Leptin gene-targeted editing in ob/ob mouse adipose tissue based on the CRISPR/Cas9 system

Gene therapy has become the most effective treatment for monogenic diseases. Congenital LEPTIN deficiency is a rare autosomal recessive monogenic obesity syndrome caused by mutations in the Leptin gene. Ob/ob mouse is a monogenic obesity model, which carries a homozygous point mutation of C to T in Exon 2 of the Leptin gene. Here, we attempted to edit the mutated Leptin gene in ob/ob mice preadipocytes and inguinal adipose tissues using CRISPR/Cas9 to correct the C to T mutation and restore the production of LEPTIN protein by adipocytes. The edited preadipocytes exhibit a correction of 5.5% of Leptin alleles and produce normal LEPTIN protein when differentiated into mature adipocytes. The ob/ob mice display correction of 1.67% of Leptin alleles, which is sufficient to restore the production and physiological functions of LEPTIN protein, such as suppressing appetite and alleviating insulin resistance. Our study suggests CRISPR/Cas9-mediated in situ genome editing as a feasible therapeutic strategy for human monogenic diseases, and paves the way for further research on efficient delivery system in potential future clinical application.     

6个品种樱花在北京地区的适应性研究

为丰富北京地区樱花种植品种,选择大渔樱(Cerasus×kanzakura ‘Tairyo-zakura’)、修善寺寒樱(Cerasus×kanzakura ‘Rubescens’)、阳春(C. spachiana ‘Yoshun’)、天之川(C. serrulata ‘Erecta’)、兰兰(C. serrulata ‘Ranran’)、红华(C. serrulata ‘Kouka’)等6个品种樱花进行实验性种植。在田间观测的基础上,测定低温胁迫下根系脱氢酶活性、枝条脯氨酸含量,高温胁迫下叶片的电导率、可溶性蛋白含量。结果表明,6个品种中,随着温度下降,6个品种樱花根系脱氢酶还原量、枝条脯氨酸含量均呈现先上升后下降的趋势。大渔樱、修善寺寒樱、兰兰在-10 ℃时脱氢酶活性与脯氨酸含量达到最大值,阳春、天之川、红华在-15 ℃脱氢酶活性与脯氨酸含量达到最大值。6个品种的叶片相对电导率,均随着温度的上升不断升高。而叶片可溶性蛋白含量变化趋势,品种间差异较大。阳春、天之川、红华在北京地区适应性良好,大渔樱、修善寺寒樱、兰兰可在小环境较好的区域栽植。     

Human cholesteryl ester transport protein transgene promotes macrophage reverse cholesterol transport in C57BL/6 mice and phospholipid transfer protein gene knockout mice

Journal of Physiology and Biochemistry - Cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) belong to the same gene family. Liver-specific expression of CETP...     

Complex Interaction and Adsorption of Glyphosate and Lead in Soil

Glyphosate [N-(phosphonomethyl)-glycine] is a herbicide widely used in large quantities in agricultural applications. It is also known to form complexes with metal ions, although its influence on metal behavior, such as lead (Pb) in soil, is not well understood. In this study, the adsorption and co-adsorption of Pb and glyphosate were determined on two soils [a red (RS) soil, Udic Ferrisol, and a yellow-brown (YB) soil, Udic Luvisol] of distinctly different chemical characteristics at varying pH conditions. Results indicate that the adsorption of lead and glyphosate strongly depends on soil types: the RS soil, characterized by a relatively high iron/aluminum content but a low pH and organic matter content, shows a much lower adsorption capacity for Pb but a higher sorption for glyphosate than the YB soil. The co-existence of Pb and glyphosate in soils resulted in complex interactions among Pb, glyphosate, Pb-glyphosate complexes, and soil minerals. The presence of glyphosate decreased Pb adsorption on the two soils, which was attributed primarily to the formation of soluble Pb-glyphosate complexes having relatively low affinities to soil surfaces. On the other hand, addition of Pb increased the adsorption of glyphosate on both soils, which was attributed to: (1) a decreased solution pH due to the ion exchange between Pb²⁺ and H⁺ on soil surfaces; and (2) increased sorption sites where Pb was adsorbed and acted as a bridge between glyphosate and the soil. The present study illustrates that the complex interactions among glyphosate, Pb, and soil may have important implications for the mobility and bioavailability of Pb in soil and should thus be considered in future environmental risk assessments.     

Overexpression of Neurospora crassa OR74A glutamate decarboxylase in Escherichia coli for efficient GABA production

This study investigated the effect of glutamate decarboxylase from Neurospora crassa OR74A on GABA production in Escherichia coli. GABA is one of the inhibitory neurotransmitters in the mammalian central nervous system, and can be used as a precursor of promising biopolymer Nylon 4. E. coli that overexpressed N. crassa glutamate decarboxylase was cultured at various pH levels and temperatures to determine optimum conditions for GABA production. When the recombinant E. coli strain was cultured at 30°C and pH 3, a final GABA concentration of 5.26 g/L was obtained from 10 g/L of monosodium glutamate (MSG), corresponding to a GABA yield of 86.23%.     

Combination of 1,4-naphthoquinone with benzothiazoles had selective algicidal effects against harmful algae

A series of naphthoquinone-benzothiazole conjugates were synthesized as algicides, and their efficacies against harmful algal blooming species, such as Chattonella marina, Heterosigma akashiwo and Cochlodinium polykrikoides, were examined. The introduction of substituted benzothiazole at the C2 position of 1,4-naphthoquinone (compounds 1–9) resulted in higher algicidal activity against C. polykrikoides than the C6 conjugates (compounds 10–20). On the other hand, of the C6 conjugates, compounds 11 and 12 exhibited better algicidal activity against H. akashiwo, C. marina, and C. polykrikoides than the C2 conjugates. Further structure-activity analysis indicated that a replacement of the methoxy groups with hydroxyl groups (compounds 21–26) decreased the algicidal activity significantly. Among the various synthetic naphthoquinonebezothiazole conjugates tested, compound 12 was found to affect the most significant decrease in the level of C. polykrikoides growth, with an IC₅₀ of 0.19 μM. Compound 11 was found to be the most potent inhibitor against H. akashiwo and C. polykrikoides, with IC₅₀ values of 0.32 and 0.12 μM, respectively. Overall, these results highlight a possible method for controlling and inhibiting red tide forming algae using NQ derivatives.     

Inhibition of tyrosinase by gastrodin: An integrated kinetic-computational simulation analysis

We studied the inhibitory effect of gastrodin on tyrosinase using inhibition kinetics and computational simulation. Gastrodin reversibly inhibited tyrosinase in a mixed-type manner with K_i = 123.8 ± 20.2 mM. Time-interval kinetics revealed the inhibition to be a first-order process with mono- and bi-phasic components. Using AutoDock Vina, we calculated a binding energy of ?6.3 kcal/mol for gastrodin and tyrosinase, and we performed a molecular dynamics simulation of the tyrosinase–gastrodin interaction. The simulation results suggested that gastrodin interacts primarily with histidine residues in the active site. A 10-ns molecular dynamics simulation showed that one copper ion in the tyrosinase active site was responsible for the interaction with gastrodin. Our study provides insight into the inhibition of tyrosinase by the hydroxyl groups of gastrodin. A combination of inhibition kinetics and computational calculations may help to confirm the inhibitory action of gastrodin on tyrosinase and define the mechanisms of inhibition.     

A pilot study of autologous CD34-depleted bone marrow mononuclear cell transplantation via the hepatic artery in five patients with liver failure

Background aimsMany rodent experiments and human studies on stem cell therapy have shown promising therapeutic approaches to liver diseases. We investigated the clinical outcomes of five patients with liver failure of various causes who received autologous CD34-depleted bone marrow-derived mononuclear cell (BM-MNC) transplantation, including mesenchymal stromal cells, through the hepatic artery.MethodsCD34-depleted BM-MNCs were obtained from five patients waiting for liver transplantation by bone marrow aspiration and using the CliniMACS CD34 Reagent System (Miltenyi Biotech, Bergisch Gladbach, Germany), and autologous hepatic artery infusion was performed. The causes of hepatic decompensation were hepatitis B virus (HBV), hepatitis C virus (HCV), propylthiouracil-induced toxic hepatitis and Wilson disease.ResultsSerum albumin levels improved 1 week after transplantation from 2.8 g/dL, 2.4 g/dL, 2.7 g/dL and 1.9 g/dL to 3.3 g/dL, 3.1 g/dL, 2.8 g/dL and 2.6 g/dL. Transient liver elastography data showed some change from 65 kPa, 33 kPa, 34.8 kPa and undetectable to 46.4 kPa, 19.8 kPa, 29.1 kPa and 67.8 kPa at 4 weeks after transplantation in a patient with Wilson disease, a patient with HCV, and two patients with HBV. Ascites decreased in two patients. One of the patients with HBV underwent liver transplantation 4 months after the infusion, and the hepatic progenitor markers (cytokeratin [CD]-7, CD-8, CD-9, CD-18, CD-19, c-Kit and epithelial cell adhesion molecule [EpCAM]) were highly expressed in the explanted liver.ConclusionsSerum albumin levels, liver stiffness, liver volume, subjective healthiness and quality of life improved in the study patients. Although these findings were observed in a small population, the results may suggest a promising future for autologous CD34-depleted BM-MNC transplantation as a bridge to liver transplantation in patients with liver failure.