The Characteristics Of Human Bone-Derived Cells (HBDCS) during osteogenesis in vitro

Background

The primary human bone-derived cell culture technique is used as a model to study human osteogenesis. Compared to cell line cultures, primary osteoprogenitor and osteoblast cultures provide more complex information about osteogenesis, bone remodeling and regeneration than cell line cultures.

Methods

In this study, we isolated human bone-derived cells (HBDCs) and promoted their differentiation into osteoblasts. The following parameters were evaluated: cell number and viability, total protein expression, alkaline phosphatase activity, collagenous matrix production and osteogenic genes expression, i.e., gene coding for type I collagen and alkaline phosphatase.

Results

It was proved the results show that HBDCs intensively proliferate during the first 7 days of culture followed by differentiation accompanied by an increase in alkaline phosphatase activity. Moreover, it was observed that during the differentiation of HBDCs, the expression of integrin β1 increased.

Conclusions

The process was also accompanied by changes in cell shape and rearrangement of the actin cytoskeleton and focal contacts containing FAK and the integrin β1 subunit. We suggest that the β1 integrin subunit may be a suitable new target in studies of the differentiation of primary human osteoblasts in culture.

     

Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy

Autophagy is an intracellular trafficking pathway sequestering cytoplasm and delivering excess and damaged cargo to the vacuole for degradation. The Atg1/ULK1 kinase is an essential component of the core autophagy machinery possibly activated by binding to Atg13 upon starvation. Indeed, we found that Atg13 directly binds Atg1, and specific Atg13 mutations abolishing this interaction interfere with Atg1 function in vivo. Surprisingly, Atg13 binding to Atg1 is constitutive and not altered by nutrient conditions or treatment with the Target of rapamycin complex 1 (TORC1)-inhibitor rapamycin. We identify Atg8 as a novel regulator of Atg1/ULK1, which directly binds Atg1/ULK1 in a LC3-interaction region (LIR)-dependent manner. Molecular analysis revealed that Atg13 and Atg8 cooperate at different steps to regulate Atg1 function. Atg8 targets Atg1/ULK1 to autophagosomes, where it may promote autophagosome maturation and/or fusion with vacuoles/lysosomes. Moreover, Atg8 binding triggers vacuolar degradation of the Atg1-Atg13 complex in yeast, thereby coupling Atg1 activity to autophagic flux. Together, these findings define a conserved step in autophagy regulation in yeast and mammals and expand the known functions of LIR-dependent Atg8 targets to include spatial regulation of the Atg1/ULK1 kinase.     

Molecular and phenotypic characterisation of Bacillus thuringiensis isolated during epizootics in Cydia pomonella L

Twelve Bacillus thuringiensis strains were isolated from intestinal tracts of Cydia pomonella larvae during epizootics in different laboratory insect culture lines. Phenotypic and genetic similarity of these isolates, together with two cultured from Leucoma salicis larvae and 14 reference B. thuringiensis strains were determined. The epizootic bacteria did not form a single group based on numerical analysis of biochemical properties. Simple RAPD method with only one primer does not allow estimating the genetic similarity of B. thuringiensis strains. We propose a novel strategy based on combining several DNA patterns obtained by RAPD technique with different primers for B. thuringiensis typing. Majority of infections in the C. pomonella culture lines were caused by bacteria with different genotypes. However, two isolates cultured from infected insects at different time (one strain was isolated in 1990 and the other in 1992) had identical DNA fingerprint that suggested a possibility of these bacteria to survive in the laboratory and to cause infections in different years. The results of SDS-PAGE of whole-cell proteins revealed a possibility to apply protein profile analysis in epidemiological investigations of infections caused by B. thuringiensis. Strains with identical DNA patterns had very similar whole-cell protein profiles.     

Prediction and preliminary validation of oncogene regulation by miRNAs

Background  

MicroRNAs (miRNAs) are one of the most abundant groups of regulatory genes in multicellular organisms, playing important roles in many fundamental cellular processes. More than four hundred miRNAs have been identified in humans and the deregulation of miRNA expression has been also shown in many cancers. Despite the postulated involvement of miRNAs in tumourigenesis, there are only a few examples where an oncogene or a tumour suppressor has been identified as a miRNA target.     

The role of Dicer protein partners in the processing of microRNA precursors

One of the cellular functions of the ribonuclease Dicer is to process microRNA precursors (pre-miRNAs) into mature microRNAs (miRNAs). Human Dicer performs this function in cooperation with its protein partners, AGO2, PACT and TRBP. The exact role of these accessory proteins in Dicer activity is still poorly understood. In this study, we used the northern blotting technique to investigate pre-miRNA cleavage efficiency and specificity after depletion of AGO2, PACT and TRBP by RNAi. The results showed that the inhibition of either Dicer protein partner substantially affected not only miRNA levels but also pre-miRNA levels, and it had a rather minor effect on the specificity of Dicer cleavage. The analysis of the Dicer cleavage products generated in vitro revealed the presence of a cleavage intermediate when pre-miRNA was processed by recombinant Dicer alone. This intermediate was not observed during pre-miRNA cleavage by endogenous Dicer. We demonstrate that AGO2, PACT and TRBP were required for the efficient functioning of Dicer in cells, and we suggest that one of the roles of these proteins is to assure better synchronization of cleavages triggered by two RNase III domains of Dicer.     

基于Nextflow构建的宏条形码自动化分析流程EPPS

基于宏条形码技术的物种快速检测有助于生物多样性的评估、预测和保护。本文介绍了常用宏条形码分析的步骤和参数设定方法。我们利用Nextflow搭建了一款宏条形码分析流程EPPS, 可以自动化地运行从原始数据的质量控制到环境多样性的比较。Nextflow软件还拥有流程监控的功能, 可视化输出每个进程所消耗的时间与内存。本文还使用测试数据和已发表数据证明该平台能够有效地分析宏条形码数据并可靠地分析环境生物多样性的相似性。     

SIRT1 shows no substrate specificity in vitro

SIR2 is a key regulator of the aging process in many model organisms. The human ortholog SIRT1 plays a pivotal role in the regulation of cellular differentiation, metabolism, cell cycle, and apoptosis. SIRT1 is an NAD(+)-dependent deacetylase, and its enzymatic activity may be regulated by cellular energy. There is a growing number of known SIRT1 substrates that contain epsilon-acetyl lysine but for which no obvious consensus sequence has been defined. In this study, we developed a novel unbiased method to identify deacetylase sequence specificity using oriented peptide libraries containing acetylated lysine. Following incubation with SIRT1, the subset of deacetylated peptides was selectively captured using a photocleavable N-hydroxysuccinimide (NHS)-biotin linker and streptavidin beads and analyzed using mass spectrometry and Edman degradation. These studies revealed that substrate recognition by SIRT1 does not depend on the amino acid sequence proximate to the acetylated lysine. This result brings us one step closer to understanding how SIRT1 and possibly other protein deacetylases chose their substrate.     

Microbial transformations of flavanone and 6-hydroxyflavanone by Aspergillus niger strains

Flavanone (1) and 6-hydroxyflavanone (2) were subjected to transformation by means of Aspergillus niger strains (one wild and three UV mutants). For both substrates the biotransformation resulted in reduction of the carbonyl group (products 5 and 7) and dehydrogenation at C-2 and C-3 (3 and 8). Additionally, for flavanone (1) reduction of C-4 together with hydroxylation at C-7 (6) and dehydrogenation at C-2, C-3 along with hydroxylation at C-3 (4) were observed.