Molecular structure discovery for untargeted metabolomics using biotransformation rules and global molecular networking.

Although untargeted mass spectrometry-based metabolomics is crucial for understanding life's molecular underpinnings, its effectiveness is hampered by low annotation rates of the generated tandem mass spectra. To address this issue, we introduce a novel data-driven approach, Biotransformation-based Annotation Method (BAM), that leverages molecular structural similarities inherent in biochemical reactions. BAM operates by applying biotransformation rules to known 'anchor' molecules, which exhibit high spectral similarity to unknown spectra, thereby hypothesizing and ranking potential structures for the corresponding 'suspect' molecule. BAM's effectiveness is demonstrated by its success in annotating suspect spectra in a global molecular network comprising hundreds of millions of spectra. BAM was able to assign correct molecular structures to 24.2 % of examined anchor-suspect cases, thereby demonstrating remarkable advancement in metabolite annotation.

Multiphoton Phosphorescence Quenching Microscopy Reveals Kinetics of Tumor Oxygenation during Antiangiogenesis and Angiotensin Signaling Inhibition.

PURPOSE: The abnormal function of tumor blood vessels causes tissue hypoxia, promoting disease progression and treatment resistance. Although tumor microenvironment normalization strategies can alleviate hypoxia globally, how local oxygen levels change is not known because of the inability to longitudinally assess vascular and interstitial oxygen in tumors with sufficient resolution. Understanding the spatial and temporal heterogeneity should help improve the outcome of various normalization strategies. EXPERIMENTAL DESIGN: We developed a multiphoton phosphorescence quenching microscopy system using a low-molecular-weight palladium porphyrin probe to measure perfused vessels, oxygen tension, and their spatial correlations in vivo in mouse skin, bone marrow, and four different tumor models. Further, we measured the temporal and spatial changes in oxygen and vessel perfusion in tumors in response to an anti-VEGFR2 antibody (DC101) and an angiotensin-receptor blocker (losartan). RESULTS: We found that vessel function was highly dependent on tumor type. Although some tumors had vessels with greater oxygen-carrying ability than those of normal skin, most tumors had inefficient vessels. Further, intervessel heterogeneity in tumors is associated with heterogeneous response to DC101 and losartan. Using both vascular and stromal normalizing agents, we show that spatial heterogeneity in oxygen levels persists, even with reductions in mean extravascular hypoxia. CONCLUSIONS: High-resolution spatial and temporal responses of tumor vessels to two agents known to improve vascular perfusion globally reveal spatially heterogeneous changes in vessel structure and function. These dynamic vascular changes should be considered in optimizing the dose and schedule of vascular and stromal normalizing strategies to improve the therapeutic outcome.

Modeling the onset of symptoms of COVID-19: Effects of SARS-CoV-2 variant.

Identifying order of symptom onset of infectious diseases might aid in differentiating symptomatic infections earlier in a population thereby enabling non-pharmaceutical interventions and reducing disease spread. Previously, we developed a mathematical model predicting the order of symptoms based on data from the initial outbreak of SARS-CoV-2 in China using symptom occurrence at diagnosis and found that the order of COVID-19 symptoms differed from that of other infectious diseases including influenza. Whether this order of COVID-19 symptoms holds in the USA under changing conditions is unclear. Here, we use modeling to predict the order of symptoms using data from both the initial outbreaks in China and in the USA. Whereas patients in China were more likely to have fever before cough and then nausea/vomiting before diarrhea, patients in the USA were more likely to have cough before fever and then diarrhea before nausea/vomiting. Given that the D614G SARS-CoV-2 variant that rapidly spread from Europe to predominate in the USA during the first wave of the outbreak was not present in the initial China outbreak, we hypothesized that this mutation might affect symptom order. Supporting this notion, we found that as SARS-CoV-2 in Japan shifted from the original Wuhan reference strain to the D614G variant, symptom order shifted to the USA pattern. Google Trends analyses supported these findings, while weather, age, and comorbidities did not affect our model's predictions of symptom order. These findings indicate that symptom order can change with mutation in viral disease and raise the possibility that D614G variant is more transmissible because infected people are more likely to cough in public before being incapacitated with fever.

Modeling the Onset of Symptoms of COVID-19.

COVID-19 is a pandemic viral disease with catastrophic global impact. This disease is more contagious than influenza such that cluster outbreaks occur frequently. If patients with symptoms quickly underwent testing and contact tracing, these outbreaks could be contained. Unfortunately, COVID-19 patients have symptoms similar to other common illnesses. Here, we hypothesize the order of symptom occurrence could help patients and medical professionals more quickly distinguish COVID-19 from other respiratory diseases, yet such essential information is largely unavailable. To this end, we apply a Markov Process to a graded partially ordered set based on clinical observations of COVID-19 cases to ascertain the most likely order of discernible symptoms (i.e., fever, cough, nausea/vomiting, and diarrhea) in COVID-19 patients. We then compared the progression of these symptoms in COVID-19 to other respiratory diseases, such as influenza, SARS, and MERS, to observe if the diseases present differently. Our model predicts that influenza initiates with cough, whereas COVID-19 like other coronavirus-related diseases initiates with fever. However, COVID-19 differs from SARS and MERS in the order of gastrointestinal symptoms. Our results support the notion that fever should be used to screen for entry into facilities as regions begin to reopen after the outbreak of Spring 2020. Additionally, our findings suggest that good clinical practice should involve recording the order of symptom occurrence in COVID-19 and other diseases. If such a systemic clinical practice had been standard since ancient diseases, perhaps the transition from local outbreak to pandemic could have been avoided.

TGF-ß inhibition combined with cytotoxic nanomedicine normalizes triple negative breast cancer microenvironment towards anti-tumor immunity.

Tumor normalization strategies aim to improve tumor blood vessel functionality (i.e., perfusion) by reducing the hyper-permeability of tumor vessels or restoring compressed vessels. Despite progress in strategies to normalize the tumor microenvironment (TME), their combinatorial antitumor effects with nanomedicine and immunotherapy remain unexplored. Methods: Here, we re-purposed the TGF-ß inhibitor tranilast, an approved anti-fibrotic and antihistamine drug, and combined it with Doxil nanomedicine to normalize the TME, increase perfusion and oxygenation, and enhance anti-tumor immunity. Specifically, we employed two triple-negative breast cancer (TNBC) mouse models to primarily evaluate the therapeutic and normalization effects of tranilast combined with doxorubicin and Doxil. We demonstrated the optimized normalization effects of tranilast combined with Doxil and extended our analysis to investigate the effect of TME normalization to the efficacy of immune checkpoint inhibitors. Results: Combination of tranilast with Doxil caused a pronounced reduction in extracellular matrix components and an increase in the intratumoral vessel diameter and pericyte coverage, indicators of TME normalization. These modifications resulted in a significant increase in tumor perfusion and oxygenation and enhanced treatment efficacy as indicated by the notable reduction in tumor size. Tranilast further normalized the immune TME by restoring the infiltration of T cells and increasing the fraction of T cells that migrate away from immunosuppressive cancer-associated fibroblasts. Furthermore, we found that combining tranilast with Doxil nanomedicine, significantly improved immunostimulatory M1 macrophage content in the tumorigenic tissue and improved the efficacy of the immune checkpoint blocking antibodies anti-PD-1/anti-CTLA-4. Conclusion: Combinatorial treatment of tranilast with Doxil optimizes TME normalization, improves immunostimulation and enhances the efficacy of immunotherapy.

Dexamethasone Increases Cisplatin-Loaded Nanocarrier Delivery and Efficacy in Metastatic Breast Cancer by Normalizing the Tumor Microenvironment.

Dexamethasone is a glucocorticoid steroid with anti-inflammatory properties used to treat many diseases, including cancer, in which it helps manage various side effects of chemo-, radio-, and immunotherapies. Here, we investigate the tumor microenvironment (TME)-normalizing effects of dexamethasone in metastatic murine breast cancer (BC). Dexamethasone normalizes vessels and the extracellular matrix, thereby reducing interstitial fluid pressure, tissue stiffness, and solid stress. In turn, the penetration of 13 and 32 nm dextrans, which represent nanocarriers (NCs), is increased. A mechanistic model of fluid and macromolecule transport in tumors predicts that dexamethasone increases NC penetration by increasing interstitial hydraulic conductivity without significantly reducing the effective pore diameter of the vessel wall. Also, dexamethasone increases the tumor accumulation and efficacy of ∼30 nm polymeric micelles containing cisplatin (CDDP/m) against murine models of primary BC and spontaneous BC lung metastasis, which also feature a TME with abnormal mechanical properties. These results suggest that pretreatment with dexamethasone before NC administration could increase efficacy against primary tumors and metastases.

Quantum dot/antibody conjugates for in vivo cytometric imaging in mice.

Multiplexed, phenotypic, intravital cytometric imaging requires novel fluorophore conjugates that have an appropriate size for long circulation and diffusion and show virtually no nonspecific binding to cells/serum while binding to cells of interest with high specificity. In addition, these conjugates must be stable and maintain a high quantum yield in the in vivo environments. Here, we show that this can be achieved using compact (∼15 nm in hydrodynamic diameter) and biocompatible quantum dot (QD) -Ab conjugates. We developed these conjugates by coupling whole mAbs to QDs coated with norbornene-displaying polyimidazole ligands using tetrazine-norbornene cycloaddition. Our QD immunoconstructs were used for in vivo single-cell labeling in bone marrow. The intravital imaging studies using a chronic calvarial bone window showed that our QD-Ab conjugates diffuse into the entire bone marrow and efficiently label single cells belonging to rare populations of hematopoietic stem and progenitor cells (Sca1(+)c-Kit(+) cells). This in vivo cytometric technique may be useful in a wide range of structural and functional imaging to study the interactions between cells and between a cell and its environment in intact and diseased tissues.

Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy.

The recent approval of a prostate cancer vaccine has renewed hope for anticancer immunotherapies. However, the immunosuppressive tumor microenvironment may limit the effectiveness of current immunotherapies. Antiangiogenic agents have the potential to modulate the tumor microenvironment and improve immunotherapy, but they often are used at high doses in the clinic to prune tumor vessels and paradoxically may compromise various therapies. Here, we demonstrate that targeting tumor vasculature with lower vascular-normalizing doses, but not high antivascular/antiangiogenic doses, of an anti-VEGF receptor 2 (VEGFR2) antibody results in a more homogeneous distribution of functional tumor vessels. Furthermore, lower doses are superior to the high doses in polarizing tumor-associated macrophages from an immune inhibitory M2-like phenotype toward an immune stimulatory M1-like phenotype and in facilitating CD4(+) and CD8(+) T-cell tumor infiltration. Based on this mechanism, scheduling lower-dose anti-VEGFR2 therapy with T-cell activation induced by a whole cancer cell vaccine therapy enhanced anticancer efficacy in a CD8(+) T-cell-dependent manner in both immune-tolerant and immunogenic murine breast cancer models. These findings indicate that vascular-normalizing lower doses of anti-VEGFR2 antibody can reprogram the tumor microenvironment away from immunosuppression toward potentiation of cancer vaccine therapies. Given that the combinations of high doses of bevacizumab with chemotherapy have not improved overall survival of breast cancer patients, our study suggests a strategy to use antiangiogenic agents in breast cancer more effectively with active immunotherapy and potentially other anticancer therapies.