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Nucleic Acid-Based, mRNA-Targeted Therapeutics for Hematologic Malignancies
Published in Gertjan J. L. Kaspers, Bertrand Coiffier, Michael C. Heinrich, Elihu Estey, Innovative Leukemia and Lymphoma Therapy, 2019
It is straightforward that without the ability to deliver material into cells, even the most cleverly designed molecule cannot be effective. As a general rule, oligonucleotides are taken up primarily through a combination of adsorptive and fluid phase endocytosis (62). After internalization, confocal and electron microscopy studies have indicated that the bulk of the oligonucleotides enter the endosome or lysosome compartment where most of the material either becomes trapped or degraded. Biological inactivity is the predictable result of these events. Nevertheless, oligonucleotides can escape from the vesicles intact, enter the cytoplasm, and then diffuse into the nucleus where they presumably acquire their mRNA target. Colocalization of effector strand and target mRNA in nucleoli (28) or cytoplasmic P-bodies (63) appears important for antisense oligonucleotide (AS ON) and siRNA, respectively. In our hands and those of others (64), lipid transfecting agents have proven toxic to hematopoietic cells. Accordingly, we have begun to develop alternate means for delivering AS ON and siRNAs to hematopoietic cells including the use of electroporation for ex vivo delivery (65). We, as are others, are also exploring the possibility of using virosomes (reconstituted viral envelopes) whose original DNA or RNA viruses have been replaced with ASNA (66), ovarious depot or protecting agents such as chitosan polymers (67,68) for systemic delivery. The subject of nucleic acid delivery to living cells has been the focus of numerous recent reviews (69–79). While there are many prospects on the horizon, delivery remains a major roadblock to this form of therapy.
Bunyaviruses
Published in Sunit K. Singh, Daniel Růžek, Neuroviral Infections, 2013
Patrik Kilian, Vlasta Danielová, Daniel Růžek
Once RNP is released into the cytoplasm, the panhandle structure of the RNA is relaxed and the N proteins dissociate from the 3′ terminus of the RNA. Primary transcription of the vRNA to mRNA occurs in the cytoplasm through the cap-snatch mechanism The endonuclease activity of the L protein is responsible for cleavage of the 5′ cap together with several nucleotides from selected host mRNAs (Figure 4.2b). The cleaved cap subsequently associates with the vRNA, and this initiates synthesis of the complementary strand. The viral protein N plays an important role in this process as it serves as a cap binding protein (similar to the eukaryotic eIF4E), and helps the L protein to cleave host mRNA (Panganiban and Mir 2009). Some specific host mRNAs are cleaved preferentially; for instance, LACY in mosquito cells utilizes preferable mRNA coding for proteins that are similar to apoptosis inhibitors from Drosophila (Borucki et al. 2002). To provide sufficient mRNAs for cleavage, hantaviruses have developed effective strategies for their storage in the cell. After the binding of N protein to the 5′ cap, the mRNAs are translocated to the so-called P-bodies where the mRNA is protected from degradation by binding to a viral nucleocapsid protein (Mir et al. 2008). At the 3′ end of the synthesized RNA, a couple of nucleotides are missed. These missed nucleotides are added by a mechanism called realign, when the L protein slides back to the start of the template vRNA after transcribing several nucleotides (Figure 4.2b) (Jin and Elliott 1993; Garcin et al. 1995). Only vRNA that is associated with an N protein can serve as a template for mRNA transcription (Dunn et al. 1995).
Internalization of trophoblastic small extracellular vesicles and detection of their miRNA cargo in P-bodies
Published in Journal of Extracellular Vesicles, 2020
Hui Li, Itziar Pinilla-Macua, Yingshi Ouyang, Elena Sadovsky, Kazuhiro Kajiwara, Alexander Sorkin, Yoel Sadovsky
We surmised that endosomal escape of cargo might take place prior to sEV degradation in lysosomes and that miRNA would reach the intracellular sites of their biological activity, likely RISC complexes at P-bodies, where miRNAs exhibit their RNA degradation or translational inhibition activities. As miRNA-517a is one of the most abundant C19MC miRNAs in trophoblastic sEVs, we tested whether this miRNA co-localizes with P-body proteins AGO2 and GW182. Immunofluorescence labelling of GW182 in PPF cells stably co-expressing FLAG-HA-AGO2 and fluorescently tagged ss-miR-517, allowed the direct comparison of subcellular localization of miR-517 and P-bodies (Figure 7a,b). Image analysis showed that FLAG-HA-AGO2 and endogenous GW182 were highly co-localized in relatively uniform puncta that were distributed throughout the cytoplasmic P-bodies, although few puncta containing a single marker were detected as well. A fraction of transfected ss-miR-517-A488 was co-localized with P-bodies while a substantial amount of this miRNA was seen in the cytoplasm and nucleus (Figure 7a,b). Furthermore, we showed that after UV-crosslinking of PPF cells expressing FLAG-HA-AGO2 and transfected with ss-miRNA-517a, HA antibody immunoprecipitates contained an increased level of miRNA-517a when compared to immunoprecipitates using non-specific IgG (Figure 8a).
Synergies of accelerating differentiation of bone marrow mesenchymal stem cells induced by low intensity pulsed ultrasound, osteogenic and endothelial inductive agent
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Ruixin He, Junlin Chen, Jingwei Jiang, Baoru Liu, Dandan Liang, Weichen Zhou, Wenzhi Chen, Yan Wang
The myeloid bodies, dilated endoplasmic reticulum, and matrix vesicles in BMSCs were observed in the cells of the bidirectional induction group under the electron microscope, indicating that the cells were differentiated into osteoblasts. Meanwhile, W-P bodies and desmosomes etc. were found in the same specimen (Figure 4), suggesting that the cells were differentiated into vascular endothelium and formed microvessels at the early angiogenesis stage. The confocal microscope images also showed that cell accumulation and red "ring-like" structures appeared at the 13th day in the bidirectional induction group (Figure 5). The cell accumulation was the mineralized bone matrix, while the red "ring-like" structures might be new microvessels. These results agree with the reported results that biomarkers of osteoblasts and vascular endothelial cells [27,28] appeared when BMSCs were induced to differentiate alone, confirming our hypothesis that construction of a bidirectional induction cell culture medium for BMSCs culture can achieve bidirectional differentiation simultaneously. In the fluorescence quantitative PCR experiment, the expression peak of osteocalcin in the bidirectional induction group was detected on day 9 and that of vWF was detected on day 13 (Figure 8). This finding is consistent with the laser confocal microscope results, indicating osteogenic differentiation may be earlier than vascular endothelial differentiation in the bidirectional induction of BMSCs. Previously, investigators have constructed animal models of bone defects that deliver osteogenesis-inducing agents and vascular endothelial-inducing agents simultaneously or sequentially to animals [29,30]. However, incubating BMSCs with two inducing agents simultaneously has not been reported. The results of this study can further explain the cytological mechanisms of these animal experiments.
Characteristics of circRNA and its approach as diagnostic tool in melanoma
Published in Expert Review of Molecular Diagnostics, 2021
Khatereh Khorsandi, HomaSadat Esfahani, Heidi Abrahamse
CircRNAs tend to be not digested by RNase R due to its particular structural conformations. But, it seems that the linear RNAs are entirely digested [21]. Studies have shown that there are various cleavage pathways for circRNA. Besides the RNase P/MRP complexes, which is defined as a main circRNAs degradation mechanism by m6A modification, another removal machinery is also known [22]. When circRNAs acted as PRK (double-stranded RNA activated protein kinase) blocker, circRNA degradation upon viral infection or poly, I: C stimulation occurs under endoribonuclease RNase L activation. They perform critical roles in the initiation of innate immunity. Also, RNA interference (RNAi) can degrade circRNA. Argonaute2 (AGO2) was identified as part of the Argonaute protein family. They are a substantial mediator for the degradation of CDR1as (or ciRS-7) and are vital for RNAi [23,24]. CDR1as shows a near perfect complementarity manner with miR-671, which can trigger AGO2-dependent cleavage. miR-7 by increasing the slicing of CDR1AS causes the improvement in this cleavage [24,25]. However, many circRNAs possess a few or even no miRNA target sites, so they do not show properties of miRNA sponges [26,27]. Recent studies have indicated that in both drosophila and human cell lines, GW182 that is often associated with the P-bodies is linked to circRNA degradation and stability but independent of P-body or RNA interference (RNAi) path. Conversely, there is a possibility that circRNA has digested by GW182 via its mid-domain [28]. The investigation established that to decrease intracellular circRNA abundance, the cell makes use of a strategy to secrete circRNA into an extracellular vesicle (EVs) [29]. On the other hand, it is claimed that circRNAs selectively transported into EVs and then delivered into recipient cells to apply its effects. Consequently, circRNAs exhibit a new way of promoting cell communication via the exchange of information between cells [30].