Continuous Flow Electrochemistry Enables Practical and Site-Selective C-H Oxidation.

The selective oxygenation of ubiquitous C(sp3 )-H bonds remains a highly sought-after method in both academia and the chemical industry for constructing functionalized organic molecules. However, it is extremely challenging to selectively oxidize a certain C(sp3 )-H bond to afford alcohols due to the presence of multiple C(sp3 )-H bonds with similar strength and steric environment in organic molecules, and the alcohol products being prone to further oxidation. Herein, we present a practical and cost-efficient electrochemical method for the highly selective monooxygenation of benzylic C(sp3 )-H bonds using continuous flow reactors. The electrochemical reactions produce trifluoroacetate esters that are resistant to further oxidation but undergo facile hydrolysis during aqueous workup to form benzylic alcohols. The method exhibits a broad scope and exceptional site selectivity and requires no catalysts or chemical oxidants. Furthermore, the electrochemical method demonstrates excellent scalability by producing 115 g of one of the alcohol products. The high site selectivity of the electrochemical method originates from its unique mechanism to cleave benzylic C(sp3 )-H bonds through sequential electron/proton transfer, rather than the commonly employed hydrogen atom transfer (HAT).

Electrochemical aromatic C-H hydroxylation in continuous flow.

The direct hydroxylation of arene C-H bonds is a highly sought-after transformation but remains an unsolved challenge due to the difficulty in efficient and regioselective C-H oxygenation and high reactivity of the phenolic products leading to overoxidation. Herein we report electrochemical C-H hydroxylation of arenes in continuous flow for the synthesis of phenols. The method is characterized by broad scope (compatible with arenes of diverse electronic properties), mild conditions without any catalysts or chemical oxidants, and excellent scalability as demonstrated by the continuous production of 1 mol (204 grams) of one of the phenol products.

Management of combined massive burn and blast injury: A 20-year experience.

INTRODUCTION: Blast injuries are complex types of physical trauma resulting from direct or indirect exposure to an explosion, which can be divided into four classes: primary, secondary, tertiary, and quaternary. Primary blast injury results in damage, principally, in gas-containing organs such as the lungs (blast lung injury, BLI). BLI is defined as radiological and clinical evidence of acute lung injury occurring within 12h of exposure to an explosion and not due to secondary or tertiary injury. BLI often combines with cutaneous thermal injury, a type of quaternary blast injury, either in terrorist bomb attacks or in civilian accidental explosions. This report summarizes our experience in the management of combined massive burn and BLI at a Shanghai Burn Center in China. METHODS: A retrospective observational analysis of clinical data was performed for massive burn patients with or without BLI during a 20-year interval. Patient characteristics, causes of injury, clinical parameters, management, and outcomes were recorded and evaluated. RESULTS: A total of 151 patients (120 males and 31 females) with severe burn injury (≥50% TBSA) treated at the Burn Center of Changhai Hospital in Shanghai between July 1997 and June 2017 were enrolled in this study. Their mean age was 38.6±17.8 (3-75) years. Among them, 28 patients had combined BLI and burn injury and 39 patients had no BLI or smoke inhalation injury (non-BLI-SII). No significant difference was observed in the burn area or full-thickness burn area between the two groups. The lowest PaO2/fraction of inspired oxygen (FiO2) ratio during the first 24h in BLI patients was significantly lower than that in non-BLI-SII patients. Exudative changes were observed by X-ray radiography in all BLI patients but not in non-BLI-SII patients within 6h after injury. A significantly higher proportion of colloids were used for fluid resuscitation in BLI patients than that in non-BLI-SII patients. A higher proportion and longer time of mechanical ventilation were needed for BLI patients than those for non-BLI-SII patients, and a higher proportion of patients received sedative agents in the BLI group than those in the non-BLI-SII group. The first escharectomy was performed relatively later in BLI patients than in non-BLI-SII patients because of more time taken by BLI patients to recover from lung injury. The length of ICU and hospital stay in BLI patients was significantly longer than that in non-BLI-SII patients. No significant difference in the overall mortality was detected between these two groups. CONCLUSION: It is a formidable challenge for clinicians to diagnose and manage massive burn patients combined with BLI. A comprehensive treatment approach is strongly recommended, including fluid resuscitation, airway management, mechanical ventilation, and surgical treatment. Given the high mortality of massive burn patients combined with BLI even in a recognized burn center, more prospective studies are encouraged to assess more effective strategies for the treatment of such patients.

Zebrafish miR-462-731 regulates hematopoietic specification and pu.1-dependent primitive myelopoiesis.

MicroRNAs (miRNAs) play significant roles in both embryonic hematopoiesis and hematological malignancy. Zebrafish miR-462-731 cluster is orthologous of miR-191-425 in human which regulates proliferation and tumorigenesis. In our previous work, miR-462-731 was found highly and ubiquitously expressed during early embryogenesis. In this study, by loss-of-function analysis (morpholino knockdown combined with CRISRP/Cas9 knockout) and mRNA profiling, we suggest that miR-462-731 is required for normal embryonic development by regulating cell survival. We found that loss of miR-462/miR-731 caused a remarkable decrease in the number of erythroid cells as well as an ectopic myeloid cell expansion at 48 hpf, suggesting a skewing of myeloid-erythroid lineage differentiation. Mechanistically, miR-462-731 provides an instructive input for pu.1-dependent primitive myelopoiesis through regulating etsrp/scl signaling combined with a novel pu.1/miR-462-731 feedback loop. On the other hand, morpholino (MO) knockdown of miR-462/miR-731 resulted in an expansion of posterior blood islands at 24 hpf, which is a mild ventralization phenotype resulted from elevation of BMP signaling. Rescue experiments with both BMP type I receptor inhibitor dorsomorphin and alk8 MO indicate that miR-462-731 acts upstream of alk8 within the BMP/Smad signaling pathway and functions as a novel endogenous BMP antagonist. Besides, an impairment of angiogenesis was observed in miR-462/miR-731 morphants. The specification of arteries and veins was also perturbed, as characterized by the irregular patterning of efnb2a and flt4 expression. Our study unveils a previously unrecognized role of miR-462-731 in BMP/Smad signaling mediated hematopoietic specification of mesodermal progenitors and demonstrates a miR-462-731 mediated regulatory mechanism driving primitive myelopoiesis in the ALPM. We also show a requirement for miR-462-731 in regulating arterial-venous specification and definitive hematopoietic stem cell (HSC) production. The current findings might provide further insights into the molecular mechanistic basis of miRNA regulation of embryonic hematopoiesis and hematological malignancy.

The molecular characterization, expression pattern and alternative initiation of Megalobrama amblycephala Hif prolyl hydroxylase Phd1.

HIF prolyl hydroxylase 1 (PHD1) functions in prolyl hydroxylation on mammal hypoxia-inducible factors (HIF), important transcription factors involved in hypoxia, however the roles of Phd1 in fish remain unclear. In this study, the full-length cDNA and promoter sequences of blunt snout bream (Megalobrama amblycephala) phd1 gene were isolated by a modified RACE strategy. The phd1 cDNA was 2672 bp for encoding 481 amino acid residues. In silico assays indicated that phd1 had 5 exons, and a 348 bp CpG island in the exon1, and several transcription factor binding sites (CAAT box, HRE, ARNT, FOX, etc) were also found on the promoter. The quantitative real-time PCR results suggested that phd1 mRNA was constitutively expressed in all detected tissues, and higher in the blood, brain and heart in normoxia, but significantly decreased after hypoxia in all detected tissues except for gill. Western blot assays indicated that two Phd1 isoforms were generated by alternative translation initiation. Moreover, these two isoforms were both localized in the nucleus, therein only the senior isoform promoted cell proliferation. Taken together, the present study firstly describes the functions of M. amblycephala two Phd1 isoforms in hypoxia and cell proliferation.

A stress response pathway in mice upregulates somatostatin level and transcription in pancreatic delta cells through Gs and ß-arrestin 1.

AIMS/HYPOTHESIS: Somatostatin secretion from islet delta cells plays an important role in regulating islet function and is tightly controlled by environmental changes. Activation of the adrenergic system promoted somatostatin secretion from islet delta cells; however, the role of the adrenergic system in regulating somatostatin content and transcription has not been defined. An imbalance between the somatostatin content and its secretion may cause dysfunctions in the islet delta cells. We have investigated the role of the adrenergic system in the modulation of somatostatin content and transcription in pancreatic delta cells and the detailed underlying mechanisms of this regulation. METHODS: The stress hormone adrenaline (epinephrine), specific adrenergic agonists or specific adrenergic antagonists were applied to islets from either wild-type or specific adrenergic receptor knockout mice and pancreatic delta cell lines to investigate their effects on somatostatin content and transcription. The GloSensor assay, quantitative real-time PCR, western blots and the dual luciferase assay were used to monitor the cAMP level, somatostatin expression, activations of kinases and transcriptional factors. Arrb1 knockout mice, specific Creb or Pax6 mutations and specific kinase inhibitors were used to dissect the signalling pathway. RESULTS: Adrenaline and isoprenaline increased somatostatin content and transcription through the activation of ß1-/ß2-adrenergic receptors (ß1-/ß2ARs). The somatostatin content in ß1AR(-/-) /ß2AR(-/-) (Adrb1/Adrb2 knockout) mice was 50% lower than in ß1AR(+/+)/ß2AR (+/+) mice. Two parallel signalling pathways, Gs-cAMP-protein kinase A (PKA)-cAMP response element binding protein (CREB) and ß-arrestin 1-extracellular signal-related kinase (ERK)-paired box protein 6 (PAX6), cooperatively regulated isoprenaline-induced somatostatin transcription. CONCLUSIONS/INTERPRETATION: A stress pathway increased somatostatin content and transcription through ß-adrenergic agonism. ß-Arrestin1, ERK and PAX6 are important pancreatic delta cell regulators in addition to cAMP, PKA and CREB. Dysfunction of ß-adrenergic agonism may impair pancreatic delta cell function.

Measurement of precursor miRNA in exosomes from human ESC-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) derived from human embryonic stem cells (ESCs) have been shown to secrete exosomes that are cardioprotective against myocardial ischemia reperfusion injury in a mouse model. To elucidate this cardioprotective mechanism, we have characterized the protein, nucleic acid, and lipid composition of MSC exosomes. Here we describe the isolation and analysis of RNA in MSC exosome. We have previously reported that RNAs in MSC exosome are primarily small RNA molecules of <300 nt and they include many miRNAs. Many of these miRNAs are in the precursor form suggesting that pre-miRNAs, and not mature miRNAs are preferentially loaded into exosomes. The protocols described here include assays to ascertain the presence of pre-miRNAs, profiling of miRNA and pre-miRNA, and quantitative estimation of mature and pre-miRNA.

Mesenchymal stem cell exosome: a novel stem cell-based therapy for cardiovascular disease.

Cardiovascular disease is a major target for many experimental stem cell-based therapies and mesenchymal stem cells (MSCs) are widely used in these therapies. Transplantation of MSCs to treat cardiac disease has always been predicated on the hypothesis that these cells would engraft, differentiate and replace damaged cardiac tissues. However, experimental or clinical observations so far have failed to demonstrate a therapeutically relevant level of transplanted MSC engraftment or differentiation. Instead, they indicate that transplanted MSCs secrete factors to reduce tissue injury and/or enhance tissue repair. Here we review the evidences supporting this hypothesis including the recent identification of exosome as a therapeutic agent in MSC secretion. In particular, we will discuss the potential and practicality of using this relatively novel entity as a therapeutic modality for the treatment of cardiac disease, particularly acute myocardial infarction.

Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs.

BACKGROUND: Exosomes or secreted bi-lipid vesicles from human ESC-derived mesenchymal stem cells (hESC-MSCs) have been shown to reduce myocardial ischemia/reperfusion injury in animal models. However, as hESC-MSCs are not infinitely expansible, large scale production of these exosomes would require replenishment of hESC-MSC through derivation from hESCs and incur recurring costs for testing and validation of each new batch. Our aim was therefore to investigate if MYC immortalization of hESC-MSC would circumvent this constraint without compromising the production of therapeutically efficacious exosomes. METHODS: The hESC-MSCs were transfected by lentivirus carrying a MYC gene. The transformed cells were analyzed for MYC transgene integration, transcript and protein levels, and surface markers, rate of cell cycling, telomerase activity, karyotype, genome-wide gene expression and differentiation potential. The exosomes were isolated by HPLC fractionation and tested in a mouse model of myocardial ischemia/reperfusion injury, and infarct sizes were further assessed by using Evans' blue dye injection and TTC staining. RESULTS: MYC-transformed MSCs largely resembled the parental hESC-MSCs with major differences being reduced plastic adherence, faster growth, failure to senesce, increased MYC protein expression, and loss of in vitro adipogenic potential that technically rendered the transformed cells as non-MSCs. Unexpectedly, exosomes from MYC-transformed MSCs were able to reduce relative infarct size in a mouse model of myocardial ischemia/reperfusion injury indicating that the capacity for producing therapeutic exosomes was preserved. CONCLUSION: Our results demonstrated that MYC transformation is a practical strategy in ensuring an infinite supply of cells for the production of exosomes in the milligram range as either therapeutic agents or delivery vehicles. In addition, the increased proliferative rate by MYC transformation reduces the time for cell production and thereby reduces production costs.

Delineating biological pathways unique to embryonic stem cell-derived insulin-producing cell lines from their noninsulin-producing progenitor cell lines.

To identify unique biochemical pathways in embryonic stem cell-derived insulin-producing cells as potential therapeutic targets to prevent or delay beta-cell dysfunction or death in diabetic patients, comparative genome-wide gene expression studies of recently derived mouse insulin-producing cell lines and their progenitor cell lines were performed using microarray technology. Differentially expressed genes were functionally clustered to identify important biochemical pathways in these insulin-producing cell lines. Biochemical or cellular assays were then performed to assess the relevance of these pathways to the biology of these cells. A total of 185 genes were highly expressed in the insulin-producing cell lines, and computational analysis predicted the pentose phosphate pathway (PPP), clathrin-mediated endocytosis, and the peroxisome proliferator-activated receptor (PPAR) signaling pathway as important pathways in these cell lines. Insulin-producing ERoSHK cells were more resistant to hydrogen peroxide (H(2)O(2))-induced oxidative stress. Inhibition of PPP by dehydroepiandrosterone and 6-aminonicotinamide abrogated this H(2)O(2) resistance with a concomitant decrease in PPP activity as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Clathrin-mediated endocytosis, which is essential in maintaining membrane homeostasis in secreting cells, was up-regulated by glucose in ERoSHK but not in their progenitor ERoSH cells. Its inhibition by chlorpromazine at high glucose concentration was toxic to the cells. Troglitazone, a PPARG agonist, up-regulated expression of Ins1 and Ins2 but not Glut2. Gene expression analysis has identified the PPP, clathrin-mediated endocytosis, and the PPAR signaling pathway as the major delineating pathways in these insulin-producing cell lines, and their biological relevance was confirmed by biochemical and cellular assays.

Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury.

Human ESC-derived mesenchymal stem cell (MSC)-conditioned medium (CM) was previously shown to mediate cardioprotection during myocardial ischemia/reperfusion injury through large complexes of 50-100 nm. Here we show that these MSCs secreted 50- to 100-nm particles. These particles could be visualized by electron microscopy and were shown to be phospholipid vesicles consisting of cholesterol, sphingomyelin, and phosphatidylcholine. They contained coimmunoprecipitating exosome-associated proteins, e.g., CD81, CD9, and Alix. These particles were purified as a homogeneous population of particles with a hydrodynamic radius of 55-65 nm by size-exclusion fractionation on a HPLC. Together these observations indicated that these particles are exosomes. These purified exosomes reduced infarct size in a mouse model of myocardial ischemia/reperfusion injury. Therefore, MSC mediated its cardioprotective paracrine effect by secreting exosomes. This novel role of exosomes highlights a new perspective into intercellular mediation of tissue injury and repair, and engenders novel approaches to the development of biologics for tissue repair.

Derivation and characterization of human fetal MSCs: an alternative cell source for large-scale production of cardioprotective microparticles.

The therapeutic effects of mesenchymal stem cells (MSCs) transplantation are increasingly thought to be mediated by MSC secretion. We have previously demonstrated that human ESC-derived MSCs (hESC-MSCs) produce cardioprotective microparticles in pig model of myocardial ischemia/reperfusion (MI/R) injury. As the safety and availability of clinical grade human ESCs remain a concern, MSCs from fetal tissue sources were evaluated as alternatives. Here we derived five MSC cultures from limb, kidney and liver tissues of three first trimester aborted fetuses and like our previously described hESC-derived MSCs; they were highly expandable and had similar telomerase activities. Each line has the potential to generate at least 10(16-19) cells or 10(7-10) doses of cardioprotective secretion for a pig model of MI/R injury. Unlike previously described fetal MSCs, they did not express pluripotency-associated markers such as Oct4, Nanog or Tra1-60. They displayed a typical MSC surface antigen profile and differentiated into adipocytes, osteocytes and chondrocytes in vitro. Global gene expression analysis by microarray and qRT-PCR revealed a typical MSC gene expression profile that was highly correlated among the five fetal MSC cultures and with that of hESC-MSCs (r(2)>0.90). Like hESC-MSCs, they produced secretion that was cardioprotective in a mouse model of MI/R injury. HPLC analysis of the secretion revealed the presence of a population of microparticles with a hydrodynamic radius of 50-65 nm. This purified population of microparticles was cardioprotective at approximately 1/10 dosage of the crude secretion.

Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs.

Intercellular exchange of protein and RNA-containing microparticles is an increasingly important mode of cell-cell communication. Here we investigate if mesenchymal stem cells (MSCs) known for secreting therapeutic paracrine factors also secrete RNA-containing microparticles. We observed that human embryonic stem cell (hESC)-derived MSC conditioned medium contained small RNAs (less than 300 nt) encapsulated in cholesterol-rich phospholipid vesicles as evidenced by their RNase sensitivity only in the presence of a sodium dodecyl sulfate-based cell lysis buffer, phospholipase A2 and a chelator of cholesterol, cyclodextrin and the restoration of their lower than expected density by detergent or phospholipase A2 treatment. MicroRNAs (miRNAs) such as hsa-let-7b and hsa-let-7g were present in a high precursor (pre)- to mature miRNA ratio by microarray analysis and quantitative reverse transcription-polymerase chain reaction. The pre-miRNAs were cleaved to mature miRNA by RNase III in vitro. High performance liquid chromatography-purified RNA-containing vesicles have a hydrodynamic radius of 55-65 nm and were readily taken up by H9C2 cardiomyocytes. This study suggests that MSCs could facilitate miRNA-mediated intercellular communication by secreting microparticles enriched for pre-miRNA.