Salvia miltiorrhiza augments endothelial cell function for ischemic hindlimb recovery.

Salvia miltiorrhiza (Salvia miltiorrhiza) root, as a traditional herb, is widely applied to pharmacotherapy for vascular system disease. In this study, we elucidate the therapy mechanism of Salvia miltiorrhiza by using a model of hindlimb ischemia. Blood perfusion measurement showed that intravenous administration of the Water Extract of Salvia miltiorrhiza (WES) could facilitate damaged hindlimb blood flow recovery and blood vessel regeneration. In vitro mRNA screen assay in cultured human umbilical vein endothelial cells (HUVECs) show that WES induced increased NOS3, VEGFA, and PLAU mRNA levels. Endothelial NOS (eNOS) promotor reporter analysis revealed that WES and the major ingredients danshensu (DSS) could enhance eNOS promoter activity. Additionally, we found that WES and its ingredients, including DSS, protocatechuic aldehyde (PAI), and salvianolic acid A (SaA), promoted HUVECs growth by the endothelial cell viability assays. A mechanistic approach confirmed that WES augments HUVECs proliferation through the activation of extracellular signal-regulated kinase (ERK) signal pathway. This study reveals that WES promotes ischemic remodeling and angiogenesis through its multiple principal ingredients, which target and regulate multiple sites of the network of the blood vessel endothelial cell regenerating process.

Polarized macrophages promote gestational beta cell growth through extracellular signal-regulated kinase 5 signalling.

AIM: To show that depletion of pancreatic macrophages impairs gestational beta cell proliferation and leads to glucose intolerance. MATERIALS AND METHODS: Genetic animal models were applied to study the effects of depletion of pancreatic macrophges on gestational beta-cell proliferaiton and glucose response. The crosstalk between macrophages and beta-cells was studied in vivo using beta-cell-specific extracellular-signal-regulated kinase 5 (ERK5) knockout and epidermal growth receptor (EGFR) knockout mice, and in vitro using a co-culture system. RESULTS: Beta cell-derived placental growth factor (PlGF) recruited naïve macrophages and polarized them towards an M2-like phenotype. These macrophages then secreted epidermal growth factor (EGF), which activated extracellular signal-regulated kinase 5 (ERK5) signalling in beta cells to promote gestational beta cell proliferation. On the other hand, activation of ERK5 signalling in beta cells likely, in turn, enhanced the production and secretion of PlGF by beta cells. CONCLUSIONS: Our study shows a regulatory loop between macrophages and beta cells through PlGF/EGF/ERK5 signalling cascades to regulate gestational beta cell growth.

Liquid Chromatographic Separation Using a 2 µm i.d. Open Tubular Column at Elevated Temperature.

We have experimentally demonstrated the extraordinarily high resolving power of liquid chromatography (LC) using a narrow open tubular (OT) column. In this work, we show that we can further increase its efficiency, peak capacity, and separation speed by elevating the operation (or column) temperature; all of these three numbers can be improved without mutual compromises. We use a mixture of five amino acids as a sample and show that we can increase the efficiency by 34%-260% and the separation speeds by 7%-10% by raising the operation temperature from 30 to 70 °C. When we use a 2 µm i.d. × 80 cm in length OT column coated with OTMS at a temperature of 70 °C, we can frequently obtain peak capacities of 700-800 within 20-30 min for separating cytochrome C digests. By increasing the column length to 160 cm, we can obtain a peak capacity of 2720 within 143 min for separating a complex peptide sample. This peak capacity is the highest peak capacity to date for one-dimensional LC separations. Importantly, heating the column is easy to implement and does not cost much, and many commercial LC systems already have compartments to control column temperatures. Running LC using a narrow OT column at an elevated temperature should broaden the applications of OT-LC in chemical and biochemical analyses.

A multi-herb-combined remedy to overcome hyper-inflammatory response by reprogramming transcription factor profile and shaping monocyte subsets.

Traditional Chinese multi-herb-combined prescriptions usually show better performance than a single agent since a group of effective compounds interfere multiple disease-relevant targets simultaneously. Huang-Lian-Jie-Du decoction is a remedy made of four herbs that are widely used to treat oral ulcers, gingivitis, and periodontitis. However, the active ingredients and underlying mechanisms are not clear. To address these questions, we prepared a water extract solution of Huang-Lian-Jie-Du decoction (HLJDD), called it as WEH (Water Extract Solution of HLJDD), and used it to treat LPS-induced systemic inflammation in mice. We observed that WEH attenuated inflammatory responses including reducing production of cytokines, chemokines and interferons (IFNs), further attenuating emergency myelopoiesis, and preventing mice septic lethality. Upon LPS stimulation, mice pretreated with WEH increased circulating Ly6C- patrolling and splenic Ly6C+ inflammatory monocytes. The acute myelopoiesis related transcriptional factor profile was rearranged by WEH. Mechanistically we confirmed that WEH interrupted LPS/TLR4/CD14 signaling-mediated downstream signaling pathways through its nine principal ingredients, which blocked LPS stimulated divergent signaling cascades, such as activation of NF-κB, p38 MAPK, and ERK1/2. We conclude that the old remedy blunts LPS-induced "danger" signal recognition and transduction process at multiple sites. To translate our findings into clinical applications, we refined the crude extract into a pure multicomponent drug by directly mixing these nine chemical entities, which completely reproduced the effect of protecting mice from lethal septic shock. Finally, we reduced a large number of compounds within a multi-herb water extract to seven-chemical combination that exhibited superior therapeutic efficacy compared with WEH.

Terahertz switching between broadband absorption and narrowband absorption.

A multilayer metamaterial with switchable functionalities is presented based on the phase-transition property of vanadium dioxide. When vanadium dioxide is in the metallic state, a broadband absorber is formed. Calculated results show that the combination of two absorption peaks enables absorptance more than 90% in the wide spectral range from 0.393 THz to 0.897 THz. Absorption performance is insensitive to polarization at the small incident angle and work well even at the larger incident angle. When vanadium dioxide is in the insulating state, the designed system behaves as a narrowband absorber at the frequency of 0.677 THz. This narrowband absorber shows the advantages of wide angle and polarization insensitivity due to the localized magnetic resonance. Furthermore, the influences of geometrical parameters on the performance of absorptance are discussed. The proposed switchable absorber can be used in various applications, such as selective heat emitter and solar photovoltaic field.

Experimentally Validating Open Tubular Liquid Chromatography for a Peak Capacity of 2000 in 3 h.

The advancements in life science research mandate effective tools capable of analyzing large numbers of samples with low quantities and high complexities. As an essential analytical tool for this research, liquid chromatography (LC) encounters an ever-increasing demand for enhanced resolving power, accelerated analysis speed, and reduced limit of detection. Although theoretical studies have indicated that open tubular (OT) columns can produce superior resolving power under comparable elution pressures and analysis times, ultrahigh-resolution and ultrahigh-speed open tubular liquid chromatography (OTLC) separations have never been reported. Here we present experimental results to demonstrate the predicted potential of this technique. We use a 2 µm i.d. × 75 cm long OT column coated with trimethoxy(octadecyl)silane for separating pepsin/trypsin digested E. coli lysates and routinely produce exceptionally high peak capacities (e.g., 1900-2000 in 3-5 h). We reduce the column length to 2.7 cm and exhibit the capability of OTLC for ultrafast separations. Under an elution pressure of 227.5 bar, we complete the separation of six amino acids in ∼800 ms and resolve these compounds within ∼400 ms. In addition, we show that OTLC has low attomole limits of detection (LOD) and each separation requires samples of only a few picoliters. Importantly, no ultrahigh elution pressures are required. With the ultrahigh resolution, ultrahigh speed, low LOD, and low sample volume requirement, OTLC can potentially be a powerful tool for biotech research, especially single cell analysis.

Integrated metamaterial with functionalities of absorption and electromagnetically induced transparency.

A switchable metamaterial with bifunctionality of absorption and electromagnetically induced transparency is proposed based on the phase-transition characteristic of phase change material-vanadium dioxide. When vanadium dioxide is in the metallic state, an isotropic narrow absorber is obtained in the terahertz region, which consists of a top metallic cross, a middle dielectric layer, and a bottom vanadium dioxide film. By adjusting structure parameters, perfect absorption is realized at the frequency of 0.498 THz. This designed narrow absorber is insensitive to polarization and incident angle. Absorptance can still reach 75% for transverse electric polarization and transverse magnetic polarization at the incident angle of 65∘. When vanadium dioxide is in the insulating state, the top metallic cross will interact with the bottom split ring resonator, and the interaction between them will lead to the appearance of electromagnetically induced transparency. The behavior of electromagnetically induced transparency works well for transverse electric polarization and transverse magnetic polarization at the small incident angle. The designed hybrid metamaterial opens possible avenues for achieving switchable functionalities in a single device.

A preliminary origin-tracking study of different densities urinary exosomes.

Based on density differences of different subpopulations of exosomes, two kinds of micro-vesicles with different densities were captured from urine by a modified sucrose density gradient ultracentrifuge separation method. Verified by transmission electron microscope (TEM) and western blot, the results showed these two kinds of micro-vesicles were all exosomes. And these two kinds of exosomes were analyzed by TEM, 2D electrophoresis (2DE), and capillary zone electrophoresis (CZE), respectively. The results of TEM showed these two exosomes with different densities have different morphological characteristics, and some tiny proteomic differences were shown in the results of 2DE of these two exosomes. At the same time, the CZE results displayed these two kinds of exosomes possessed different retention times, indicated that they may have different electrification property and particle weight. These results may attribute to their different origins. This work may provide a preliminary experience for the origin-tracking study for urinary exosomes, and would be more useful for future targeted biomarker discovery.

Tunable Electroosmosis-Based Femto-Liter Pipette: A Promising Tool toward Living-Cell Surgery.

Single-cell analysis has attracted increasing attention because of cell heterogeneities. Various strategies have been developed for analyzing single cells, but most of these analytical processes kill the cells. Tools that can qualitatively and quantitatively measure the cellular contents without killing the cell are highly demanding because they enable us to conduct single-cell time-course studies (e.g., to examine how a cell responds to a therapy before, during, and after a treatment). Here we develop a femto-liter (fL) pipet to serve this purpose. To ensure that we can accurately and precisely pipet fL solutions, we fill all conduits with liquid and use an electroosmotic pump (EOP) as the driving force to facilitate withdrawal of cellular contents from single cells. We tentatively term this device an EOP-driven pipette or EDP. We characterize the EDP for accurately and precisely withdrawing solution from ∼250 fL to 80 nL; a volume range that covers the applications for most types of cells. To demonstrate the feasibility of utilizing the EDP for a single-cell time-course study, we utilize the EDP to take the cellular contents out at different times during the course of a zebrafish embryo development for cholesterol measurements. More than 50% of the embryos survive after each pipetting and analysis step, and this number will increase considerably as we improve our cell manipulation skills and reduce the pipet-tip diameter. We expect this EDP to become an effective tool for single-cell time-course studies.

High-performance liquid chromatographic cartridge with gradient elution capability coupled with UV absorbance detector and mass spectrometer for peptide and protein analysis.

We discuss the construction and performance of a high-performance liquid chromatography cartridge that we developed that resulted from a culmination of previous research. We have recently developed an innovative approach to creating gradient elutions using dual electroosmotic pumps and a series of three valves. This method has been proved to be the most reproducible and robust in producing gradients compared to our previously tested methods. Using this approach, we have assembled a high-performance liquid chromatography cartridge powered and controlled via a computer. We have successfully coupled the cartridge with an ultraviolet absorbance detector and a mass spectrometer for separating complex protein/peptide samples. The cartridge is readily coupled with other detectors such as electrochemical detector and laser-induced fluorescence detector.

Simultaneously sizing and quantitating zeptomole-level DNA at high throughput in free solution.

Determining the sizes and measuring the quantities of DNA molecules are fundamental tasks in molecular biology. DNA sizes are usually evaluated by gel electrophoresis, but this method cannot simultaneously size and quantitate a DNA at low zeptomole (zmol) levels of concentration. We have recently developed a new technique, called bare-narrow-capillary/hydrodynamic-chromatography or BaNC-HDC, for resolving DNA based on their sizes without using any sieving matrices. In this report, we utilize BaNC-HDC for measuring the sizes and quantities of DNA fragments at zmol to several-molecule levels of concentration. DNA ranging from a few base pairs to dozens of kilo base pairs are accurately sized and quantitated at a throughput of 15 samples per hour, and each sample contains dozens of DNA strands of different lengths. BaNC-HDC can be a cost-effective means and an excellent tool for high-throughput DNA sizing and quantitation at extremely low quantity level.

Protocol to assess the antitumor efficacy of an immunotherapeutic peptide in syngeneic orthotopic glioma mouse models.

Understanding the glioblastoma (GBM) immune microenvironment and development of clinical treatment drugs rely on suitable preclinical GBM models. Here, we present a protocol to establish syngeneic orthotopic glioma mouse models. We also describe the steps to intracranially deliver immunotherapeutic peptides and monitor the treatment response. Finally, we show how to assess the tumor immune microenvironment with treatment outcomes. For complete details on the use and execution of this protocol, please refer to Chen et al. (2021).1.

Development of a human glioblastoma model using humanized DRAG mice for immunotherapy.

Glioblastoma (GBM) is the most common and lethal primary brain tumor with high mortality rates and a short median survival rate of about 15 months despite intensive multimodal treatment of maximal surgical resection, radiotherapy, and chemotherapy. Although immunotherapies have been successful in the treatment of various cancers, disappointing results from clinical trials for GBM immunotherapy represent our incomplete understanding. The development of alternative humanized mouse models with fully functional human immune cells will potentially accelerate the progress of GBM immunotherapy. In this study, we developed a humanized DRAG (NOD.Rag1KO.IL2RγcKO) mouse model, in which the human hematopoietic stem cells (HSCs) were well-engrafted and subsequently differentiated into a full lineage of immune cells. Using this humanized DRAG mouse model, GBM patient-derived tumorsphere lines were successfully engrafted to form xenografted tumors, which can recapitulate the pathological features and the immune cell composition of human GBM. Importantly, the administration of anti-human PD-1 antibodies in these DRAG mice bearing a GBM patient-derived tumorsphere line resulted in decreasing the major tumor-infiltrating immunosuppressive cell populations, including CD4 + PD-1 + and CD8 + PD-1 + T cells, CD11b + CD14 + HLA-DR + macrophages, CD11b + CD14 + HLA-DR - CD15 - and CD11b + CD14 - CD15 + myeloid-derived suppressor cells, indicating the humanized DRAG mouse model as a useful model to test the efficacy of immune checkpoint inhibitors in GBM immunotherapy. Together, these results suggest that humanized DRAG mouse models are a reliable preclinical platform for brain cancer immunotherapy and beyond.

Development of a human glioblastoma model using humanized DRAG mice for immunotherapy.

Glioblastoma (GBM) is the most common and lethal primary brain tumor. The development of alternative humanized mouse models with fully functional human immune cells will potentially accelerate the progress of GBM immunotherapy. We successfully generated humanized DRAG (NOD.Rag1KO.IL2RγcKO) mouse model by transplantation of human DR4+ hematopoietic stem cells (hHSCs), and effectively grafted GBM patient-derived tumorsphere cells to form xenografted tumors intracranially. The engrafted tumors recapitulated the pathological features and the immune cell composition of human GBM. Administration of anti-human PD-1 antibodies in these tumor-bearing humanized DRAG mice decreased the major tumor-infiltrating immunosuppressive cell populations, including CD4+PD-1+ and CD8+PD-1+ T cells, CD11b+CD14+HLA-DR+ macrophages, CD11b+CD14+HLA-DR-CD15- and CD11b+CD14-CD15+ myeloid-derived suppressor cells, indicating the humanized DRAG mice as a useful model to test the efficacy of GBM immunotherapy. Taken together, these results suggest that the humanized DRAG mouse model is a reliable preclinical platform for studying brain cancer immunotherapy and beyond.

TP53-PTEN-NF1 depletion in human brain organoids produces a glioma phenotype in vitro.

Glioblastoma (GBM) is fatal and the study of therapeutic resistance, disease progression, and drug discovery in GBM or glioma stem cells is often hindered by limited resources. This limitation slows down progress in both drug discovery and patient survival. Here we present a genetically engineered human cerebral organoid model with a cancer-like phenotype that could provide a basis for GBM-like models. Specifically, we engineered a doxycycline-inducible vector encoding shRNAs enabling depletion of the TP53, PTEN, and NF1 tumor suppressors in human cerebral organoids. Designated as inducible short hairpin-TP53-PTEN-NF1 (ish-TPN), doxycycline treatment resulted in human cancer-like cerebral organoids that effaced the entire organoid cytoarchitecture, while uninduced ish-TPN cerebral organoids recapitulated the normal cytoarchitecture of the brain. Transcriptomic analysis revealed a proneural GBM subtype. This proof-of-concept study offers a valuable resource for directly investigating the emergence and progression of gliomas within the context of specific genetic alterations in normal cerebral organoids.

A neurodevelopmental epigenetic programme mediated by SMARCD3-DAB1-Reelin signalling is hijacked to promote medulloblastoma metastasis.

How abnormal neurodevelopment relates to the tumour aggressiveness of medulloblastoma (MB), the most common type of embryonal tumour, remains elusive. Here we uncover a neurodevelopmental epigenomic programme that is hijacked to induce MB metastatic dissemination. Unsupervised analyses of integrated publicly available datasets with our newly generated data reveal that SMARCD3 (also known as BAF60C) regulates Disabled 1 (DAB1)-mediated Reelin signalling in Purkinje cell migration and MB metastasis by orchestrating cis-regulatory elements at the DAB1 locus. We further identify that a core set of transcription factors, enhancer of zeste homologue 2 (EZH2) and nuclear factor I X (NFIX), coordinates with the cis-regulatory elements at the SMARCD3 locus to form a chromatin hub to control SMARCD3 expression in the developing cerebellum and in metastatic MB. Increased SMARCD3 expression activates Reelin-DAB1-mediated Src kinase signalling, which results in a MB response to Src inhibition. These data deepen our understanding of how neurodevelopmental programming influences disease progression and provide a potential therapeutic option for patients with MB.

Open tubular liquid chromatographic system for using columns with inner diameter of 2 µm. A tutorial.

We have recently demonstrated the remarkable performances of liquid chromatography (LC) using 2-µm-i.d. open tubular (OT) columns; peak capacities of 2000+ within less than three hours have been routinely obtained at an elution pressure of around 100 bar or less. However, only a small number of researchers have been involved in the research in this area; part of the reason is due to the issues associated with setting up open tubular liquid chromatography (OTLC) systems. While cautions should be taken here and there in carrying out separations, but none of the issues can inhibit us from performing OTLC separations. Therefore, we feel it desirable to write a tutorial on how to build an OTLC system. In this tutorial, we introduce the key components for the apparatus, how to construct/prepare them or where to purchase them, and how to assemble them together into a complete system. We further discuss the advantages and disadvantages of the system; we mention particularly the practical issues from using the narrow (2-µm-i.d.) columns and how to mitigate these issues.

Natural Coevolution of Tumor and Immunoenvironment in Glioblastoma.

Isocitrate dehydrogenase (IDH) wild-type glioblastoma (GBM) has a dismal prognosis. A better understanding of tumor evolution holds the key to developing more effective treatment. Here we study GBM's natural evolutionary trajectory by using rare multifocal samples. We sequenced 61,062 single cells from eight multifocal IDH wild-type primary GBMs and defined a natural evolution signature (NES) of the tumor. We show that the NES significantly associates with the activation of transcription factors that regulate brain development, including MYBL2 and FOSL2. Hypoxia is involved in inducing NES transition potentially via activation of the HIF1A-FOSL2 axis. High-NES tumor cells could recruit and polarize bone marrow-derived macrophages through activation of the FOSL2-ANXA1-FPR1/3 axis. These polarized macrophages can efficiently suppress T-cell activity and accelerate NES transition in tumor cells. Moreover, the polarized macrophages could upregulate CCL2 to induce tumor cell migration. SIGNIFICANCE: GBM progression could be induced by hypoxia via the HIF1A-FOSL2 axis. Tumor-derived ANXA1 is associated with recruitment and polarization of bone marrow-derived macrophages to suppress the immunoenvironment. The polarized macrophages promote tumor cell NES transition and migration. This article is highlighted in the In This Issue feature, p. 2711.

Loss of MAT2A compromises methionine metabolism and represents a vulnerability in H3K27M mutant glioma by modulating the epigenome.

Diffuse midline gliomas (DMGs) bearing driver mutations of histone 3 lysine 27 (H3K27M) are incurable brain tumors with unique epigenomes. Here, we generated a syngeneic H3K27M mouse model to study the amino acid metabolic dependencies of these tumors. H3K27M mutant cells were highly dependent on methionine. Interrogating the methionine cycle dependency through a short-interfering RNA screen identified the enzyme methionine adenosyltransferase 2A (MAT2A) as a critical vulnerability in these tumors. This vulnerability was not mediated through the canonical mechanism of MTAP deletion; instead, DMG cells have lower levels of MAT2A protein, which is mediated by negative feedback induced by the metabolite decarboxylated S-adenosyl methionine. Depletion of residual MAT2A induces global depletion of H3K36me3, a chromatin mark of transcriptional elongation perturbing oncogenic and developmental transcriptional programs. Moreover, methionine-restricted diets extended survival in multiple models of DMG in vivo. Collectively, our results suggest that MAT2A presents an exploitable therapeutic vulnerability in H3K27M gliomas.

A resource of high-quality and versatile nanobodies for drug delivery.

Therapeutic and diagnostic efficacies of small biomolecules and chemical compounds are hampered by suboptimal pharmacokinetics. Here, we developed a repertoire of robust and high-affinity antihuman serum albumin nanobodies (NbHSA) that can be readily fused to small biologics for half-life extension. We characterized the thermostability, binding kinetics, and cross-species reactivity of NbHSAs, mapped their epitopes, and structurally resolved a tetrameric HSA-Nb complex. We parallelly determined the half-lives of a cohort of selected NbHSAs in an HSA mouse model by quantitative proteomics. Compared to short-lived control nanobodies, the half-lives of NbHSAs were drastically prolonged by 771-fold. NbHSAs have distinct and diverse pharmacokinetics, positively correlating with their albumin binding affinities at the endosomal pH. We then generated stable and highly bioactive NbHSA-cytokine fusion constructs "Duraleukin" and demonstrated Duraleukin's high preclinical efficacy for cancer treatment in a melanoma model. This high-quality and versatile Nb toolkit will help tailor drug half-life to specific medical needs.