Targeting the oncogenic m6A demethylase FTO suppresses tumourigenesis and potentiates immune response in hepatocellular carcinoma.

OBJECTIVE: Fat mass and obesity-associated protein (FTO), an eraser of N 6-methyadenosine (m6A), plays oncogenic roles in various cancers. However, its role in hepatocellular carcinoma (HCC) is unclear. Furthermore, small extracellular vesicles (sEVs, or exosomes) are critical mediators of tumourigenesis and metastasis, but the relationship between FTO-mediated m6A modification and sEVs in HCC is unknown. DESIGN: The functions and mechanisms of FTO and glycoprotein non-metastatic melanoma protein B (GPNMB) in HCC progression were investigated in vitro and in vivo. Neutralising antibody of syndecan-4 (SDC4) was used to assess the significance of sEV-GPNMB. FTO inhibitor CS2 was used to examine the effects on anti-PD-1 and sorafenib treatment. RESULTS: FTO expression was upregulated in patient HCC tumours. Functionally, FTO promoted HCC cell proliferation, migration and invasion in vitro, and tumour growth and metastasis in vivo. FTO knockdown enhanced the activation and recruitment of tumour-infiltrating CD8+ T cells. Furthermore, we identified GPNMB to be a downstream target of FTO, which reduced the m6A abundance of GPNMB, hence, stabilising it from degradation by YTH N 6-methyladenosine RNA binding protein F2. Of note, GPNMB was packaged into sEVs derived from HCC cells and bound to the surface receptor SDC4 of CD8+ T cells, resulting in the inhibition of CD8+ T cell activation. A potential FTO inhibitor, CS2, suppresses the oncogenic functions of HCC cells and enhances the sensitivity of anti-PD-1 and sorafenib treatment. CONCLUSION: Targeting the FTO/m6A/GPNMB axis could significantly suppress tumour growth and metastasis, and enhance immune activation, highlighting the potential of targeting FTO signalling with effective inhibitors for HCC therapy.

Sorted-Cell Sequencing on HCC Specimens Reveals EPS8L3 as a Key Player in CD24/CD13/EpCAM-Triple Positive, Stemness-Related HCC Cells.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a heterogeneous cancer with varying levels of liver tumor initiating or cancer stem cells in the tumors. We aimed to investigate the expression of different liver cancer stem cell (LCSC) markers in human HCCs and identify their regulatory mechanisms in stemness-related cells. METHODS: We used an unbiased, single-marker sorting approach by flow cytometry, fluorescence-activated cell sorting, and transcriptomic analyses on HCC patients' resected specimens. Knockdown approach was used, and relevant functional assays were conducted on the identified targets of interest. RESULTS: Flow cytometry on a total of 60 HCC resected specimens showed significant heterogeneity in the expression of LCSC markers, with CD24, CD13, and EpCAM mainly contributing to this heterogeneity. Concomitant expression of CD24, CD13, and EpCAM was detected in 32 HCC samples, and this was associated with advanced tumor stages. Transcriptomic sequencing on the HCC cells sorted for these individual markers identified epidermal growth factor receptor kinase substrate 8-like protein 3 (EPS8L3) as a common gene associated with the 3 markers and was functionally validated in HCC cells. Knocking down EPS8L3 suppressed the expression of all 3 markers. To search for the upstream regulation of EPS8L3, we found SP1 bound to EPS8L3 promoter to drive EPS8L3 expression. Furthermore, using Akt inhibitor MK2206, we showed that Akt signaling-driven SP1 drove the expression of the 3 LCSC markers. CONCLUSIONS: Our findings suggest that Akt signaling-driven SP1 promotes EPS8L3 expression, which is critical in maintaining the downstream expression of CD24, CD13, and EpCAM. The findings provide insight into potential LCSC-targeting therapeutic strategies.

Temporal and spatial analysis of over 7,000 measles cases outbreak from 2018 to 2019 in the Brazilian Amazon.

OBJECTIVE: This study aimed to present a temporal and spatial analysis of the 2018 measles outbreak in Brazil, particularly in the metropolitan city of Manaus in the Amazon region, and further introduce a new tool for spatial analysis. METHODS: We analyzed the geographical data of the residences of over 7,000 individuals with measles in Manaus during 2018 and 2019. Spatial and temporal analyses were conducted to characterize various aspects of the outbreak, including the onset and prevalence of symptoms, demographics, and vaccination status. A visualization tool was also constructed to display the geographical and temporal distribution of the reported measles cases. RESULTS: Approximately 95% of the included participants had not received vaccination within the past decade. Heterogeneity was observed across all facets of the outbreak, including variations in the incubation period and symptom presentation. Age distribution exhibited two peaks, occurring at one year and 18 years of age, and the potential implications of this distribution on predictive analysis were discussed. Additionally, spatial analysis revealed that areas with the highest case densities tended to have the lowest standard of living. CONCLUSION: Understanding the spatial and temporal spread of measles outbreaks provides insights for decision-making regarding measures to mitigate future epidemics.

Unraveling the impact of cancer-associated fibroblasts on hypovascular pancreatic neuroendocrine tumors.

BACKGROUND: Pancreatic neuroendocrine tumors (PNETs) with low microvessel density and fibrosis often exhibit clinical aggressiveness. Given the contribution of cancer-associated fibroblasts (CAFs) to the hypovascular fibrotic stroma in pancreatic ductal adenocarcinoma, investigating whether CAFs play a similar role in PNETs becomes imperative. In this study, we investigated the involvement of CAFs in PNETs and their effects on clinical outcomes. METHODS: We examined 79 clinical PNET specimens to evaluate the number and spatial distribution of α-smooth muscle actin (SMA)-positive cells, which are indicative of CAFs. Then, the findings were correlated with clinical outcomes. In vitro and in vivo experiments were conducted to assess the effects of CAFs (isolated from clinical specimens) on PNET metastasis and growth. Additionally, the role of the stromal-cell-derived factor 1 (SDF1)-AGR2 axis in mediating communication between CAFs and PNET cells was investigated. RESULTS: αSMA-positive and platelet-derived growth factor-α-positive CAFs were detected in the hypovascular stroma of PNET specimens. A higher abundance of α-SMA-positive CAFs within the PNET stroma was significantly associated with a higher level of clinical aggressiveness. Notably, conditioned medium from PNET cells induced an inflammatory phenotype in isolated CAFs. These CAFs promoted PNET growth and metastasis. Mechanistically, PNET cells secreted interleukin-1, which induced the secretion of SDF1 from CAFs. This cascade subsequently elevated AGR2 expression in PNETs, thereby promoting tumor growth and metastasis. The downregulation of AGR2 in PNET cells effectively suppressed the CAF-mediated promotion of PNET growth and metastasis. CONCLUSION: CAFs drive the growth and metastasis of aggressive PNETs. The CXCR4-SDF1 axis may be a target for antistromal therapy in the treatment of PNET. This study clarifies mechanisms underlying PNET aggressiveness and may guide future therapeutic interventions targeting the tumor microenvironment.

Reciprocal interactions between malignant cells and macrophages enhance cancer stemness and M2 polarization in HBV-associated hepatocellular carcinoma.

Background: The tumor microenvironment of cancers has emerged as a crucial component in regulating cancer stemness and plays a pivotal role in cell-cell communication. However, the specific mechanisms underlying these phenomena remain poorly understood. Methods: We performed the single-cell RNA sequencing (scRNA-seq) on nine HBV-associated hepatocellular carcinoma (HCC) patients. The heterogeneity of the malignant cells in pathway functions, transcription factors (TFs) regulation, overall survival, stemness, as well as ligand-receptor-based intercellular communication with macrophages were characterized. The aggressive and stemness feature for the target tumor subclone was validated by the conduction of in vitro assays including sphere formation, proliferation, Annexin V apoptosis, flow cytometry, siRNA library screening assays, and multiple in vivo preclinical mouse models including mouse hepatoma cell and human HCC cell xenograft models with subcutaneous or orthotopic injection. Results: Our analysis yielded a comprehensive atlas of 31,664 cells, revealing a diverse array of malignant cell subpopulations. Notably, we identified a stemness-related subclone of HCC cells with concurrent upregulation of CD24, CD47, and ICAM1 expression that correlated with poorer overall survival. Functional characterization both in vitro and in vivo validated S100A11 as one of the top downstream mediators for tumor initiation and stemness maintenance of this subclone. Further investigation of cell-cell communication within the tumor microenvironment revealed a propensity for bi-directional crosstalk between this stemness-related subclone and tumor-associated macrophages (TAMs). Co-culture study showed that this interaction resulted in the maintenance of the expression of cancer stem cell markers and driving M2-like TAM polarization towards a pro-tumorigenic niche. We also consolidated an inverse relationship between the proportions of TAMs and tumor-infiltrating T cells. Conclusions: Our study highlighted the critical role of stemness-related cancer cell populations in driving an immunosuppressive tumor microenvironment and identified the S100A11 gene as a key mediator for stemness maintenance in HCC. Moreover, our study provides support that the maintenance of cancer stemness is more attributed to M2 polarization than the recruitment of the TAMs.

LANCL1, a cell surface protein, promotes liver tumor initiation through FAM49B-Rac1 axis to suppress oxidative stress.

BACKGROUND AND AIMS: HCC is an aggressive cancer with a poor clinical outcome. Understanding the mechanisms that drive tumor initiation is important for improving treatment strategy. This study aimed to identify functional cell membrane proteins that promote HCC tumor initiation. APPROACH AND RESULTS: Tailor-made siRNA library screening was performed for all membrane protein-encoding genes that are upregulated in human HCC (n = 134), with sphere formation as a surrogate readout for tumor initiation. Upon confirmation of membranous localization by immunofluorescence and tumor initiation ability by limiting dilution assay in vivo, LanC-like protein-1 (LANCL1) was selected for further characterization. LANCL1 suppressed intracellular reactive oxygen species (ROS) and promoted tumorigenicity both in vitro and in vivo. Mechanistically, with mass spectrometry, FAM49B was identified as a downstream binding partner of LANCL1. LANCL1 stabilized FAM49B by blocking the interaction of FAM49B with the specific E3 ubiquitin ligase TRIM21, thus protecting FAM49B from ubiquitin-proteasome degradation. The LANCL1-FAM49B axis suppressed the Rac1-NADPH oxidase-driven ROS production, but this suppression of ROS was independent of the glutathione transferase function of LANCL1. Clinically, HCCs with high co-expression of LANCL1 and FAM49B were associated with more advanced tumor stage, poorer overall survival, and disease-free survival. In addition, anti-LANCL1 antibodies targeting the extracellular N-terminal domain were able to suppress the self-renewal ability, as demonstrated by the sphere formation ability of HCC cells. CONCLUSIONS: Our data showed that LANCL1 is a cell surface protein and a key contributor to HCC initiation. Targeting the LANCL1-FAM49B-Rac1-NADPH oxidase-ROS signaling axis may be a promising therapeutic strategy for HCC.

Temporal and spatial analysis of over 7,000 measles cases outbreak from 2018 to 2019 in the Brazilian Amazon

ABSTRACT Objective: This study aimed to present a temporal and spatial analysis of the 2018 measles outbreak in Brazil, particularly in the metropolitan city of Manaus in the Amazon region, and further introduce a new tool for spatial analysis. Methods: We analyzed the geographical data of the residences of over 7,000 individuals with measles in Manaus during 2018 and 2019. Spatial and temporal analyses were conducted to characterize various aspects of the outbreak, including the onset and prevalence of symptoms, demographics, and vaccination status. A visualization tool was also constructed to display the geographical and temporal distribution of the reported measles cases. Results: Approximately 95% of the included participants had not received vaccination within the past decade. Heterogeneity was observed across all facets of the outbreak, including variations in the incubation period and symptom presentation. Age distribution exhibited two peaks, occurring at one year and 18 years of age, and the potential implications of this distribution on predictive analysis were discussed. Additionally, spatial analysis revealed that areas with the highest case densities tended to have the lowest standard of living. Conclusion: Understanding the spatial and temporal spread of measles outbreaks provides insights for decision-making regarding measures to mitigate future epidemics.

Midline 1 interacting protein 1 promotes cancer metastasis through FOS-like 1-mediated matrix metalloproteinase 9 signaling in HCC.

BACKGROUND AND AIMS: Understanding the mechanisms of HCC progression and metastasis is crucial to improve early diagnosis and treatment. This study aimed to identify key molecular targets involved in HCC metastasis. APPROACH AND RESULTS: Using whole-transcriptome sequencing of patients' HCCs, we identified and validated midline 1 interacting protein 1 (MID1IP1) as one of the most significantly upregulated genes in metastatic HCCs, suggesting its potential role in HCC metastasis. Clinicopathological correlation demonstrated that MID1IP1 upregulation significantly correlated with more aggressive tumor phenotypes and poorer patient overall survival rates. Functionally, overexpression of MID1IP1 significantly promoted the migratory and invasive abilities and enhanced the sphere-forming ability and expression of cancer stemness-related genes of HCC cells, whereas its stable knockdown abrogated these effects. Perturbation of MID1IP1 led to significant tumor shrinkage and reduced pulmonary metastases in an orthotopic liver injection mouse model and reduced pulmonary metastases in a tail-vein injection model in vivo . Mechanistically, SP1 transcriptional factor was found to be an upstream driver of MID1IP1 transcription. Furthermore, transcriptomic sequencing on MID1IP1-overexpressing HCC cells identified FOS-like 1 (FRA1) as a critical downstream mediator of MID1IP1. MID1IP1 upregulated FRA1 to subsequently promote its transcriptional activity and extracellular matrix degradation activity of matrix metalloproteinase MMP9, while knockdown of FRA1 effectively abolished the MID1IP1-induced migratory and invasive abilities. CONCLUSIONS: Our study identified MID1IP1 as a regulator in promoting FRA1-mediated-MMP9 signaling and demonstrated its role in HCC metastasis. Targeting MID1IP1-mediated FRA1 pathway may serve as a potential therapeutic strategy against HCC progression.

S100A10 promotes HCC development and progression via transfer in extracellular vesicles and regulating their protein cargos.

OBJECTIVE: Growing evidence indicates that tumour cells exhibit characteristics similar to their lineage progenitor cells. We found that S100 calcium binding protein A10 (S100A10) exhibited an expression pattern similar to that of liver progenitor genes. However, the role of S100A10 in hepatocellular carcinoma (HCC) progression is unclear. Furthermore, extracellular vesicles (EVs) are critical mediators of tumourigenesis and metastasis, but the extracellular functions of S100A10, particularly those related to EVs (EV-S100A10), are unknown. DESIGN: The functions and mechanisms of S100A10 and EV-S100A10 in HCC progression were investigated in vitro and in vivo. Neutralising antibody (NA) to S100A10 was used to evaluate the significance of EV-S100A10. RESULTS: Functionally, S100A10 promoted HCC initiation, self-renewal, chemoresistance and metastasis in vitro and in vivo. Of significance, we found that S100A10 was secreted by HCC cells into EVs both in vitro and in the plasma of patients with HCC. S100A10-enriched EVs enhanced the stemness and metastatic ability of HCC cells, upregulated epidermal growth factor receptor (EGFR), AKT and ERK signalling, and promoted epithelial-mesenchymal transition. EV-S100A10 also functioned as a chemoattractant in HCC cell motility. Of significance, S100A10 governed the protein cargos in EVs and mediated the binding of MMP2, fibronectin and EGF to EV membranes through physical binding with integrin αⅤ. Importantly, blockage of EV-S100A10 with S100A10-NA significantly abrogated these enhancing effects. CONCLUSION: Altogether, our results uncovered that S100A10 promotes HCC progression significantly via its transfer in EVs and regulating the protein cargoes of EVs. EV-S100A10 may be a potential therapeutic target and biomarker for HCC progression.

Evidence of assortative mating for theory of mind via facial expressions but not language.

Assortative mating is a phenomenon in which romantic partners typically resemble each other at a level greater than chance. There is converging evidence that social behaviours are subject to assortative mating, though less is known regarding social cognition. Social functioning requires the ability to identify and understand the mental states of others, i.e., theory of mind. The present study recruited a sample of 102 heterosexual couples via an online survey to test if theory of mind as measured using facial expressions (Reading the Mind in the Eyes Test) or language (Stiller-Dunbar Stories Task) is associated with assortative mating. Results provide evidence of assortative mating for theory of mind via facial expressions, though there was no such effect for theory of mind via language. Assortative mating for theory of mind via facial expressions was not moderated by length of relationship nor by partner similarity in age, educational attainment, or religiosity, all variables relevant to social stratification. This suggests assortative mating for theory of mind via facial expressions is better explained by partners being alike at the start of their relationship (initial assortment) rather than becoming similar through sustained social interaction (convergence), and by people seeking out partners that are like themselves (active assortment) rather than simply pairing with those from similar demographic backgrounds (social homogamy).

Intra-arc binary collimation with dynamic axes trajectory optimization for the SRS treatment of multiple metastases with multiple prescriptions.

PURPOSE: This work generates multi-metastases cranial stereotactic radiosurgery/radiotherapy (SRS/SRT) plans using a novel treatment planning technique in which dynamic couch, collimator, and gantry trajectories are used with periodic binary target collimation. The performance of this planning technique is evaluated against conventional volumetric arc therapy (VMAT) planning in terms of various dose and plan quality metrics. METHODS: A 3D cost space (referred to herein as the combined optimization of dynamic axes or CODA cube) was calculated based on an overlap between targets and organs-at-risk (OARs) and uncollimated areas between targets (island blocking) for all combinations of couch, gantry, and collimator angles. Gradient descent through the cube was applied to determine dynamic trajectories. At each control point (CP), each target can either be conformally treated or blocked by the multi-leaf collimator (referred to as intra-arc binary collimation, iABC). Simulated annealing was used to optimize the collimation patterns throughout the arcs as well as the monitor units (MUs) delivered at each CP. Seven previously treated VMAT plans were selected for comparison against the CODA-iABC planning technique. Two CODA-iABC plans were developed: a single gantry arc plan and a plan with one gantry arc and two couch arcs. Plan quality comparison metrics included maximum and mean dose to OARs (brainstem, chiasm, optic nerves, eyes, and lenses), the volume of normal brain receiving 12 Gy (V12Gy), total MUs, target conformity, and dose-gradient index. RESULTS: Treatment plans generated with 1-arc CODA-iABC plans delivered an average of 21% and 30% higher maximum and mean doses to brainstem, respectively, when compared to VMAT plans. Treatment plans generated with 3-arc CODA-iABC used an average of 24% fewer MUs and resulted in an average reduction of 48% maximum dose and 50% mean dose to the OARs, when compared to VMAT. Target conformity values were worse in both CODA-iABC plans than VMAT by an average of 35% and 15%, respectively. There are no significant differences in V12Gy for all three planning techniques; however, 3-arc CODA-iABC is more effective at reducing dose to normal brain in the low-dose region (<12 Gy). CONCLUSION: CODA-iABC is a novel planning technique that has been developed to automatically generate patient-specific multi-axis trajectories for multiple metastases cranial SRS/SRT. This work has demonstrated the feasibility of planning with this novel method. The 1-arc CODA-iABC planning technique is slightly dosimetric inferior to VMAT. With an increased sampling of a three-dimensional CODA cube by using a 3-arc CODA-iABC technique, there was improved total dose sparing to all the OARs and increased MU efficiency, but with a cost in target conformity, when compared to VMAT.

Ephrin-A3/EphA2 axis regulates cellular metabolic plasticity to enhance cancer stemness in hypoxic hepatocellular carcinoma.

BACKGROUND & AIMS: The highly proliferative nature of hepatocellular carcinoma (HCC) frequently results in a hypoxic intratumoural microenvironment, which creates a therapeutic challenge owing to a lack of mechanistic understanding of the phenomenon. We aimed to identify critical drivers of HCC development and progression in the hypoxic microenvironment. METHODS: We performed integrative analysis of multiple transcriptomic and genomic profiles specific for HCC and hypoxia and identified the Ephrin-A3/Eph receptor A2 (EphA2) axis as a clinically relevant and hypoxia-inducible signalling axis in HCC. The functional significance and mechanistic consequences of the Ephrin-A3/EphA2 axis were examined in EFNA3- and EPHA2- knockdown/overexpressing HCC cells. The potential downstream pathways were investigated by transcriptome sequencing, quantitative reverse-transcription PCR, western blotting analysis and metabolomics. RESULTS: EFNA3 was frequently upregulated in HCC and its overexpression was associated with more aggressive tumour behaviours. HIF-1α directly and positively regulated EFNA3 expression under hypoxia. EFNA3 functionally contributed to self-renewal, proliferation and migration in HCC cells. EphA2 was identified as a key functional downstream mediator of EFNA3. Functional characterisation of the Ephrin-A3/EphA2 forward-signalling axis demonstrated a promotion of self-renewal ability and tumour initiation. Mechanistically, the Ephrin-A3/EphA2 axis promoted the maturation of SREBP1 and expression of its transcriptional target, ACLY, was significantly associated with the expression of EFNA3 and hypoxia markers in clinical cohorts. The metabolic signature of EPHA2 and ACLY stable knockdown HCC cells demonstrated significant overlap in fatty acid, cholesterol and tricarboxylic acid cycle metabolite profiles. ACLY was confirmed to mediate the self-renewal function of the Ephrin-A3/EphA2 axis. CONCLUSIONS: Our findings revealed the novel role of the Ephrin-A3/EphA2 axis as a hypoxia-sensitive modulator of HCC cell metabolism and a key contributor to HCC initiation and progression. LAY SUMMARY: Hepatocellular carcinoma (HCC) is a fast-growing tumour; hence, areas of the tumour often have insufficient vasculature and become hypoxic. The presence of hypoxia within tumours has been shown to negatively impact on the survival of patients with tumours, including HCC. Herein, we identified the Ephrin-A3/EphA2 axis as a key functional driver of tumour initiation and progression in response to hypoxia. Additionally, we showed that SREBP1-ACLY-mediated metabolic rewiring was an important downstream effector that induced cancer stemness in response to Ephrin-A3/EphA2 forward-signalling.

New PK/PD model directly links diabetes drug dose to blood glucose level for personalized care.

The management of diabetes mellitus focuses on close monitoring of a patient's blood glucose level while the clinician experiments with a dosing strategy using clinical guidelines and his/her own experience. We propose a pharmacokinetic and pharmacodynamics model that characterizes the dose-response of patients receiving anti-diabetic drug therapy. We derive and establish a direct relationship between drug dosage and blood glucose level. This new drug-dose drug-effect model, combined with a linear disease progression model, is used to fit the patient's daily self-monitored blood glucose (SMBG) data to obtain the personalized treatment effect for each patient. The model predicts the long-term drug effect using the prescribed dose, thus allowing for dose optimization. The model is evaluated on patients with gestational diabetes mellitus. SMBG data collected during the first month of treatment is used to train the model. The model is able to characterize the personalized dose-response and disease progression. Moreover, when compared to a descriptive autoregression model, our model gives a better long-term prediction of the drug effect on the trend of the blood glucose level. This mechanism-based treatment effect model utilizes daily recorded blood glucose data to estimate and predict a patient's personalized dose-response and disease progression. Such evidence can be used by clinicians to individualize and optimize dose regimens to achieve better treatment outcomes.

Analyzing Strategies for Containing Avian Influenza.

In this paper, we couple a general-purpose infectious disease theory with a computational modeling framework to analyze strategies for avian influenza containment. We focus on virus transmission among domestic poultry populations to optimize and evaluate the effectiveness of three containment strategies and their combinations: reducing the contact rate among domestic birds, reducing the population of infected birds, and reducing the transportation of infected birds. We illustrate their usage during a two-wave avian flu outbreak in Nigeria. Our findings show that reducing contacts by 20% via cluster isolation early in the first wave can achieve containment rapidly. It also helps avert the second wave. Slaughtering infected birds is not as effective, requiring scheduled killings of over 80% of the poultry while failing to avert the second wave. This practice also risks damaging the local economy and potential secondary infections from the carcasses of infected birds. Reducing transportation between northern and southern Nigeria does not offer good containment since the disease spread began in both regions simultaneously. Reducing transportation has an impact when applied to neighboring regions and cities, or when the initial incidence of the disease is localized. Combination strategies prove to be the most practical and cost-effective to implement. The use of 3D-effectiveness visualized plots allows policymakers to evaluate multiple combination strategies and choose the one that optimizes containment while also adhering to budget constraints, resource availability, and management preference. The generalized mathematical theory and modeling framework is highly flexible and can be applied to other diseases, including those with multiple hosts, multiple species involvement, and across a broad array of heterogeneous regions.

Antioxidant supplements promote tumor formation and growth and confer drug resistance in hepatocellular carcinoma by reducing intracellular ROS and induction of TMBIM1.

BACKGROUND: Controversy over the benefits of antioxidants supplements in cancers persists for long. Using hepatocellular carcinoma (HCC) as a model, we investigated the effects of exogenous antioxidants N-acetylcysteine (NAC) and glutathione (GSH) on tumor formation and growth. METHODS: Multiple mouse models, including diethylnitrosamine (DEN)-induced and Trp53KO/C-MycOE-induced HCC models, mouse hepatoma cell and human HCC cell xenograft models with subcutaneous or orthotopic injection were used. In vitro assays including ROS assay, colony formation, sphere formation, proliferation, migration and invasion, apoptosis, cell cycle assays were conducted. Western blot was performed for protein expression and RNA-sequencing to identify potential gene targets. RESULTS: In these multiple different mouse and cell line models, we observed that NAC and GSH promoted HCC tumor formation and growth, accompanied with significant reduction of intracellular reactive oxygen species (ROS) levels. Moreover, NAC and GSH promoted cancer stemness, and abrogated the tumor-suppressive effects of Sorafenib both in vitro and in vivo. Exogenous supplementation of NAC or GSH reduced the expression of NRF2 and GCLC, suggesting the NRF2/GCLC-related antioxidant production pathway might be desensitized. Using transcriptomic analysis to identify potential gene targets, we found that TMBIM1 was significantly upregulated upon NAC and GSH treatment. Both TCGA and in-house RNA-sequence databases showed that TMBIM1 was overexpressed in HCC tumors. Stable knockdown of TMBIM1 increased the intracellular ROS; it also abolished the promoting effects of the antioxidants in HCC cells. On the other hand, BSO and SSA, inhibitors targeting NAC and GSH metabolism respectively, partially abrogated the pro-oncogenic effects induced by NAC and GSH in vitro and in vivo. CONCLUSIONS: Our data implicate that exogenous antioxidants NAC and GSH, by reducing the intracellular ROS levels and inducing TMBIM expression, promoted HCC formation and tumor growth, and counteracted the therapeutic effect of Sorafenib. Our study provides scientific insight regarding the use of exogenous antioxidant supplements in cancers.

Strategies for Vaccine Prioritization and Mass Dispensing.

We propose a system that helps decision makers during a pandemic find, in real time, the mass vaccination strategies that best utilize limited medical resources to achieve fast containments and population protection. Our general-purpose framework integrates into a single computational platform a multi-purpose compartmental disease propagation model, a human behavior network, a resource logistics model, and a stochastic queueing model for vaccination operations. We apply the modeling framework to the current COVID-19 pandemic and derive an optimal trigger for switching from a prioritized vaccination strategy to a non-prioritized strategy so as to minimize the overall attack rate and mortality rate. When vaccine supply is limited, such a mixed vaccination strategy is broadly effective. Our analysis suggests that delays in vaccine supply and inefficiencies in vaccination delivery can substantially impede the containment effort. Employing an optimal mixed strategy can significantly reduce the attack and mortality rates. The more infectious the virus, the earlier it helps to open the vaccine to the public. As vaccine efficacy decreases, the attack and mortality rates rapidly increase by multiples; this highlights the importance of early vaccination to reduce spreading as quickly as possible to lower the chances for further mutations to evolve and to reduce the excessive healthcare burden. To maximize the protective effect of available vaccines, of equal importance are determining the optimal mixed strategy and implementing effective on-the-ground dispensing. The optimal mixed strategy is quite robust against variations in model parameters and can be implemented readily in practice. Studies with our holistic modeling framework strongly support the urgent need for early vaccination in combating the COVID-19 pandemic. Our framework permits rapid custom modeling in practice. Additionally, it is generalizable for different types of infectious disease outbreaks, whereby a user may determine for a given type the effects of different interventions including the optimal switch trigger.

Characterization of initial key steps of IL-17 receptor B oncogenic signaling for targeted therapy of pancreatic cancer.

The members of the interleukin-17 (IL-17) cytokine family and their receptors were identified decades ago. Unlike IL-17 receptor A (IL-17RA), which heterodimerizes with IL-17RB, IL-17RC, and IL-17RD and mediates proinflammatory gene expression, IL-17RB plays a distinct role in promoting tumor growth and metastasis upon stimulation with IL-17B. However, the molecular basis by which IL-17RB promotes oncogenesis is unknown. Here, we report that IL-17RB forms a homodimer and recruits mixed-lineage kinase 4 (MLK4), a dual kinase, to phosphorylate it at tyrosine-447 upon treatment with IL-17B in vitro. Higher amounts of phosphorylated IL-17RB in tumor specimens obtained from patients with pancreatic cancer correlated with worse prognosis. Phosphorylated IL-17RB recruits the ubiquitin ligase tripartite motif containing 56 to add lysine-63-linked ubiquitin chains to lysine-470 of IL-17RB, which further assembles NF-κB activator 1 (ACT1) and other factors to propagate downstream oncogenic signaling. Consequentially, IL-17RB mutants with substitution at either tyrosine-447 or lysine-470 lose their oncogenic activity. Treatment with a peptide consisting of amino acids 403 to 416 of IL-17RB blocks MLK4 binding, tyrosine-477 phosphorylation, and lysine-470 ubiquitination in vivo, thereby inhibiting tumorigenesis and metastasis and prolonging the life span of mice bearing pancreatic tumors. These results establish a clear pathway of how proximal signaling of IL-17RB occurs and provides insight into how this pathway provides a therapeutic target for pancreatic cancer.

Redox sensor NPGPx restrains ZAP70 activity and modulates T cell homeostasis.

Emerging evidences implicate the contribution of ROS to T cell activation and signaling. The tyrosine kinase, ζ-chain-associated protein of 70 kDa (ZAP70), is essential for T cell development and activation. However, it remains elusive whether a direct redox regulation affects ZAP70 activity upon TCR stimulation. Here, we show that deficiency of non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), a redox sensor, results in T cell hyperproliferation and elevated cytokine productions. T cell-specific NPGPx-knockout mice reveal enhanced T-dependent humoral responses and are susceptible to experimental autoimmune encephalomyelitis (EAE). Through proteomic approaches, ZAP70 is identified as the key interacting protein of NPGPx through disulfide bonding. NPGPx is activated by ROS generated from TCR stimulation, and modulates ZAP70 activity through redox switching to reduce ZAP70 recruitment to TCR/CD3 complex in membrane lipid raft, therefore subduing TCR responses. These results reveal a delicate redox mechanism that NPGPx serves as a modulator to curb ZAP70 functions in maintaining T cell homeostasis.

Strategies for Disease Containment: A Biological-Behavioral-Intervention Computational Informatics Framework.

In this study, we describe the development and use of a biological-behavior-intervention computational informatics framework that combines disease modelling for infectious virus with stratifications for social behavior and employment, and resource logistics. The framework incorporates heterogeneous group behavior and interaction dynamics, and optimizes intervention and resources for effective containment. We demonstrate its usage by analyzing and optimizing containment strategies for the 2014-2016 West Africa Ebola outbreak, and its implementation for responses to the 2020 COVID-19 pandemic in the United States. Our analysis shows that timely action within 1.5 months from the onset of confirmed cases can cut down 90% of overall infections and bring rapid containment within 6-8 months. The additional medical resources required are minor and would ensure proper treatment and quarantine of patients while reducing the risk of infections among healthcare workers. The benefit (in infection / death control) would be reduced by 10 to over 100 fold and time to containment would increase by 2-4 fold when intervention and medical resources are injected within 5 months. In contrast, the additional resources needed to bring down the overall infection in a delayed intervention are significant, with inferior results. The disease module can be tailored for different pathogens. It expands the well-used SEIR model to include social and intervention activities, asymptomatic and post-recovery transmission, hospitalization, outcome of recovery, and funeral events. The model also examines the transmission rate of health care workers and allows for heterogenous infection factors among different groups. It also captures time-variant human behavior during the horizon of the outbreak. The framework optimizes the intervention timeline and resource allocation during an infectious disease outbreak and offers insights on how resource availability in time and quantity can affect the disease trends and containment significantly. This can inform policy, disease management and resource allocation. While focusing on bed availability for quarantine and treatment appears to be simplistic, their necessity for Ebola responses cannot be overemphasized. We link these insights to a web-based tool to provide quick and intuitive observations for decision making and investigation of the disease outbreak situation. Subsequent use of the system to determine the optimal timing and effectiveness and tradeoffs analysis of various non-pharmaceutical intervention strategies for COVID-19 provide a foundation for policy makers to execute the first-step response. These results have been implemented on the ground since March 2020. The web-based tool pinpoints accurately the import of disease from global travels and associated disease spread and health burdens. This prospectively affirms the importance of such a real-time computational system, and its availability before onset of a pandemic.

A systems approach to examine hospital-acquired infections in a paediatric CICU.

OBJECTIVE: We aimed to apply systems engineering principles to address hospital-acquired infections in the paediatric intensive care setting. DESIGN: Mixed method approach involving four steps: perform time-motion study of cardiac intensive care unit (CICU) care processes, establish a meaningful schema to classify observations, design a web-based system to manage and analyse data, and design a prototypical computer-based training system to assist with hygiene compliance. SETTING: Paediatric CICU at the Children's Healthcare of Atlanta. PATIENTS: Paediatric patients undergoing congenital heart surgery. INTERVENTIONS: Extensive time-motion study of CICU care processes. MEASUREMENTS: Non-compliances were recorded for each care process observed during the time-motion study. RESULTS: Guided by our observations, we introduced a novel categorisation schema with action types, observation categories, severity classes, procedure classifications, and personnel categories that offer a systematic and efficient mechanism for reporting and classifying non-compliance and violations. Utilising these categories, a web-based database management system was designed that allows observers to input their data. This web analytic tool offers easy summarisation, data analysis, and visualisation of findings. A computer-based training system with modules to educate visitors in hospital-acquired infections hygiene was also created. CONCLUSION: Our study offers a checklist of non-compliance situations and potential development of a proactive surveillance system of awareness of infection-prone situations. Working with quality improvement experts and stakeholders, recommendations and actionable practice will be synthesised for implementation in clinical settings. Careful design of the implementation protocol is needed to measure and quantify the potential improvements in outcomes.