Molecular Determinants for RNA Release into Extracellular Vesicles

Extracellular vesicles (EVs) are important for intercellular communication and act as vehicles for biological material, such as various classes of coding and non-coding RNAs, a few of which were shown to selectively target into vesicles. However, protein factors, mechanisms, and sequence elements contributing to this specificity remain largely elusive. Here, we use a reporter system that results in different types of modified transcripts to decipher the specificity determinants of RNAs released into EVs. First, we found that small RNAs are more efficiently packaged into EVs than large ones, and second, we determined absolute quantities for several endogenous RNA transcripts in EVs (U6 snRNA, U1 snRNA, Y1 RNA, and GAPDH mRNA). We show that RNA polymerase III (pol III) transcripts are more efficiently secreted into EVs compared to pol II-derived transcripts. Surprisingly, our quantitative analysis revealed no RNA accumulation in the vesicles relative to the total cellular levels, based on both overexpressed reporter transcripts and endogenous RNAs. RNA appears to be EV-associated only at low copy numbers, ranging between 0.02 and 1 molecule per EV. This RNA association may reflect internal EV encapsulation or a less tightly bound state at the vesicle surface.

Osteoblast iron genes: real time PCR and microarray hybridization approach under hyperoxia

Objectives Iron is essential for cell growth, differentiation, electron transfer, and oxygen transport. Hyperoxia may increase the turnover of bone matrix components with a net effect of accelerated bone growth. Although hyperoxia was claimed could increase osteoblast activity, but expression level in possible genes which play role in proliferation is still unclear. This research aims to prove the differences of expression level of transferrin receptor gene and iron regulated transporter and other genes of 7F2 under 24 h normoxia, 24 h hyperoxia, and 48 h hyperoxia and the effect of hyperoxia by using osteoblast cell culture 7F2. Methods Reverse transcriptase, real time Polymerase Chain Reaction (PCR), and microarray is used to qualitatively detect gene expression. The computer softwares such as National Center for Biotechnology Information (NCBI) data base, Software Affymetrix, DNA Strider program, Genomatix – DiAlign program, Oligo 5.0 program (Software primer design) from Wojciech & Piotr Rychlik, and Genetyx-Mac version 8.0 have been used to analyze the PCR result. Results Under 24 h hyperoxia, there were 3,884 copies of transferrin receptor mRNA per 1,000,000 copies of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. After 24 h hyperoxia, 8,325 copies of transferrin receptor mRNA per 1,000,000 GAPDH mRNA copies were found showing 2.1-fold up regulation. After 48 h hyperoxia, there was no significant increase at the level of expression of transferrin receptor mRNA, 8,079 mRNA copies per 1,000,000 copies of mRNA were found (2.0-fold up regulation compared with 24 h normoxia). Conclusions It can be concluded that hyperoxia might have an effect on upregulating the expression of some osteoblast genes which might have an impact on osteoblast activity.

Complexation design of cationized gelatin and molecular beacon to visualize intracellular mRNA

The objective of this study is to prepare cationized gelatin-molecular beacon (MB) complexes for the visualization of intracellular messenger RNA (mRNA). The complexes were prepared from cationized gelatins with different extents of cationization and different mixing ratios of MB to cationized gelatin. The apparent size of complexes was almost similar, while the zeta potential was different among the complexes. Irrespective of the preparation conditions, the complexes had a sequence specificity against the target oligonucleotides in hybridization. The cytotoxicity and the amount of complexes internalized into cells increased with an increase in the cationization extent and the concentration of cationized gelatin. After the incubation with complexes prepared from cationized gelatin with the highest extent of cationization and at mixing ratios of 10 and 20 pmole MB/μg cationized gelatin, a high fluorescent intensity was detected. On the other hand, the complex prepared with the mixing ratio at 20 pmole/μg did not show any cytotoxicity. The complex was the most effective to visualize the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA endogenously present. In addition, even for enhanced green fluorescent protein (EGFP) mRNA exogenously transfected, the complex permitted to effectively detect it as well. It is concluded that both the endogenous and exogenous mRNA can be visualized in living cells by use of cationized gelatin-MB complexes designed.

Sekuen DNA Parsial Dari Gen GAPDH Pada Sirsak (Annona muricata L.)

AbstrakGen glyceraldehyde-3-phosphate dehydrogenase (GAPDH) merupakan salah satu gen referensi yang sering bertindak sebagai kontrol internal pada analisis ekspresi gen di beberapa spesies tumbuhan. Penelitian ini bertujuan menganalisis sekuen gen GAPDH parsial pada sirsak (Annona muricata L.). Metode meliputi persiapan sampel tanaman, isolasi DNA total menggunakan Genomic DNA mini kit Plant (Geneaid), amplifikasi gen GAPDH dengan teknik polymerase chain reaction (PCR), elektroforesis pada 1% gel agarose dan analisis data sekuen DNA. Studi ini telah memperoleh sekuen DNA dari gen GAPDH parsial sirsak sepanjang 961 pb. Sekuen tersebut memiliki kemiripan sekitar 68,93–84,35% dengan sekuen mRNA gen GAPDH pada beberapa spesies tumbuhan. Sekuen ini diprediksi terdiri dari 5 ekson dan 4 intron. Total ekson diprediksi terdiri dari 429 pb. Sekuen ini adalah yang pertama kali dilaporkan dari genus Annona dan juga dari famili Annonaceae. Sekuen ini dapat dimanfaatkan untuk analisis ekspresi gen pada sirsak dan dapat menjadi dasar untuk mengisolasi gen GAPDH spesies lain di dalam genus Annona dan famili Annonaceae. Abstract GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene is one of reference genes that is frequently became an internal control in any plant species. This study reports a DNA sequence of parsial GAPDH gene on soursop (Annona muricata L.). Methods included sample preparation, total DNA isolation using Genomic DNA mini kit Plant (Geneaid), amplification of GAPDH gene using PCR (polymerase chain reaction) technique, electrophoresis using 1% agarose gel and data analysis. This study had been obtained the DNA sequence of soursop partial GAPDH gene sizing 961 bp. The sequence had 68.93–84.35% similarity to GAPDH mRNA of some plants species. The soursop partial GAPDH gene was predicted consisting of 5 exons and 4 introns. The total exons length was 429 bp. The sequence is the first reported from Annona genus and also Annonaceae family. The sequence can be used for gene expression in soursop and also can be used to isolate GAPDH gene of other species in Annona genus and Annonaceae family.

Aberrant Expression of Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) in Warthin Tumors

The Warthin tumor represents the second most frequent benign tumor of the parotid gland and is characterized by the presence of oncocytes rich in structurally and functionally altered mitochondria. Next to its role in metabolism, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also implicated in cellular mitophagy. Immunohistochemistry was carried out on Warthin tumor and normal control (parotid gland with striated ducts) tissues, using anti-GAPDH specific antibodies followed by digital image analysis. Laser capture microdissection was used to isolate the oncocytic tumor cell and normal control striated duct compartments for RNA extraction and qPCR. Warthin tumor oncocytes exhibited a markedly spotted GAPDH staining pattern exhibiting cells with cytoplasmic and nuclear, only nuclear or none GAPDH staining. A significantly lower (p < 0.0001) total GAPDH signal was detected in Warthin tumor oncocytes. Similarly, significantly lower (p < 0.005) GAPDH mRNA levels were seen in oncocytes compared with normal ductal cells. To exclude the possibility of this GAPDH staining pattern being a general feature of oncocytic neoplasms of different organs, we tested a cohort of renal oncocytoma and oncocytic chromophobe carcinoma; none showed this type of staining. The observed progressive GAPDH loss in Warthin tumor oncocytes could be implicated in the pathogenesis of Warthin tumors.

Comparison between Lipofectamine RNAiMAX and GenMute transfection agents in two cellular models of human hepatoma

RNA interference is a powerful approach to understand gene function both for therapeutic and experimental purposes. Since the lack of knowledge in the gene silencing of various hepatic cell lines, this work was aimed to compare two transfection agents, the liposome-based Lipofectamine™ RNAiMAX and the HepG2-specific, polymer-based GenMute™, in two cellular models of human hepatoma, HepG2 and Huh7.5. In the first part, we assessed transfection efficiency of a fluorescent Cy3-labeled negative control siRNA by cell imaging analysis; we found that cells treated with GenMute present a higher uptake of the fluorescent negative control siRNA when compared to Lipofectamine RNAiMAX-transfected cells, both in HepG2 and in Huh7.5 cells. In the second part, we evaluated GAPDH silencing with the two transfection reagents by RT-PCR similar GAPDH mRNA expression after each transfection treatment. Finally, we measured cell viability by the MTT assay, observing that cells transfected with GenMute have higher viability with respect to Lipofectamine RNAiMAX-administered cells. These results suggest that GenMute reagent might be considered the most suitable transfection agent for hepatic gene silencing.

RANKL EXPRESSION AND METABOLIC CHANGES IN OVARIECTOMIZED RATS AND THE POSSIBLE PROTECTION BY VEGETABLE FORMULA.

Objective: This study was to evaluate complications of osteoporosis in ovariectomized rats and the possibility to ameliorate these changes by consumption of vegetable formula. Furthermore, transcription of mRNA of RANKL gene was matched with bone mass density (BMD) and bone formation marker (human procollagen 1 N terminal peptide [PINP]).Methods: Thirty rats were divided into three groups. The first is non ovariectomized control group (NOVXC), the second is ovariectomized control group (OVXC), and the third is ovariectomized rats supplemented with the vegetable formula (OVXT). Animals were fed for 8 successive weeks. Animals were treated and sacrificed under the recommended ethics of laboratory animal’s treatment. The vegetable mixture was formulated with the purpose to correct the bone compromise and supply all the presumed deficient elements and hormone.Results: Chemical analysis showed that the formulated vegetable mixture had a high amount of flavonoids as catechin (100 mg/100 g of dry weight) and polyphenols as tannic acid (1000 mg/100 g dry weight. Furthermore, it had high reducing power (1,1-diphenyl-2-picrylhydrazyl radical showed inhibition percentage of 91.81%.). Several phytochemicals necessary for bone health were demonstrated in the vegetable mixture using high-performance liquid chromatography. RANKL/GAPDH mRNA transcription ratio showed marked an increase in OVXC versus the control NOVXC rats (1.00 vs. 0.199, respectively) accompanied with a drop in BMD (0.157 vs. 0.25 mg/cm2, respectively) and PINP values (27.9±2.8 compared to NOVXC 34±2.4 μ/L, respectively). The vegetable mixture supplementation showed better values of BMD and PINP in OVXT group directed back toward normal (0.183 mg/cm2 and 29.35±3.4 μ/L, respectively). Furthermore, analysis of blood plasma of supplemented group showed lower blood glucose, lipid profile, and oxidative markers if compared to that in OVXC group.Conclusion: It may be concluded that the plant formula was effective to minimize health hazards in ovariectomized rats and maybe for postmenopause women. Perhaps longer time may be needed for more significant and clear effect.

RANKL EXPRESSION AND METABOLIC CHANGES IN OVARIECTOMIZED RATS AND THE POSSIBLE PROTECTION BY VEGETABLE FORMULA.

Objective: This study was to evaluate complications of osteoporosis in ovariectomized rats and the possibility to ameliorate these changes by consumption of vegetable formula. Furthermore, transcription of mRNA of RANKL gene was matched with bone mass density (BMD) and bone formation marker (human procollagen 1 N terminal peptide [PINP]).Methods: Thirty rats were divided into three groups. The first is non ovariectomized control group (NOVXC), the second is ovariectomized control group (OVXC), and the third is ovariectomized rats supplemented with the vegetable formula (OVXT). Animals were fed for 8 successive weeks. Animals were treated and sacrificed under the recommended ethics of laboratory animal’s treatment. The vegetable mixture was formulated with the purpose to correct the bone compromise and supply all the presumed deficient elements and hormone.Results: Chemical analysis showed that the formulated vegetable mixture had a high amount of flavonoids as catechin (100 mg/100 g of dry weight) and polyphenols as tannic acid (1000 mg/100 g dry weight. Furthermore, it had high reducing power (1,1-diphenyl-2-picrylhydrazyl radical showed inhibition percentage of 91.81%.). Several phytochemicals necessary for bone health were demonstrated in the vegetable mixture using high-performance liquid chromatography. RANKL/GAPDH mRNA transcription ratio showed marked an increase in OVXC versus the control NOVXC rats (1.00 vs. 0.199, respectively) accompanied with a drop in BMD (0.157 vs. 0.25 mg/cm2, respectively) and PINP values (27.9±2.8 compared to NOVXC 34±2.4 μ/L, respectively). The vegetable mixture supplementation showed better values of BMD and PINP in OVXT group directed back toward normal (0.183 mg/cm2 and 29.35±3.4 μ/L, respectively). Furthermore, analysis of blood plasma of supplemented group showed lower blood glucose, lipid profile, and oxidative markers if compared to that in OVXC group.Conclusion: It may be concluded that the plant formula was effective to minimize health hazards in ovariectomized rats and maybe for postmenopause women. Perhaps longer time may be needed for more significant and clear effect.