Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.     

基于遗传算法特征选择的~(31)P磁共振波谱图数据分类

基于31P磁共振波谱图(31Phosphorus Magnetic Resonance Spectroscopy,31P-MRS)对肝脏数据进行诊断,共分为三种类型:肝癌,肝硬化和正常肝。本文在线性分类器分类前先用遗传算法进行特征选择,选择出最优特征子集。实验中,用线性分类器分别对经过遗传算法特征选择后的最优特征子集分类和对提取的全波谱数据进行分类。实验结果证明,前者方法不仅明显提高了分类的准确率,而且减少了分类器运行的时间,其中31P-MR波谱对活体肝细胞癌的诊断正确率从62.50%提高到89.35%。     

Molecular Determinants of the CaVβ-induced Plasma Membrane Targeting of the CaV1.2 Channel

Ca_Vβ subunits modulate cell surface expression and voltage-dependent gating of high voltage-activated (HVA) Ca_V1 and Ca_V2 α1 subunits. High affinity Ca_Vβ binding onto the so-called α interaction domain of the I-II linker of the Ca_Vα1 subunit is required for Ca_Vβ modulation of HVA channel gating. It has been suggested, however, that Ca_Vβ-mediated plasma membrane targeting could be uncoupled from Ca_Vβ-mediated modulation of channel gating. In addition to Ca_Vβ, Ca_Vα2δ and calmodulin have been proposed to play important roles in HVA channel targeting. Indeed we show that co-expression of Ca_Vα2δ caused a 5-fold stimulation of the whole cell currents measured with Ca_V1.2 and Ca_Vβ3. To gauge the synergetic role of auxiliary subunits in the steady-state plasma membrane expression of Ca_V1.2, extracellularly tagged Ca_V1.2 proteins were quantified using fluorescence-activated cell sorting analysis. Co-expression of Ca_V1.2 with either Ca_Vα2δ, calmodulin wild type, or apocalmodulin (alone or in combination) failed to promote the detection of fluorescently labeled Ca_V1.2 subunits. In contrast, co-expression with Ca_Vβ3 stimulated plasma membrane expression of Ca_V1.2 by a 10-fold factor. Mutations within the α interaction domain of Ca_V1.2 or within the nucleotide kinase domain of Ca_Vβ3 disrupted the Ca_Vβ3-induced plasma membrane targeting of Ca_V1.2. Altogether, these data support a model where high affinity binding of Ca_Vβ to the I-II linker of Ca_Vα1 largely accounts for Ca_Vβ-induced plasma membrane targeting of Ca_V1.2.     

Bioacoustics for species management: two case studies with a Hawaiian forest bird

The management of animal endangered species requires detailed information on their distribution and abundance, which is often hard to obtain. When animals communicate using sounds, one option is to use automatic sound recorders to gather information on the species for long periods of time with low effort. One drawback of this method is that processing all the information manually requires large amounts of time and effort. Our objective was to create a relatively “user‐friendly” (i.e., that does not require big programming skills) automatic detection algorithm to improve our ability to get basic data from sound‐emitting animal species. We illustrate our algorithm by showing two possible applications with the Hawai'i ‘Amakihi, Hemignathus virens virens, a forest bird from the island of Hawai'i. We first characterized the ‘Amakihi song using recordings from areas where the species is present in high densities. We used this information to train a classification algorithm, the support vector machine (SVM), in order to identify ‘Amakihi songs from a series of potential songs. We then used our algorithm to detect the species in areas where its presence had not been previously confirmed. We also used the algorithm to compare the relative abundance of the species in different areas where management actions may be applied. The SVM had an accuracy of 86.5% in identifying ‘Amakihi. We confirmed the presence of the ‘Amakihi at the study area using the algorithm. We also found that the relative abundance of ‘Amakihi changes among study areas, and this information can be used to assess where management strategies for the species should be better implemented. Our automatic song detection algorithm is effective, “user‐friendly” and can be very useful for optimizing the management and conservation of those endangered animal species that communicate acoustically.     

Sorting protein-related receptor SorLA controls regulated secretion of glial cell line-derived neurotrophic factor

Glial cell line-derived neurotrophic factor (GDNF), after secreted from cells, plays a critical role in central and peripheral neuron survival and function. The secretion of GDNF can be either constitutive or regulated by physiological stimuli; however, the detailed mechanism driving GDNF secretion is still unknown. Here, we report that sorting protein-related receptor with A-type repeats (SorLA), a member of the mammal Vps10p domain receptor, interacts with GDNF and is localized to GDNF-containing vesicles. Overexpression of SorLA significantly increases, and knockdown of SorLA by siRNA decreases, the regulated secretion of GDNF in PC12 and MN9D cells but has no effect on GDNF constitutive secretion. In addition, overexpression of a truncated form of SorLA also impairs GDNF-regulated secretion. Finally, we found that the prodomain of GDNF mediates the interaction of GDNF with SorLA under acidic conditions. Moreover, overexpression of SorLA could enhance the regulated secretion of the GDNF prodomain-GFP fusion protein, suggesting that the prodomain of GDNF is responsible for its regulated secretion. Together, these findings will advance our understanding of the molecular mechanism underlying GDNF-regulated secretion.     

Validation of an algorithm for automatic quantification of nucleic acid copy numbers by real-time polymerase chain reaction

Real-time quantitative polymerase chain reaction (PCR) with on-line fluorescence detection has become an important technique not only for determination of the absolute or relative copy number of nucleic acids but also for mutation detection, which is usually done by measuring melting curves. Optimum assay conditions have been established for a variety of targets and experimental setups, but only limited attention has been directed to data evaluation and validation of the results. In this work, algorithms for the processing of real-time PCR data are evaluated for several target sequences (p53, IGF-1, PAI-1, Factor VIIc) and compared to the results obtained by standard procedures. The algorithms are implemented in software called SoFAR, which allows fully automatic analysis of real-time PCR data obtained with a Roche LightCycler instrument. The software yields results with considerably increased precision and accuracy of quantifications. This is achieved mainly by the correction of amplification-independent signal trends and a robust fit of the exponential phase of the signal curves. The melting curve data are corrected for signal changes not due to the melting process and are smoothed by fitting cubic splines. Therefore, sensitivity, resolution, and accuracy of melting curve analyses are improved.     

Molecular and structural analysis of C4-specific PEPC isoform from Pennisetum glaucum plays a role in stress adaptation

As an activator of adenylate cyclase, the neuropeptide Pituitary Adenylate Cyclase Activating Peptide (PACAP) impacts levels of cyclic AMP, a key second messenger available in brain cells. PACAP is involved in certain adult behaviors. To elucidate PACAP interactions, a compendium of microarrays representing mRNA expression in the adult mouse whole brain was pooled from the Phenogen database for analysis. A regulatory network was computed based on mutual information between gene pairs using gene expression data across the compendium. Clusters among genes directly linked to PACAP, and probable interactions between corresponding proteins were computed. Database “experts” affirmed some of the inferred relationships. The findings suggest ADCY7 is probably the adenylate cyclase isoform most relevant to PACAP's action. They also support intervening roles for kinases including GSK3B, PI 3-kinase, SGK3 and AMPK. Other high-confidence interactions are hypothesized for future testing. This new information has implications for certain behavioral and other disorders.     

In silico structural, functional and pathogenicity evaluation of a novel mutation: an overview of HSD3B2 gene mutations

Mutations of 3 beta hydroxysteroid dehydrogenase type II (HSD3B2) gene result in different clinical consequences. We explain a patient who demonstrated a salt wasting form of 3βHSD deficiency in infancy. Signs of hyponatremia and hyperkalemia were recognized in the infant with ambiguous genitalia and perineal hypospadias. The 46,XY male was genotyped by direct sequencing of HSD3B2 gene. Steroid profiles showed elevated concentration of 17 hydroxyprogesterone, and decrease in concentration of cortisol, and testosterone. Dehydroepiandrotone (DHEA) to androstenedione ratio had 6 fold increases. Direct sequencing of the patient revealed homozygous missense A82P mutation in exon 3. This mutation was confirmed by segregation analysis of the parents. Bioinformatic tools were used for in silico structural and functional analyses. Also, the pathological effect of the mutation was validated by different software. Alanine is a conserved amino acid in the membrane binding domain of the enzyme and proline substitution was predicted to destabilize the protein. This report may highlight the importance of the screening programs of the disorder in Iran.     

Diverse effects of phospholipids on lipoprotein sorting and ATP hydrolysis by the ABC transporter LolCDE complex

The LolCDE complex of Escherichia coli releases outer membrane-specific lipoproteins from the inner membrane. Lipoproteins with Asp at + 2 remain in the inner membrane since this residue functions as a LolCDE avoidance signal depending on phosphatidylethanolamine. We examined the effects of other phospholipids on lipoprotein sorting in proteoliposomes reconstituted with LolCDE and various synthetic phospholipids. The lipoprotein release and ATP hydrolysis were both low at 2 mM Mg²⁺ but very high at 10 mM Mg²⁺ in proteoliposomes containing cardiolipin alone. However, the Lol avoidance function was abolished at 10 mM Mg²⁺, and the release of lipoproteins with Asp at + 2 was as efficient as that of outer membrane-specific lipoproteins. The addition of phosphatidylethanolamine to cardiolipin stimulated the ATP hydrolysis and increased the Lol avoidance function of Asp at + 2 at 2 mM Mg²⁺. The addition of phosphatidylglycerol to cardiolipin nearly completely inhibited the release of lipoproteins with Asp at + 2 even at 10 mM Mg²⁺, while that of outer membrane-specific lipoproteins was not. Taken together, these results indicate that three major phospholipids of E. coli differently affect lipoprotein sorting and the activity of LolCDE.