ENHANCED PROTEIN LOADING INTO LIPOSOMES BY THE MULTIPLE CROSSFLOW INJECTION TECHNIQUE

ABSTRACT

Methods for encapsulation of a drug into liposomes should preferably result in a high encapsulation efficiency and a high encapsulation capacity. Our studies were focussed on the establishment of an efficient encapsulation procedure of the radical scavenging protein, rh-Cu/Zn-SOD, into liposomes with the cross flow injection method. Limitations to increase the encapsulation efficiency are caused by the enclosed aqueous volume, by the lipid concentration, the aspired vesicle size and the final ethanol concentration. Our research was performed to maximize the encapsulation following several strategies of injecting higher lipid concentrations into the aqueous phase. The one way triple technique, a sophisticated preparation procedure is presented, which enables three times higher encapsulation rates in comparison to standard procedures. Additionally, scalability studies demonstrate reproducibility independent of the preparation volume. Vesicle size distribution and encapsulation efficiency remain constant. Furthermore, special attention is paid on reproducibility of prepared liposomes, scale-up and on long term stability of the lipid vesicles.     

Glioma-derived mutations in isocitrate dehydrogenase 2 beneficial to traditional chemotherapy

Sonoporation is a promising drug delivery technique with great potential in medicine. However, its applications have been limited mostly by the lack of understanding its underlying biophysical mechanism, partly due to the inadequacy of the existing models for coupling with highly sensitive imaging techniques to directly observe the actual precursor events of cell–microbubble interaction under low intensity ultrasound. Here, we introduce a new in vitro method utilizing capillary-microgripping system and micro-transducer to achieve maximum level of experimental flexibility for capturing real time highly magnified images of cell–microbubble interaction, hitherto unseen in this context. Insonation of isolated single cells and microbubbles parallel with high speed microphotography and fluorescence microscopy allowed us to identify dynamic responses of cell-membrane/microbubble in correlation with sonoporation. Our results showed that bubble motion and linear oscillation in close contact with the cell membrane can cause local deformation and transient porosity in the cell membrane without rupturing it. This method can also be used as an in situ gene/drug delivery system of targeted cells for non-invasive clinical applications.     

Asymptotic Conditional Singular Value Decomposition for High‐Dimensional Genomic Data

Summary High‐dimensional data, such as those obtained from a gene expression microarray or second generation sequencing experiment, consist of a large number of dependent features measured on a small number of samples. One of the key problems in genomics is the identification and estimation of factors that associate with many features simultaneously. Identifying the number of factors is also important for unsupervised statistical analyses such as hierarchical clustering. A conditional factor model is the most common model for many types of genomic data, ranging from gene expression, to single nucleotide polymorphisms, to methylation. Here we show that under a conditional factor model for genomic data with a fixed sample size, the right singular vectors are asymptotically consistent for the unobserved latent factors as the number of features diverges. We also propose a consistent estimator of the dimension of the underlying conditional factor model for a finite fixed sample size and an infinite number of features based on a scaled eigen‐decomposition. We propose a practical approach for selection of the number of factors in real data sets, and we illustrate the utility of these results for capturing batch and other unmodeled effects in a microarray experiment using the dependence kernel approach of Leek and Storey (2008, Proceedings of the National Academy of Sciences of the United States of America 105 , 18718–18723) .     

A high-throughput screening fluorescence polarization assay for fatty acid adenylating enzymes in Mycobacterium tuberculosis

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), encodes for an astonishing 34 fatty acid adenylating enzymes (FadDs), which play key roles in lipid metabolism. FadDs involved in lipid biosynthesis are functionally nonredundant and serve to link fatty acid and polyketide synthesis to produce some of the most architecturally complex natural lipids including the essential mycolic acids as well as the virulence-conferring phthiocerol dimycocerosates, phenolic glycolipids, and mycobactins. Here we describe the systematic development and optimization of a fluorescence polarization assay to identify small molecule inhibitors as potential antitubercular agents. We fluorescently labeled a bisubstrate inhibitor to generate a fluorescent probe/tracer, which bound with a K_D of 245 nM to FadD28. Next, we evaluated assay performance by competitive binding experiments with a series of known ligands and assessed the impact of control parameters including incubation time, stability of the signal, temperature, and DMSO concentration. As a final level of validation the LOPAC1280 library was screened in a 384-well plate format and the assay performed with a Z-factor of 0.75, demonstrating its readiness for high-throughput screening.     

Effect of 1,25(OH)(2)D(3) on rat peritoneal mesothelial cells treated with high glucose plus lipopolysaccharide

1,25(OH)₂D₃, the active metabolite of vitamin D₃, its activity is not limited to mineral and skeletal homeostasis. In recent years, there has been increasing evidence pointing to the role of its activity in the regulation of cell proliferation, cell differentiation and immunomodulation. Here we report lipopolysaccharide (LPS), a glycolipid that is produced and secreted by gram-negative bacteria during peritonitis, plus high glucose (HG) can significantly inhibit mesothelial cell viability while induce more apoptosis in rat peritoneal mesothelial cells (RPMC). Pretreatment with 1,25(OH)₂D₃ can reverse the above effect in a concentration dependent manner. HG plus LPS can down-regulate the levels of both mRNA and protein of VDR, and up-regulate the expression of TGF-β1 and TNF-α in RPMC, which can also be effectively reversed by pretreatment with 1,25(OH)₂D₃. The above results suggest that HG plus LPS may induce changes in RPMC’s viability and apoptosis, leading to peritoneal injury. 1,25(OH)₂D₃ can reverse the inhibition of cell viability, the increase of apoptotic rate and induction of fibrosis related cytokine TGF-β1 and TNF-α by HG plus LPS in RPMC, thus protect peritoneal membrane.     

The cytoplasmic domain of TGFβR3 through its interaction with the scaffolding protein, GIPC, directs epicardial cell behavior

The epicardium is a major contributor of the cells that are required for the formation of coronary vessels. Mice lacking both copies of the gene encoding the Type III Transforming Growth Factor β Receptor (TGFβR3) fail to form the coronary vasculature, but the molecular mechanism by which TGFβR3 signals coronary vessel formation is unknown. We used intact embryos and epicardial cells from E11.5 mouse embryos to reveal the mechanisms by which TGFβR3 signals and regulates epicardial cell behavior. Analysis of E13.5 embryos reveals a lower rate of epicardial cell proliferation and decreased epicardially derived cell invasion in Tgfbr3^−/− hearts. Tgfbr3^−/− epicardial cells in vitro show decreased proliferation and decreased invasion in response to TGFβ1 and TGFβ2. Unexpectedly, loss of TGFβR3 also decreases responsiveness to two other important regulators of epicardial cell behavior, FGF2 and HMW-HA. Restoring full length TGFβR3 in Tgfbr3^−/− cells rescued deficits in invasion in vitro in response TGFβ1 and TGFβ2 as well as FGF2 and HMW-HA. Expression of TGFβR3 missing the 3 C-terminal amino acids that are required to interact with the scaffolding protein GIPC1 did not rescue any of the deficits. Overexpression of GIPC1 alone in Tgfbr3^−/− cells did not rescue invasion whereas knockdown of GIPC1 in Tgfbr3^+/+ cells decreased invasion in response to TGFβ2, FGF2, and HMW-HA. We conclude that TGFβR3 interaction with GIPC1 is critical for regulating invasion and growth factor responsiveness in epicardial cells and that dysregulation of epicardial cell proliferation and invasion contributes to failed coronary vessel development in Tgfbr3^−/− mice.     

高原鼢鼠肝脏组织细胞周期相关基因的进化和表达

高原鼢鼠Myospalax baileyi是一种世居青藏高原的地下鼠,对严重的低氧环境有很强的适应性。低氧诱导细胞周期G1、G2期阻滞。为了探讨高原鼢鼠适应低氧环境的分子机制,应用生物信息学方法对p53下游细胞周期基因p21、CyclinD1、CyclinE、CDK6、CDK2、14-3-3-σ、Gadd45α、B99和CyclinB1的序列和编码的氨基酸序列进行了进化分析,并以SD大鼠Rattus norvegicus为对照,研究了这些基因在不同海拔(3300 m、2260 m)条件下的表达模式。结果表明:(1)高原鼢鼠细胞周期相关基因的序列与以色列鼹鼠Nannospalax galili同源性最高,达到90%以上;p21、CyclinD1、CyclinE和CyclinB1编码蛋白与以色列鼹鼠存在明显的趋同进化位点;SIFT评估发现,p21和CyclinB1氨基酸序列分别在第27号位点和第105号位点的变异对细胞周期调控功能有显著影响;(2)与低海拔条件相比,在高海拔条件下,高原鼢鼠肝脏组织中与G1期相关的基因p21表达水平显著上升,p21下游基因CyclinD1、CyclinE、CDK6和CDK2表达水平显著下降,而在SD大鼠中没有显著变化;与G2期相关的基因Gadd45α、B99、14-3-3-δ和CyclinB1在高原鼢鼠和SD大鼠中随海拔变化不发生明显变化。在不同海拔条件下,高原鼢鼠肝脏组织中的上述细胞周期相关基因的表达水平均极显著高于SD大鼠(P<0.01)。以上结果提示,高原鼢鼠经过长期的低氧适应,通过上调p21基因的表达抑制下游CyclinD1、CyclinE、CDK6和CDK2基因的表达,导致细胞周期G1期阻滞,从而提供充足的时间进行DNA修复,保证了DNA复制的准确性;同时高原鼢鼠肝脏组织中细胞周期的调控不仅与细胞周期基因的表达水平有关,而且可能与细胞周期因子p21的第27号位点和CyclinB1的第105号位点的变异有关。     

山西吕梁山草本群落物种多样性的海拔梯度格局及与环境因子的关系

山地草本群落物种多样性的海拔梯度格局及其与环境因子的关系一直是生态学和地理学研究的热点问题。为探究吕梁山草本群落不同尺度物种多样性(α、β、γ多样性)的海拔梯度格局及其与环境因子的关系,该研究采用样方法在吕梁山从北向南选取管涔山、关帝山、五鹿山,并对每个山地划分出高、中、低3个海拔梯度,对草本群落物种多样性和环境因子进行调查分析。结果表明:(1)α多样性随海拔升高呈先升后减的单峰变化格局,峰值出现在中海拔; β多样性中Cody指数在1 900～2 000 m间出现峰值,而Bray-Curtis 指数出现最小值,表明1 900～2 000 m海拔带是草本群落物种更新速率和组成变化较快的过渡地带; γ多样性随海拔升高也呈先升后减的单峰变化格局(R²=0.406,P<0.01)。(2)海拔对温度、湿度变化的影响极其显著(P<0.01); 海拔、温度、湿度等环境因子共同影响物种的α多样性,其中海拔是主要影响因素(P<0.05),温度、湿度影响不显著(P>0.05)。综上所述,吕梁山草本群落多样性在海拔梯度上多表现为 “中间膨胀”变化格局,海拔变化是影响多样性变化的主要因素。     

山西省参考作物蒸散量的时空变化特征

基于山西省境内70个地面气象观测站1960—2019年的逐日降水量、气温、日照时数、相对湿度、风速、水汽压等气象资料,应用Penman-Monteith公式计算参考作物蒸散量(ET₀),对山西省ET₀的时空变化特征及不同气候带和海拔的蒸散特征进行定量分析。结果表明: 1960—2019年,研究区年均ET₀在空间上呈现由西向东逐渐递减的趋势;以1982年为拐点,前后两个时段均呈逐年增加趋势,月际、旬际波动为单峰变化曲线。不同气候带ET₀的差异性表现为:温带半干旱气候区的年、春、夏、秋季ET₀高于暖温带半湿润气候区和暖温带半干旱气候区;冬季,暖温带半湿润气候区ET₀最高。不同海拔ET₀的差异性表现为:＜660 m海拔区的年、夏、秋、冬季ET₀高于其他海拔区域。