Stromal HIF2 Regulates Immune Suppression in the Pancreatic Cancer Microenvironment.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) has a hypoxic, immunosuppressive stroma that contributes to its resistance to immune checkpoint blockade therapies. The hypoxia-inducible factors (HIFs) mediate the cellular response to hypoxia, but their role within the PDAC tumor microenvironment remains unknown. METHODS: We used a dual recombinase mouse model to delete Hif1α or Hif2α in α-smooth muscle actin-expressing cancer-associated fibroblasts (CAFs) arising within spontaneous pancreatic tumors. The effects of CAF HIF2α expression on tumor progression and composition of the tumor microenvironment were evaluated by Kaplan-Meier analysis, reverse transcription quantitative real-time polymerase chain reaction, histology, immunostaining, and by both bulk and single-cell RNA sequencing. CAF-macrophage crosstalk was modeled ex vivo using conditioned media from CAFs after treatment with hypoxia and PT2399, an HIF2 inhibitor currently in clinical trials. Syngeneic flank and orthotopic PDAC models were used to assess whether HIF2 inhibition improves response to immune checkpoint blockade. RESULTS: CAF-specific deletion of Hif2α, but not Hif1α, suppressed PDAC tumor progression and growth, and improved survival of mice by 50% (n = 21-23 mice/group, Log-rank P = .0009). Deletion of CAF-HIF2 modestly reduced tumor fibrosis and significantly decreased the intratumoral recruitment of immunosuppressive M2 macrophages and regulatory T cells. Treatment with the clinical HIF2 inhibitor PT2399 significantly reduced in vitro macrophage chemotaxis and M2 polarization, and improved tumor responses to immunotherapy in both syngeneic PDAC mouse models. CONCLUSIONS: Together, these data suggest that stromal HIF2 is an essential component of PDAC pathobiology and is a druggable therapeutic target that could relieve tumor microenvironment immunosuppression and enhance immune responses in this disease.

Soft - sensing modeling based on ABC - MLSSVM inversion for marine low - temperature alkaline protease MP fermentation process.

BACKGROUND: Aiming at the characteristics of nonlinear, multi-parameter, strong coupling and difficulty in direct on-line measurement of key biological parameters of marine low-temperature protease fermentation process, a soft-sensing modeling method based on artificial bee colony (ABC) and multiple least squares support vector machine (MLSSVM) inversion for marine protease fermentation process is proposed. METHODS: Firstly, based on the material balance and the characteristics of the fermentation process, the dynamic "grey box" model of the fed-batch fermentation process of marine protease is established. The inverse model is constructed by analyzing the inverse system existence and introducing the characteristic information of the fermentation process. Then, the inverse model is identified off-line using MLSSVM. Meanwhile, in order to reduce the model error, the ABC algorithm is used to correct the inverse model. Finally, the corrected inverse model is connected in series to the marine alkaline protease MP fermentation process to form a composite pseudo-linear system, thus, real-time on-line prediction of key biological parameters in fermentation process can be realized. RESULTS: Taking the alkaline protease MP fermentation process as an example, the simulation results demonstrate that the soft-sensing modeling method can solve the real-time prediction problem of key biological parameters in the fermentation process on-line, and has higher accuracy and generalization ability than the traditional soft-sensing method of support vector machine. CONCLUSIONS: The research provides a new method for soft-sensing modeling of key biological parameters in fermentation process, which can be extended to soft-sensing modeling of general nonlinear systems.

Effects of air pollution control measures on air quality improvement in Guangzhou, China.

The ambient air quality of Guangzhou in 2016 has significantly improved since Guangzhou and its surrounding cities implemented a series of air pollution control measures from 2014 to 2016. This study not only estimated the effects of meteorology and emission control measures on air quality improvement in Guangzhou but also assessed the contributions of emissions reduction from various sources through the combination of observation data and simulation results from Weather Research and Forecasting - Community Multiscale Air Quality (WRF-CMAQ) modeling system. Results showed that the favorable meteorological conditions in 2016 alleviated the air pollution. Compared to change in meteorology, implementing emission control measures in Guangzhou and surrounding cities was more beneficial for air quality improvement, and it could reduce the concentrations of SO2, NO2, PM2.5, PM10, and O3 by 9.7 µg m-3 (48.4%), 9.2 µg m-3 (17.7%), 7.7 µg m-3 (14.6%), 9.7 µg m-3 (13.4%), and 12.0 µg m-3 (7.7%), respectively. Furthermore, emission control measures that implemented in Guangzhou contributed most to the concentration reduction of SO2, NO2, PM2.5, and PM10 (46.0% for SO2, 15.2% for NO2, 9.4% for PM2.5, and 9.1% for PM10), and it increased O3 concentration by 2.4%. With respect to the individual contributions of source emissions reduction, power sector emissions reduction showed the greatest contribution in reducing the concentrations of SO2, NO2, PM2.5, and PM10 due to the implementation of Ultra-Clean control technology. As for O3 mitigation, VOCs product-related source emissions reduction was most effective, and followed by transportation source emissions reduction, while the reductions of power sector, industrial boiler, and industrial process source might not be as effective. Our findings provide scientific advice for the Guangzhou government to formulate air pollution prevention and control policies in the future.

Soft-sensing method based on FDLS-SVM in marine alkaline protease fermentation process.

To overcome the problem that soft-sensing model cannot be updated with the bioprocess changes, this article proposed a soft-sensing modeling method which combined fuzzy c-means clustering (FCM) algorithm with least squares support vector machine theory (LS-SVM). FCM is used for separating a whole training data set into several clusters with different centers, each subset is trained by LS-SVM and sub-models are developed to fit different hierarchical property of the process. The new sample data that bring new operation information is introduced in the model, and the fuzzy membership function of the sample to each clustering is first calculated by the FCM algorithm. Then, a corresponding LS-SVM sub-model of the clustering with the largest fuzzy membership function is used for performing dynamic learning so that the model can update online. The proposed method is applied to predict the key biological parameters in the marine alkaline protease MP process. The simulation result indicates that the soft-sensing modeling method increases the model's adaptive abilities in various operation conditions and can improve its generalization ability.

Enhanced humoral and cellular immune responses to PRRS virus GP5 glycoprotein by DNA prime-adenovirus boost vaccination in mice.

In order to investigate the induction of humoral and cellular immune responses against porcine reproductive and respiratory syndrome virus (PRRSV), BALB/c mice were immunized in a pcDNA3-GP5 prime-rAd-GP5 boost regimen. After humoral and cellular immune response detection, levels of PRRSV-specific antibodies, neutralizing antibodies, lymphocyte proliferation response, and cytotoxic T-lymphocyte response were significantly increased as compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. Significant difference was observed between heterogeneous and homologous vaccination. Induction of anti-GP5 antibody response was higher in all heterogeneous combinations than those of the homologous combinations. In the induction of lymphocyte proliferation response and CTL response, the homologous combination of pcDNA3-GP5/pcDNA3-GP5/pcDNA3-GP5was significantly stronger than that of rAd-GP5/rAd-GP5/rAd-GP5, but was relatively weaker than the heterogeneous combination of pcDNA3-GP5/pcDNA3-GP5/rAd-GP5 and pcDNA3-GP5/rAd-GP5/rAd-GP5. This heterogeneous combination was a most efficient immunization regimen in induction of PRRSV-specific cellular immune response just as the antibody response. These results suggested that DNA immunization followed by recombinant adenovirus boosting could be used as a potential PRRSV vaccine.

[Effects of different gene segments of influenza virus on the growth of recombinants].

OBJECTIVE: We explored which internal genes of influenza virus that affect the titer of recombinant viruses and contribute to the high yield of Influenza A seed virus in ovo. METHODS: Six internal genes or mutant or polymerase complex of A/Puerto Rico/8/1934 (H1N1) (PR8) virus genes were replaced individually by corresponding gene of A/ chicken/ZJ/China/2013 ( H5N1) virus, and the hemagglutination titers of recombinant viruses were compared by HA assay. RESULTS: PB2 gene had the greatest influence, its replace failed to generate recombinant virus. When PB1, PA, or M gene was replaced, the titers of recombinant viruses dropped by 3.7, 3.4, 3.0 (log2), respectively. NS gene had little influence upon HA titer. When polymerase complex genes were replaced, virus titer dropped slightly to 7.6 log2, and it did not confer the same growth characteristics (8.4 log2) found when a complete polymerase complex was of PR8 origin. When amino acids of position 627 of PR8 PB2 gene were mutated to glutamic acid, virus titer rose from 8.4 log2 to 8.7 log2. CONCLUSION: The optimal gene combinations may facilitate replication through viral RNA and protein interaction with cellular components as well as interaction of viral RNA and protein or protein-protein interactions within the virus. These multi-factorial contributions resulted in selection of a high replication competent reassortant in embryonated chicken eggs in comparison to the respective low yield wild type viruses, and laid the foundation for high

Simultaneous real-time detection of fractional exhaled nitric oxide and end-tidal carbon dioxide by quantum cascade laser absorption spectroscopy.

The simultaneous detection of fractional exhaled nitric oxide (FeNO) and end-tidal carbon dioxide (ETCO2) is of great importance for the distinguishing and diagnosis of asthma and chronic obstructive pulmonary disease (COPD), providing more comprehensive information on respiratory disorders. This work demonstrates a simultaneous ETCO2 and FeNO detection system based on quantum cascade laser absorption spectroscopy (QCLAS) technology was presented. The system employs wavelength modulation spectroscopy (WMS) technology and the Herriott multi-pass cell, achieving a detection limit of 2.82 ppb for nitric oxide (NO) and 0.05 % for carbon dioxide (CO2). Real-time exhalation measurements were performed on volunteers with varying ETCO2 and FeNO levels, and the results of the test can accurately distinguish whether the corresponding volunteer was healthy, had asthma or COPD. The effect of exhalation flow rate on the concentration of the two gases was explored. A range of expiratory flow rates were tested in the flow rate interval from 1 to 4 L/min, and there was always an inverse relationship between expiratory flow rate and FeNO concentration, but flow rate changes did not affect ETCO2 concentration. The results indicate that this detection system can simultaneously and effectively measure ETCO2 and FeNO concentrations in real-time.

Single-cell multiomics decodes regulatory programs for mouse secondary palate development.

Perturbations in gene regulation during palatogenesis can lead to cleft palate, which is among the most common congenital birth defects. Here, we perform single-cell multiome sequencing and profile chromatin accessibility and gene expression simultaneously within the same cells (n = 36,154) isolated from mouse secondary palate across embryonic days (E) 12.5, E13.5, E14.0, and E14.5. We construct five trajectories representing continuous differentiation of cranial neural crest-derived multipotent cells into distinct lineages. By linking open chromatin signals to gene expression changes, we characterize the underlying lineage-determining transcription factors. In silico perturbation analysis identifies transcription factors SHOX2 and MEOX2 as important regulators of the development of the anterior and posterior palate, respectively. In conclusion, our study charts epigenetic and transcriptional dynamics in palatogenesis, serving as a valuable resource for further cleft palate research.

Single-nucleus multiomics reveals the disrupted regulatory programs in three brain regions of sporadic early-onset Alzheimer's disease.

Sporadic early-onset Alzheimer's disease (sEOAD) represents a significant but less-studied subtype of Alzheimer's disease (AD). Here, we generated a single-nucleus multiome atlas derived from the postmortem prefrontal cortex, entorhinal cortex, and hippocampus of nine individuals with or without sEOAD. Comprehensive analyses were conducted to delineate cell type-specific transcriptomic changes and linked candidate cis-regulatory elements (cCREs) across brain regions. We prioritized seven conservative transcription factors in glial cells in multiple brain regions, including RFX4 in astrocytes and IKZF1 in microglia, which are implicated in regulating sEOAD-associated genes. Moreover, we identified the top 25 altered intercellular signaling between glial cells and neurons, highlighting their regulatory potential on gene expression in receiver cells. We reported 38 cCREs linked to sEOAD-associated genes overlapped with late-onset AD risk loci, and sEOAD cCREs enriched in neuropsychiatric disorder risk loci. This atlas helps dissect transcriptional and chromatin dynamics in sEOAD, providing a key resource for AD research.

Single-nucleus multiomics reveals the disrupted regulatory programs in three brain regions of sporadic early-onset Alzheimer's disease.

Sporadic early-onset Alzheimer's disease (sEOAD) represents a significant but less-studied subtype of Alzheimer's disease (AD). Here, we generated a single-nucleus multiome atlas derived from the postmortem prefrontal cortex, entorhinal cortex, and hippocampus of nine individuals with or without sEOAD. Comprehensive analyses were conducted to delineate cell type-specific transcriptomic changes and linked candidate cis- regulatory elements (cCREs) across brain regions. We prioritized seven conservative transcription factors in glial cells in multiple brain regions, including RFX4 in astrocytes and IKZF1 in microglia, which are implicated in regulating sEOAD-associated genes. Moreover, we identified the top 25 altered intercellular signaling between glial cells and neurons, highlighting their regulatory potential on gene expression in receiver cells. We reported 38 cCREs linked to sEOAD-associated genes overlapped with late-onset AD risk loci, and sEOAD cCREs enriched in neuropsychiatric disorder risk loci. This atlas helps dissect transcriptional and chromatin dynamics in sEOAD, providing a key resource for AD research.

Comparative Untargeted Metabolomic Profiling of Induced Mitochondrial Fusion in Pancreatic Cancer.

Mitochondria are dynamic organelles that constantly alter their shape through the recruitment of specialized proteins, like mitofusin-2 (Mfn2) and dynamin-related protein 1 (Drp1). Mfn2 induces the fusion of nearby mitochondria, while Drp1 mediates mitochondrial fission. We previously found that the genetic or pharmacological activation of mitochondrial fusion was tumor suppressive against pancreatic ductal adenocarcinoma (PDAC) in several model systems. The mechanisms of how these different inducers of mitochondrial fusion reduce pancreatic cancer growth are still unknown. Here, we characterized and compared the metabolic reprogramming of these three independent methods of inducing mitochondrial fusion in KPC cells: overexpression of Mfn2, genetic editing of Drp1, or treatment with leflunomide. We identified significantly altered metabolites via robust, orthogonal statistical analyses and found that mitochondrial fusion consistently produces alterations in the metabolism of amino acids. Our unbiased methodology revealed that metabolic perturbations were similar across all these methods of inducing mitochondrial fusion, proposing a common pathway for metabolic targeting with other drugs.

APOBEC3A drives deaminase domain-independent chromosomal instability to promote pancreatic cancer metastasis.

Despite efforts in understanding its underlying mechanisms, the etiology of chromosomal instability (CIN) remains unclear for many tumor types. Here, we identify CIN initiation as a previously undescribed function for APOBEC3A (A3A), a cytidine deaminase upregulated across cancer types. Using genetic mouse models of pancreatic ductal adenocarcinoma (PDA) and genomics analyses in human tumor cells we show that A3A-induced CIN leads to aggressive tumors characterized by enhanced early dissemination and metastasis in a STING-dependent manner and independently of the canonical deaminase functions of A3A. We show that A3A upregulation recapitulates numerous copy number alterations commonly observed in patients with PDA, including co-deletions in DNA repair pathway genes, which in turn render these tumors susceptible to poly (ADP-ribose) polymerase inhibition. Overall, our results demonstrate that A3A plays an unexpected role in PDA as a specific driver of CIN, with significant effects on disease progression and treatment.

Selective EGLN Inhibition Enables Ablative Radiotherapy and Improves Survival in Unresectable Pancreatic Cancer.

When pancreatic cancer cannot be removed surgically, patients frequently experience morbidity and death from progression of their primary tumor. Radiation therapy (RT) cannot yet substitute for an operation because radiation causes fatal bleeding and ulceration of the nearby stomach and intestines before achieving tumor control. There are no FDA-approved medications that prevent or reduce radiation-induced gastrointestinal injury. Here, we overcome this fundamental problem of anatomy and biology with the use of the oral EGLN inhibitor FG-4592, which selectively protects the intestinal tract from radiation toxicity without protecting tumors. A total of 70 KPC mice with autochthonous pancreatic tumors received oral FG-4592 or vehicle control ± ablative RT to a cumulative 75 Gy administered in 15 daily fractions to a limited tumor field. Although ablative RT reduced complications from local tumor progression, fatal gastrointestinal bleeding was observed in 56% of mice that received high-dose RT with vehicle control. However, radiation-induced bleeding was completely ameliorated in mice that received high-dose RT with FG-4592 (0% bleeding, P < 0.0001 compared with vehicle). Furthermore, FG-4592 reduced epithelial apoptosis by half (P = 0.002) and increased intestinal microvessel density by 80% compared with vehicle controls. EGLN inhibition did not stimulate cancer growth, as treatment with FG-4592 alone, or overexpression of HIF2 within KPC tumors independently improved survival. Thus, we provide a proof of concept for the selective protection of the intestinal tract by the EGLN inhibition to enable ablative doses of cytotoxic therapy in unresectable pancreatic cancer by reducing untoward morbidity and death from radiation-induced gastrointestinal bleeding. SIGNIFICANCE: Selective protection of the intestinal tract by EGLN inhibition enables potentially definitive doses of radiation therapy. This might allow radiation to be a surgical surrogate for unresectable pancreatic cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/9/2327/F1.large.jpg.

Mitochondrial fusion exploits a therapeutic vulnerability of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) requires mitochondrial oxidative phosphorylation (OXPHOS) to fuel its growth, however, broadly inhibiting this pathway might also disrupt essential mitochondrial functions in normal tissues. PDAC cells exhibit abnormally fragmented mitochondria that are essential to its oncogenicity, but it was unclear if this mitochondrial feature was a valid therapeutic target. Here, we present evidence that normalizing the fragmented mitochondria of pancreatic cancer via the process of mitochondrial fusion reduces OXPHOS, which correlates with suppressed tumor growth and improved survival in preclinical models. Mitochondrial fusion was achieved by genetic or pharmacologic inhibition of dynamin related protein-1 (Drp1) or through overexpression of mitofusin-2 (Mfn2). Notably, we found that oral leflunomide, an FDA-approved arthritis drug, promoted a two-fold increase in Mfn2 expression in tumors and was repurposed as a chemotherapeutic agent, improving the median survival of mice with spontaneous tumors by 50% compared to vehicle. We found that the chief tumor suppressive mechanism of mitochondrial fusion was enhanced mitophagy, which proportionally reduced mitochondrial mass and ATP production. These data suggest that mitochondrial fusion is a specific and druggable regulator of pancreatic cancer growth that could be rapidly translated to the clinic.

The inhibition of migration and invasion of cancer cells by graphene via the impairment of mitochondrial respiration.

Graphene and its derivatives have become important nanomaterials worldwide and have potential medical applications including in vivo diagnosis, drug delivery, and photothermal therapy of cancer. However, little is known about their effect on the metastasis of cancer cells, which is the cause of over 90% of patient deaths. In the present investigation, we provide direct evidence that low concentrations of pristine graphene and graphene oxide show no apparent influence on the viability of MDA-MB-231 human breast cancer cells, PC3 human prostate cancer cells, as well as B16F10 mouse melanoma cells. However, both pristine graphene and graphene oxide can effectively inhibit the migration and invasion of these cancer cells. Further studies indicate that exposure of cells to graphene led to the direct inhibition of the electron transfer chain complexes I, II, III and IV, most likely by disrupting electron transfer between iron-sulfur centers, which is due to its stronger ability to accept electrons compared to iron-sulfur clusters through theoretical calculations. The decreased electron transfer chain activity caused a reduction in the production of ATP and subsequent impairment of F-actin cytoskeleton assembly, which is crucial for the migration and invasion of metastatic cancer cells. The inhibition of cancer cell metastasis by graphene and graphene oxide might provide new insights into specific cancer treatment.

Energy metabolism analysis reveals the mechanism of inhibition of breast cancer cell metastasis by PEG-modified graphene oxide nanosheets.

Recent advances in nanomedicine provide promising alternatives for cancer treatment that may improve the survival of patients with metastatic disease. The goal of the present study was to evaluate graphene oxide (GO) as a potential anti-metastatic agent. For this purpose, GO was modified with polyethylene glycol (PEG) to form PEG-modified GO (PEG-GO), which improves its aqueous stability and biocompatibility. We show here that PEG-GO exhibited no apparent effects on the viability of breast cancer cells (MDA-MB-231, MDA-MB-436, and SK-BR-3) or non-cancerous cells (MCF-10A), but inhibited cancer cell migration in vitro and in vivo. Analysis of cellular energy metabolism revealed that PEG-GO significantly impaired mitochondrial oxidative phosphorylation (OXPHOS) in breast cancer cells; however, PEG-GO showed no effect on OXPHOS in non-cancerous cells. To explore the underlying mechanisms, a SILAC (Stable Isotope Labeling by Amino acids in Cell culture) labeling strategy was used to quantify protein expression in PEG-GO-exposed breast cancer versus non-cancerous cells. The results indicated that PEG-GO selectively down-regulated PGC-1α in breast cancer cells and thus modified the expression of diverse energy generation-related proteins, which accounts for the inhibition of OXPHOS. The inhibition of OXPHOS by PEG-GO significantly reduced ATP production and impaired assembly of the F-actin cytoskeleton in breast cancer cells, which is required for the migratory and invasive phenotype of cancer cells. Taken together, these effects of PEG-GO on cancer cell metastasis may allow the development of a new approach to treat metastatic breast cancer.