Autophagy in hematopoietic stem cell development of the embryo.

Hematopoietic stem cells (HSC) emerge from hemogenic endothelial cells (HEC) in the aorta-gonad-mesonephros (AGM) region of embryos, which go through the pre-HSC process. Various intrinsic and extrinsic factors are involved in this process. We recently discovered that the existence of distinct macroautophagic/autophagic statuses in hematopoietic precursors is related to the hematopoietic potential of pre-HSCs and the depletion of the Atg5 (autophagy related 5) gene specifically in endothelial cells impaired in the transition of endothelial to pre-HSCs, by hampering the autophagic process, likely via the NCL (nucleolin) pathway.Abbreviation: Atg5: autophagy related 5; EGFP: enhanced green fluorescent protein; EHT: endothelial-to-hematopoietic transition; HEC: hemogenic endothelial cell; HSC: hematopoietic stem cell; NCL: nucleolin; RFP: red fluorescent protein.

A subset of megakaryocytes regulates development of hematopoietic stem cell precursors.

Understanding the regulatory mechanisms facilitating hematopoietic stem cell (HSC) specification during embryogenesis is important for the generation of HSCs in vitro. Megakaryocyte emerged from the yolk sac and produce platelets, which are involved in multiple biological processes, such as preventing hemorrhage. However, whether megakaryocytes regulate HSC development in the embryonic aorta-gonad-mesonephros (AGM) region is unclear. Here, we use platelet factor 4 (PF4)-Cre;Rosa-tdTomato+ cells to report presence of megakaryocytes in the HSC developmental niche. Further, we use the PF4-Cre;Rosa-DTA (DTA) depletion model to reveal that megakaryocytes control HSC specification in the mouse embryos. Megakaryocyte deficiency blocks the generation and maturation of pre-HSCs and alters HSC activity at the AGM. Furthermore, megakaryocytes promote endothelial-to-hematopoietic transition in a OP9-DL1 coculture system. Single-cell RNA-sequencing identifies megakaryocytes positive for the cell surface marker CD226 as the subpopulation with highest potential in promoting the hemogenic fate of endothelial cells by secreting TNFSF14. In line, TNFSF14 treatment rescues hematopoietic cell function in megakaryocyte-depleted cocultures. Taken together, megakaryocytes promote production and maturation of pre-HSCs, acting as a critical microenvironmental control factor during embryonic hematopoiesis.

Autophagy regulates the maturation of hematopoietic precursors in the embryo.

An understanding of the mechanisms regulating embryonic hematopoietic stem cell (HSC) development would facilitate their regeneration. The aorta-gonad-mesonephros region is the site for HSC production from hemogenic endothelial cells (HEC). While several distinct regulators are involved in this process, it is not yet known whether macroautophagy (autophagy) plays a role in hematopoiesis in the pre-liver stage. Here, we show that different states of autophagy exist in hematopoietic precursors and correlate with hematopoietic potential based on the LC3-RFP-EGFP mouse model. Deficiency of autophagy-related gene 5 (Atg5) specifically in endothelial cells disrupts endothelial to hematopoietic transition (EHT), by blocking the autophagic process. Using combined approaches, including single-cell RNA-sequencing (scRNA-seq), we have confirmed that Atg5 deletion interrupts developmental temporal order of EHT to further affect the pre-HSC I maturation, and that autophagy influences hemogenic potential of HEC and the formation of pre-HSC I likely via the nucleolin pathway. These findings demonstrate a role for autophagy in the formation/maturation of hematopoietic precursors.

Rapid identification and isolation of neuraminidase inhibitors from mockstrawberry (Duchesnea indica Andr.) based on ligand fishing combined with HR-ESI-Q-TOF-MS.

Neuraminidase inhibitors (NAIs) are the mainstay antiviral drugs against influenza infection. In this study, a ligand fishing protocol was developed to screen NAIs using neuraminidase immobilized magnetic beads (NA-MB). After verifying the feasibility of NA-MB with an artificial mixture including NA inhibitors and non-inhibitors, the developed ligand fishing protocol was applied to screen NAIs from the crude extracts of Duchesnea indica Andr. Twenty-four NA binding compounds were identified from the normal butanol (n-BuOH) extract of D. indica as potential NAIs by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS) assisted with Compound Structure Identification (CSI):FingerID, including 12 ellagitannins, 4 brevifolin derivatives, 3 ellagic acid derivatives, and 4 flavonoids. Among them, 9 compounds were isolated and tested for in vitro NA inhibitory activities against NA from Clostridium perfringens, and from oseltamivir sensitive and resistant influenza A virus strains. The results indicate that compound B23 has the NA inhibitory activities in both the oseltamivir sensitive and resistant viral NA, with half maximal inhibitory concentration (IC50) values of 197.9 and 125.4 µmol/L, respectively. Moreover, B23 can obviously reduce the replication of oseltamivir sensitive and resistant viruses in Madin-Darby canine kidney (MDCK) cells at the concentrations of 40 and 200 µmol/L.

Magnetic beads-based neuraminidase enzyme microreactor as a drug discovery tool for screening inhibitors from compound libraries and fishing ligands from natural products.

Neuraminidase (NA) is a glycoside hydrolase that has been proposed as a potential therapeutic target for influenza. Thus, the identification of compounds that modulate NA activity could be of great therapeutic importance. The aim of this study is to develop a drug discovery tool for the identification of novel modulators of NA from both compound libraries and natural plant extracts. NA was immobilized onto the surface of magnetic beads and the inherent catalytic activity of NA-functionalized magnetic beads was characterized. Based on the enzymatic activity (hydrolysis ratio), the inhibitory activities of 12 compounds from plant secondary metabolites were screened, and the desired anti-NA activities of flavonoids were certified. Ligand fishing with the immobilized enzyme was optimized using an artificial test mixture consisting of oseltamivir, lycorine and matrine prior to carrying out the proof-of-concept experiment with the crude extract of Flos Lonicerae. The combination of ligand fishing and HPLC-MS/MS identified luteolin-7-O-ß-D-glucoside, luteolin, 3,5-di-O-caffeoylquinic acid and 3,4-di-O-caffeoylquinic acid as neuraminidase inhibitory ligands in Flos Lonicerae. This is the first report on the use of neuraminidase functionalized magnetic beads for the identification of active ligands from a botanical matrix, and it sets the basis for the de novo identification of NA modulators from complex biological mixtures.

Isolation and identification of l/d-lactate-conjugated bufadienolides from toad eggs revealing lactate racemization in amphibians.

Three pairs of bufadienolide l/d-lactate epimers (1-6) were isolated from the eggs of the toad Bufo bufo gargarizans. The structures were elucidated by using spectroscopic methods, X-ray diffraction analysis and a modified Mosher's method. Compounds 1-6 represent the first occurrence of lactate-conjugated bufadienolides in nature, and illustrate the existence of an enzyme-controlled epimerization from l- to d-lactate in amphibians. The biosynthetic pathways, in which two key enzymes might be involved (i.e., lactate racemase and acyltransferase), were proposed. In addition, the biological assays revealed that compounds 1-4 are potent cytotoxic agents against human gastric cancer cells BGC-823 and human lung cancer cells A549 with IC50 values in a range of 8.0 to 80.0 nM.