Embryo Microinjection for Transgenesis in Drosophila.

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope. Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

Glycometabolism reprogramming: Implications for cardiovascular diseases.

Glycometabolism is well known for its roles as the main source of energy, which mainly includes three metabolic pathways: oxidative phosphorylation, glycolysis and pentose phosphate pathway. The orderly progress of glycometabolism is the basis for the maintenance of cardiovascular function. However, upon exposure to harmful stimuli, the intracellular glycometabolism changes or tends to shift toward another glycometabolism pathway more suitable for its own development and adaptation. This shift away from the normal glycometabolism is also known as glycometabolism reprogramming, which is commonly related to the occurrence and aggravation of cardiovascular diseases. In this review, we elucidate the physiological role of glycometabolism in the cardiovascular system and summarize the mechanisms by which glycometabolism drives cardiovascular diseases, including diabetes, cardiac hypertrophy, heart failure, atherosclerosis, and pulmonary hypertension. Collectively, directing GMR back to normal glycometabolism might provide a therapeutic strategy for the prevention and treatment of related cardiovascular diseases.

MCU-dependent mitochondrial calcium uptake-induced mitophagy contributes to apelin-13-stimulated VSMCs proliferation.

Apelin is an endogenous ligand of the G protein-coupled receptor APJ. Both apelin and APJ receptors, which are expressed in vascular smooth muscle cells (VSMCs), play important roles in the cardiovascular system. Our previous studies researches indicated that mitophagy mediated apelin-13-induced VSMCs proliferation. However, little is known about how apelin-13 regulates mitophagy to participate in VSMC proliferation. The results of the present study demonstrated that mitochondrial calcium uniporter (MCU) uptake-dependent mitochondrial calcium-induced mitophagy is involved in apelin-13-induced VSMCs proliferation. Apelin-13 promoted the expression of MCU which increases mitochondrial calcium uptake. Apelin-13-induced MCU-dependent mitochondrial calcium uptake further increased mitochondrial ROS (mtROS) concentrations and promoted mitophagy, which can be evidenced through the upregulation of the Dynamin-related protein 1(Drp1), PTEN-induced kinase 1 (PINK1), and Parkin. The clearance of mtROS by Mito-TEMPO significantly reversed apelin-13-induced mitophagy. Moreover, both the Drp1 inhibitor mdivi-1 and siRNA-Drp1 inhibited apelin-13-induced mitophagy. Furthermore, the APJ receptor antagonist F13A, MCU inhibitor Ru360, mitochondria-targeted antioxidant Mito-TEMPO, Drp1 inhibitor Mdivi-1, siRNA-Drp1, siRNA-PINK1, and siRNA-Parkin inhibited the proliferation of VSMCs induced by apelin-13. In ApoE-/- mice, intraperitoneal administration of apelin-13 induced the expression of MCU, Drp1, PINK1, Parkin, and α-SMA and increased atherosclerotic plaque lesions. However, F13A and Ru360 decreased the expression of MCU, Drp1, PINK1, Parkin, and α-SMA and reduced atherosclerotic plaque lesions in ApoE-/- mice injected with apelin-13. Collectively, our results demonstrate that MCU-dependent mitochondrial calcium uptake-induced mitophagy is involved in apelin-13-stimulated VSMCs proliferation.

M6 A modification: A mechanism for protecting hematopoietic development in mammals.

N6 -methyladenosine (m6 A) is one of the most common internal modifications in messenger RNA, which is necessary for cell physiological activities. A recent study shows that during mammalian hematopoietic development, loss of m6 A modification leads to the aberrant production of double-stranded RNA, which results in the abnormal activation of innate immune response, and ultimately leads to hematopoietic failure. Accordingly, m6 A modification provide us an attractive direction for us to understand mammalian hematopoietic development and innate immune response.

VDAC oligomer pores: A mechanism in disease triggered by mtDNA release.

A recent study suggests that voltage-dependent anion channel (VDAC) oligomer pores promote mitochondrial outer membrane permeabilization and allow mitochondrial DNA (mtDNA) to be released into the cytosol in live cells. It challenges the notion that only occurs in apoptotic cells via BAX/BAK macropores. Cytosolic mtDNA activates cyclic GMP-AMP synthase (cGAS)-stimulator of IFN gene (STING) pathway and triggers type I interferon (IFN) response thereafter, which ultimately causes systemic lupus erythematosus. Mechanistically, mtDNA can interact with three positively charged residues (Lys12, Arg15, and Lys20) at the N-terminus of VDAC1, thereby strengthening VDAC1 oligomerization and facilitating mtDNA release. In addition, there are other pathways that can mediate mtDNA release, such as BAX/BAK macropores and virus-derived pores. The mtDNA released into the cytosol also triggers type I IFN response via the generally accepted cGAS-STING-TANK-binding kinase 1-IFN regulatory factor 3 axis. Collectively, VDAC oligomer pores provide us an attractive direction for us to understand mtDNA release-related diseases.

Apelin/Elabela-APJ: a novel therapeutic target in the cardiovascular system.

Apelin and Elabela (ELA) are endogenous ligands of angiotensin domain type 1 receptor-associated proteins (APJ). Apelin/ELA-APJ signal is widely distributed in the cardiovascular system of fetuse and adult. The signal is involved in the development of the fetal heart and blood vessels and regulating vascular tension in adults. This review described the effects of apelin/ELA-APJ on fetal (vasculogenesis and angiogenesis) and adult cardiovascular function [vascular smooth muscle cell (VSMC) proliferation, vasodilation, positive myodynamia], and relative diseases [eclampsia, hypertension, pulmonary hypertension, heart failure (HF), myocardial infarction (MI), atherosclerosis, etc.] in detail. The pathways of apelin/ELA-APJ regulating cardiovascular function and cardiovascular-related diseases are summarized. The drugs developed based on apelin and ELA suggests APJ is a prospective strategy for cardiovascular disease therapy.

Apelin/APJ system: an emerging therapeutic target for respiratory diseases.

Apelin is an endogenous ligand of G protein-coupled receptor APJ. It is extensively expressed in many tissues such as heart, liver, and kidney, especially in lung tissue. A growing body of evidence suggests that apelin/APJ system is closely related to the development of respiratory diseases. Therefore, in this review, we focus on the role of apelin/APJ system in respiratory diseases, including pulmonary arterial hypertension (PAH), pulmonary embolism (PE), acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), obstructive sleep apnoea syndrome (OSAS), non-small cell lung cancer (NSCLC), pulmonary edema, asthma, and chronic obstructive pulmonary diseases. In detail, apelin/APJ system attenuates PAH by activating AMPK-KLF2-eNOS-NO signaling and miR424/503-FGF axis. Also, apelin protects against ALI/ARDS by reducing mitochondrial ROS-triggered oxidative damage, mitochondria apoptosis, and inflammatory responses induced by the activation of NF-κB and NLRP3 inflammasome. Apelin/APJ system also prevents the occurrence of pulmonary edema via activating AKT-NOS3-NO pathway. Moreover, apelin/APJ system accelerates NSCLC cells' proliferation and migration via triggering ERK1/2-cyclin D1 and PAK1-cofilin signaling, respectively. Additionally, apelin/APJ system may act as a predictor in the development of OSAS and PE. Considering the pleiotropic actions of apelin/APJ system, targeting apelin/APJ system may be a potent therapeutic avenue for respiratory diseases.

Mitochondrial superoxide/hydrogen peroxide: An emerging therapeutic target for metabolic diseases.

Mitochondria are well known for their roles as energy and metabolic factory. Mitochondrial reactive oxygen species (mtROS) refer to superoxide anion radical (•O2-) and hydrogen peroxide (H2O2). They are byproducts of electron transport in mitochondrial respiratory chain and are implicated in the regulation of physiological and pathological signal transduction. Especially when mitochondrial •O2-/H2O2 production is disturbed, this disturbance is closely related to the occurrence and development of metabolic diseases. In this review, the sources of mitochondrial •O2-/H2O2 as well as mitochondrial antioxidant mechanisms are summarized. Furthermore, we particularly emphasize the essential role of mitochondrial •O2-/H2O2 in metabolic diseases. Specifically, perturbed mitochondrial •O2-/H2O2 regulation aggravates the progression of metabolic diseases, including diabetes, gout and nonalcoholic fatty liver disease (NAFLD). Given the deleterious effect of mitochondrial •O2-/H2O2 in the development of metabolic diseases, antioxidants targeting mitochondrial •O2-/H2O2 might be an attractive therapeutic approach for the prevention and treatment of metabolic diseases.