present tumor-associated antigens to the host immune system. However, the immunosuppressive conditions within the TME restrict and limit the anti-tumor response of tumor associated macrophages (TAMs), including their ability to recruit and activate other immune cells against the tumor.Engineered CAR-Monocytes can serve a unique function in cell therapy by bridging a key gap in the treatment of solid tumors. We are developing an autologous engineered CAR-M cell therapy product targeting Glypican-3 to treat hepatocellular carcinoma. The CAR serves as a homing ‘GPS’ signal for trafficking directly to the tumor site, and directs phagocytosis specifically at targeted tumor cells. Our proprietary M83.CAR molecule contains a macrophage-specific costimulatory domain that significantly increases the phagocytosis function of the CAR-M cells. Furthermore, we have demonstrated that the M83.CAR-M cells are not inhibited by the prevalent CD47 ‘do not eat me’ checkpoint, which is known to restrict myeloid function in the TME.MethodsPrimary hematopoietic stem cells (HSCs) were harvested and engineered to express a CAR molecule by lentivirus transduction generating CAR-HSCs. The CAR-HSCs underwent our proprietary ex-vivo HSC differentiation process to yield CAR-Monocytes.ResultsEffective phagocytosis of engineered CAR-M cells is central to subsequent mechanisms of actions that can elicit robust anti-tumor immunity against patient-specific neoantigens. However, the first barrier to overcome is effective infiltration of engineered CAR-M into the tumor from the periphery. We have demonstrated that our engineered CAR-Monocyte drug product can successfully home to the targeted tumor specifically, from the periphery. Subsequent in situ differentiation of CAR-monocytes into CAR-macrophages enables robust tumor cell phagocytosis, the central mechanism that results in the following: 1) proinflammatory shift in the TME, 2) recruitment of APCs and immune cells, 3) activation of T-cells against tumor neoantigens.ConclusionsThe above summarizes a unique outcome of the CAR-M mechanism of action that is not capitulated by CAR-T or CAR-NK cells. Leveraging and further enhancing CAR-M function could lead to the rejection of the tumor and its metastases, particularly in combination with other immune-modulating therapies. Our proprietary HSC-derived CAR-M platform yields a unique product that demonstrates durability and superior function, which we expect to translate to the clinic.