Trehalose Promotes Clearance of Proteotoxic Aggregation of Neurodegenerative Disease-Associated Aberrant Proteins.

Accumulation of misfolded proteins compromises overall cellular health and fitness. The failure to remove misfolded proteins is a critical reason for their unwanted aggregation in dense cellular protein pools. The accumulation of various inclusions serves as a clinical feature for neurodegenerative diseases. Previous findings suggest that different cellular compartments can store these abnormal inclusions. Studies of transgenic mice and cellular models of neurodegenerative diseases indicate that depleted chaperone capacity contributes to the aggregation of damaged or aberrant proteins, which consequently disturb proteostasis and cell viability. However, improving these abnormal proteins' selective elimination is yet to be well understood. Still, molecular strategies that can promote the effective degradation of abnormal proteins without compromising cellular viability are unclear. Here, we reported that the trehalose treatment elevates endogenous proteasome levels and enhances the activities of the proteasome. Trehalose-mediated proteasomal activation elevates the removal of both bona fide misfolded and various neurodegenerative disease-associated proteins. Our current study suggests that trehalose may retain a proteasome activation potential, which seems helpful in the solubilization of different mutant misfolded proteins, improving cell viability. These results reveal a possible molecular approach to reduce the overload of intracellular misfolded proteins, and such cytoprotective functions may play a critical role against protein conformational diseases.

Significance of TLR2 signaling during megakaryocyte development: regulatory cross-talk of miR-125b, cytokine induction, and MAPK pathway during dengue infection.

OBJECTIVE: Dengue is a viral infection endemic in more than 100 countries as per the WHO reports with approximately 5.2 million patients worldwide that spreads from mosquitoes to humans. Severe form of dengue fever can cause serious bleeding (low platelets) and death. Megakaryocytes are the immune cells responsible for the production of platelets. The molecular drivers behind platelet defects are mostly ambiguous. Here, we attempted to understand the distinct pathogen-elicited toll-like receptors (TLRs) functions in megakaryocyte biology. To understand the TLR induction and the molecular events that are governed in the mammalian system during dengue infection and to study TLR2-mediated cellular signaling-associated mechanisms with respect to their dimerization partners during dengue infection. METHODS: In this study, we used the human Megakaryoblastic cells, DAMI, and treated them with TLR agonists (LPS and Zymosan) and Dengue virus (DNV-II). RESULTS AND DISCUSSION: TLR2 could play an important role by dimerizing with TLR1, TLR4, and TLR6, which we induced for functional characterization. We observed that megakaryocyte maturation markers CD-41 and CD-61 were elevated. This augmentation under the LPS and Zymosan system along with DNV Infection was further confirmed. Our analysis also suggested that activation of miR-125b and MAPK signaling led to lipid droplet elevation. This led us to analyze TLR-mediated consequences and their impact on megakaryocyte development under diverse pathogen-elicited conditions. CONCLUSION: Pathogenic challenges associated with toll-like receptor system activation could further our understanding of the platelet biogenesis mechanistic pathways under various pathogenic circumstances.

Disturb mitochondrial associated proteostasis: Neurodegeneration and imperfect ageing.

The disturbance in mitochondrial functions and homeostasis are the major features of neuron degenerative conditions, like Parkinson's disease, Amyotrophic Lateral Sclerosis, and Alzheimer's disease, along with protein misfolding. The aberrantly folded proteins are known to link with impaired mitochondrial pathways, further contributing to disease pathogenesis. Despite their central significance, the implications of mitochondrial homeostasis disruption on other organelles and cellular processes remain insufficiently explored. Here, we have reviewed the dysfunction in mitochondrial physiology, under neuron degenerating conditions. The disease misfolded proteins impact quality control mechanisms of mitochondria, such as fission, fusion, mitophagy, and proteasomal clearance, to the detriment of neuron. The adversely affected mitochondrial functional roles, like oxidative phosphorylation, calcium homeostasis, and biomolecule synthesis as well as its axes and contacts with endoplasmic reticulum and lysosomes are also discussed. Mitochondria sense and respond to multiple cytotoxic stress to make cell adapt and survive, though chronic dysfunction leads to cell death. Mitochondria and their proteins can be candidates for biomarkers and therapeutic targets. Investigation of internetworking between mitochondria and neurodegeneration proteins can enhance our holistic understanding of such conditions and help in designing more targeted therapies.

Long Non-Coding RNAs as Cellular Metabolism and Haematopoiesis Regulators.

Long non-coding RNAs (lncRNAs) are a category of non-coding RNAs (ncRNAs) that are more than 200 bases long and play major regulatory roles in a wide range of biologic processes, including hematopoeisis and metabolism. Metabolism in cells is an immensely complex process that involves the interconnection and unification of numerous signaling pathways. A growing body of affirmation marks that lncRNAs do participate in metabolism, both directly and indirectly, via metabolic regulation of enzymes and signaling pathways, respectively. The complexities are disclosed by the latest studies demonstrating how lncRNAs could indeed alter tissue-specific metabolism. We have entered a new realm for discovery that is both intimidating and intriguing. Understanding the different functions of lncRNAs in various cellular pathways aids in the advancement of predictive and therapeutic capabilities for a wide variety of myelodysplastic and metabolic disorders. This review has tried to give an overview of the different ncRNAs and their effects on hematopoiesis and metabolism. We have focused on the pathway of action of several lncRNAs and have also delved into their prognostic value. Their use as biomarkers and possible therapeutic targets has also been discussed. SIGNIFICANCE STATEMENT: This review has tried to give an overview of the different ncRNAs and their effects on hematopoiesis and metabolism. The pathway of action of several lncRNAs and their prognostic value was discussed. Their use as biomarkers and possible therapeutic targets has also been elaborated.

Role of Long Non-Coding RNAs in Human-Induced Pluripotent Stem Cells Derived Megakaryocytes: A p53, HOX Antisense Intergenic RNA Myeloid 1, and miR-125b Interaction Study.

Megakaryocytes (MKs) are rare polyploid cells found in the bone marrow and produce platelets. Platelets are small cell fragments that are essential during wound healing and vascular hemostasis. In vitro differentiation of MKs from human-induced pluripotent stem cell-derived CD34+ hematopoietic stem cells (hiPSC-HSCs) could provide an alternative treatment option for thrombocytopenic patients as a platelet source. In this approach, we developed a method to produce functional MKs from hiPSC-HSCs using a xeno-free and feeder-free condition and minimize the variation and risk from animal-derived products in cell culture. We have also investigated the genome-wide expression as well as functional significance of long noncoding RNAs (lncRNAs) in hiPSC-HSC-derived MKs to get insight into MK biology. We have performed lncRNAs expression profiling by using the Human LncProfilers qPCR Array Kit and identified 26 differentially regulated lncRNAs in hiPSC-HSC-derived MKs as compared with those in hiPSC-HSCs. HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) was the most highly upregulated lncRNA in hiPSC-HSC-derived MKs and phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic-differentiating K562 cells. Furthermore, we have studied the potential mechanism of HOTAIRM1 based on the interactions between HOTAIRM1, p53, and miR-125b in PMA-induced K562 cells. Our results demonstrated that during MK maturation, HOTAIRM1 might be associated with the transcriptional regulation of p53 via acting as a decoy for miR-125b. Thus, the interaction between HOTAIRM1, p53, and miR-125b is likely involved in controlling cell cycling (cyclin D1), reactive oxygen species production, and apoptosis to support terminal maturation of MKs. SIGNIFICANCE STATEMENT: In vitro generation of megakaryocytes (MKs) from human-induced pluripotent stem cell-derived hematopoietic stem cells (hiPSC-HSCs) could provide an alternative source of platelets for treating thrombocytopenic patients. This study has investigated the functional significance of long non-coding RNAs in hiPSC-HSC-derived MKs, which remains unclear. This study's findings suggest that the regulatory role of HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) in p53-mediated regulation of cyclin D1 during megakaryocytopoiesis is to promote MK maturation by decoying miR-125b.

Resveratrol Promotes LRSAM1 E3 Ubiquitin Ligase-Dependent Degradation of Misfolded Proteins Linked with Neurodegeneration.

BACKGROUND/AIMS: Cells require regular maintenance of proteostasis. Synthesis of new polypeptides and elimination of damaged or old proteins is an uninterrupted mechanism essential for a healthy cellular environment. Impairment in the removal of misfolded proteins can disturb proteostasis; such toxic aggregation of misfolded proteins can act as a primary risk factor for neurodegenerative diseases and imperfect ageing. The critical challenge is to design effective protein quality control (PQC) based molecular tactics that could potentially eliminate aggregation-prone protein load from the cell. Still, targeting specific components of the PQC pathway for the suppression of proteotoxic insults retains several challenges. Earlier, we had observed that LRSAM1 promotes the degradation of aberrant proteins. Here, we examined the effect of resveratrol, a stilbenoid phytoalexin compound, treatment on LRSAM1 E3 ubiquitin ligase, involved in the spongiform neurodegeneration. METHODS: In this study, we reported induction of mRNA and protein levels of LRSAM1 in response to resveratrol treatment via RT-PCR, immunoblotting, and immunofluorescence analysis. The LRSAM1-mediated proteasomal-based clearance of misfolded proteins was also investigated via proteasome activity assays, immunoblotting and immunofluorescence analysis. The increased stability of LRSAM1 by resveratrol was demonstrated by cycloheximide chase analysis. RESULTS: Here, we show that resveratrol treatment induces LRSAM1 E3 ubiquitin ligase expression levels. Further, our findings suggest that overexpression of LRSAM1 significantly elevates proteasome activities and improves the degradation of bona fide heat-denatured luciferase protein. Exposure of resveratrol not only slows down the turnover of LRSAM1 but also effectively degrades abnormal proteinaceous inclusions, which eventually promotes cell viability. CONCLUSION: Our findings suggest that resveratrol facilitates LRSAM1 endogenous establishment, which consequently promotes the proteasome machinery for effective removal of intracellular accumulated misfolded or proteasomal-designated substrates. Altogether, our study proposes a promising molecular approach to specifically trigger PQC signaling for efficacious rejuvenation of defective proteostasis via activation of overburdened proteolytic machinery.

Long Non-coding RNA Therapeutics: Recent Advances and Challenges.

The discovery of the roles of RNA other than just as a messenger, such as a ribozyme, and regulatory RNAs, such as microRNA and long noncoding RNAs, is fascinating. RNA is now recognized as an important regulator involved in practically every biological process. Research in the field of non-coding RNAs, specifically microRNAs (miRNAs) and long non-coding RNAs (LncRNAs) have developed immensely over the years. Recent studies identified diverse RNAs, including non-coding RNAs such as LncRNA and their various modes of action in the cells. These RNAs are anticipated to be key targets for the treatment of various diseases since they control a broad array of biological pathways. LncRNA-targeted drug platform delivers the pharmaceutical industry a myriad of opportunities and has the potential to modulate diseases at the genetic level while also overcoming the limitations of inconsistent proteins. This article focuses on the recent advancement as well as the major challenges in the field and describes the various RNA-based therapeutics that alter the quality of healthcare for many diseases and bring personalized medicines to fruition. The article also summarizes RNA-based therapeutics that are undergoing testing in clinical trials or have been granted FDA approval.

Development and Challenges of Diclofenac-Based Novel Therapeutics: Targeting Cancer and Complex Diseases.

Diclofenac is a highly prescribed non-steroidal anti-inflammatory drug (NSAID) that relieves inflammation, pain, fever, and aches, used at different doses depending on clinical conditions. This drug inhibits cyclooxygenase-1 and cyclooxygenase-2 enzymes, which are responsible for the generation of prostaglandin synthesis. To improve current diclofenac-based therapies, we require new molecular systematic therapeutic approaches to reduce complex multifactorial effects. However, the critical challenge that appears with diclofenac and other drugs of the same class is their side effects, such as signs of stomach injuries, kidney problems, cardiovascular issues, hepatic issues, and diarrhea. In this article, we discuss why defining diclofenac-based mechanisms, pharmacological features, and its medicinal properties are needed to direct future drug development against neurodegeneration and imperfect ageing and to improve cancer therapy. In addition, we describe various advance molecular mechanisms and fundamental aspects linked with diclofenac which can strengthen and enable the better designing of new derivatives of diclofenac to overcome critical challenges and improve their applications.

Non-coding RNAs: are they the protagonist or antagonist in the regulation of leukemia?

The idea of functional non-coding RNAs is taking precedence over the previous notion which believed that they only comprise the auxiliary and junk material of the genome. Newer technologies and studies have proven their importance in regulating and affecting several cellular processes. One such area of research wherein their importance has started to take light is in cancer research, particularly leukemia. Myeloid leukemia is a blood malignancy birthed from mutations in hematopoiesis that disable myeloid progenitor cells from proper differentiation. This review will compile the most recent findings regarding the effects of these regulatory non-coding RNAs on the two types of myeloid leukemia. In particular, the effects of circular RNAs, micro RNAs and long non-coding RNAs, on the pathogenesis and proliferation of Acute and Chronic myeloid leukemia will be revealed in a molecular, cellular and prognostic light. The mechanisms of proliferation, gene-to-gene interactions and possible therapeutic effects will also be discussed. Finally, an understanding of the overall "goodness" and "badness" of these non-coding RNAs will be summarised. This review hopes to provide a platform for easy access to data regarding the current non-coding RNAs in myeloid leukemia, for faster and easier research. Finally, the review will summarize a few key players that have protagonistic and antagonistic functions, and those that regulate multiple pathways in leukemia simultaneously.

Megakaryoblastic leukemia: a study on novel role of clinically significant long non-coding RNA signatures in megakaryocyte development during treatment with phorbol ester.

Acute megakaryocytic leukemia (AMKL) is one of the rarest sub-types of acute myeloid leukemia (AML). AMKL is characterized by high proliferation of megakaryoblasts and myelofibrosis of bone marrow, this disease is also associated with poor prognosis. Previous analyses have reported that the human megakaryoblastic cells can be differentiated into cells with megakaryocyte (MK)-like characteristics by phorbol 12-myristate 13-acetate (PMA). However, little is known about the mechanism responsible for regulating this differentiation process. We performed long non-coding RNA (lncRNA) profiling to investigate the differently expressed lncRNAs in megakaryocyte blast cells treated with and without PMA and examined those that may be responsible for the PMA-induced differentiation of megakaryoblasts into MKs. We found 30 out of 90 lncRNA signatures to be differentially expressed after PMA treatment of megakaryoblast cells, including the highly expressed JPX lncRNA. Further, in silico lncRNA-miRNA and miRNA-mRNA interaction analysis revealed that the JPX is likely involved in unblocking the expression of TGF-ß receptor (TGF-ßR) by sponging oncogenic miRNAs (miR-9-5p, miR-17-5p, and miR-106-5p) during MK differentiation. Further, we report the activation of TGF-ßR-induced non-canonical ERK1/2 and PI3K/AKT pathways during PMA-induced MK differentiation and ploidy development. The present study demonstrates that TGF-ßR-induced non-canonical ERK1/2 and PI3K/AKT pathways are associated with PMA-induced MK differentiation and ploidy development; in this molecular mechanism, JPX lncRNA could act as a decoy for miR-9-5p, miR-17-5p, and miR-106-5p, titrating them away from TGF-ßR mRNAs. Importantly, this study reveals the activation of ERK1/2 and PI3K/AKT pathway in PMA-induced Dami cell differentiation into MK. The identified differentially expressed lncRNA signatures may facilitate further study of the detailed molecular mechanisms associated with MK development. Thus, our data provide numerous targets with therapeutic potential for the modulation of the differentiation of megakaryoblastic cells in AMKL.

Endocannabinoid system: Role in blood cell development, neuroimmune interactions and associated disorders.

The endocannabinoid system (ECS) is a complex physiological network involved in creating homeostasis and maintaining human health. Studies of the last 40 years have shown that endocannabinoids (ECs), a group of bioactive lipids, together with their set of receptors, function as one of the most important physiologic systems in human body. ECs and cannabinoid receptors (CBRs) are found throughout the body: in the brain tissues, immune cells, and in the peripheral organs and tissues as well. In recent years, ECs have emerged as key modulators of affect, neurotransmitter release, immune function, and several other physiological functions. This modulatory homoeostatic system operates in the regulation of brain activity and states of physical health and disease. In several research studies and patents the ECS has been recognised with neuro-protective properties thus it might be a target in neurodegenerative diseases. Most immune cells express these bioactive lipids and their receptors, recent data also highlight the immunomodulatory effects of endocannabinoids. Interplay of immune and nervous system has been recognized in past, recent studies suggest that ECS function as a bridge between neuronal and immune system. In several ongoing clinical trial studies, the ECS has also been placed in the anti-cancer drugs spotlight. This review summarizes the literature of cannabinoid ligands and their biosynthesis, cannabinoid receptors and their distribution, and the signaling pathways initiated by the binding of cannabinoid ligands to cannabinoid receptors. Further, this review highlights the functional role of cannabinoids and ECS in blood cell development, neuroimmune interactions and associated disorders. Moreover, we highlight the current state of knowledge of cannabinoid ligands as the mediators of neuroimmune interactions, which can be therapeutically effective for neuro-immune disorders and several diseases associated with neuroinflammation.

Comparative hematopoiesis and signal transduction in model organisms.

Hematopoiesis is a continuous phenomenon involving the formation of hematopoietic stem cells (HSCs) giving rise to diverse functional blood cells. This developmental process of hematopoiesis is evolutionarily conserved, yet comparably different in various model organisms. Vertebrate HSCs give rise to all types of mature cells of both the myeloid and the lymphoid lineages sequentially colonizing in different anatomical tissues. Signal transduction in HSCs facilitates their potency and specifies branching of lineages. Understanding the hematopoietic signaling pathways is crucial to gain insights into their deregulation in several blood-related disorders. The focus of the review is on hematopoiesis corresponding to different model organisms and pivotal role of indispensable hematopoietic pathways. We summarize and discuss the fundamentals of blood formation in both invertebrate and vertebrates, examining the requirement of key signaling nexus in hematopoiesis. Knowledge obtained from such comparative studies associated with developmental dynamics of hematopoiesis is beneficial to explore the therapeutic options for hematopoietic diseases.

Co-stimulatory effect of TLR2 and TLR4 stimulation on megakaryocytic development is mediated through PI3K/NF-ĸB and XBP-1 loop.

Toll-like receptors (TLRs) are a class of proteins (patterns recognition receptors-PRRs) capable of recognizing molecules frequently found in pathogens (that are so-called pathogen-associated molecular patterns-PAMPs), they play a key role in the initiation of innate immune response by detecting PAMPs. Our findings show that the functional effects of TLRs co-stimulation on megakaryocytopoiesis. A single cell may receive multiple signal inputs and we consider that multiple TLRs are likely triggered during infection by multiple PAMPs that, in turn, might be involved in infection driven megakaryocytopoiesis, and the present study provide the evidence for the megakaryocytic effects of TLRs co-stimulation.

Virodhamine, an endocannabinoid, induces megakaryocyte differentiation by regulating MAPK activity and function of mitochondria.

Endocannabinoids are well-known regulators of neurotransmission by activating the cannabinoid (CB) receptors. Endocannabinoids are being used extensively for the treatment of various neurological disorders such as Alzheimer's and Parkinson's diseases. Although endocannabinoids are well studied in cell survival, proliferation, and differentiation in various neurological disorders and several cancers, the functional role in the regulation of blood cell development is less examined. In the present study, virodhamine, which is an agonist of CB receptor-2, was used to examine its effect on megakaryocytic development from a megakaryoblastic cell. We observed that virodhamine increases cell adherence, cell size, and cytoplasmic protrusions. Interestingly, we have also observed large nucleus and increased expression of megakaryocytic marker (CD61), which are the typical hallmarks of megakaryocytic differentiation. Furthermore, the increased expression of CB2 receptor was noticed in virodhamine-induced megakaryocytic cells. The effect of virodhamine on megakaryocytic differentiation could be mediated through CB2 receptor. Therefore, we have studied virodhamine induced molecular regulation of megakaryocytic differentiation; mitogen-activated protein kinase (MAPK) activity, mitochondrial function, and reactive oxygen species (ROS) production were majorly affected. The altered mitochondrial functions and ROS production is the crucial event associated with megakaryocytic differentiation and maturation. In the present study, we report that virodhamine induces megakaryocytic differentiation by triggering MAPK signaling and ROS production either through MAPK effects on ROS-generating enzymes or by the target vanilloid receptor 1-mediated regulation of mitochondrial function.

RUNX1 and TGF-ß signaling cross talk regulates Ca2+ ion channels expression and activity during megakaryocyte development.

Thrombocytopenia is characterized by low platelet count and is typically observed among all preterm and low birthweight neonates admitted to the neonatal intensive care unit. Although the underlying cause for this predisposition is unclear, recent studies have proposed that the intrinsic inability of neonatal hematopoietic stem/progenitor cells to produce mature polyploid megakaryocytes (MKs) may result in delayed platelet engraftment. The developmental and molecular differences between neonatal and adult MKs are not yet fully understood. Previously, we had reported that the key MK transcription factor RUNX1, which is crucial for the regulation of MK specification and maturation, is down-regulated in neonatal MKs when compared with adult MKs. In humans, loss-of-function mutations in RUNX1 cause familial platelet disorder, which is characterized by thrombocytopenia, indicating its crucial role in MK development. However, information about its cross talk with developmentally regulated signaling pathways in MKs is lacking. In this study, we performed a differential gene expression analysis in MKs derived from human cord blood (CB) and adult peripheral blood (PB) CD34+ cells. Further, validation and correlation studies between RUNX1 and transforming growth factor beta (TGF-ß) were performed in a differentiating megakaryocytic cell line model. The analysis revealed that TGF-ß pathway was the main pathway affected between CB- and PB-MKs. RUNX1 is reported to be a modulator of TGF-ß signaling in several studies. The correlation between RUNX1 and TGF-ß pathway was analyzed in the PMA-induced megakaryocytic differentiating K562 cells, which exhibit mature megakaryocytic features. The RT2 profiler PCR array analysis revealed that TGF-ß pathway components were up-regulated in the PMA-induced megakaryocytic differentiating cells. Furthermore, our study indicated that human TGF-ß1 promotes cytosolic calcium (Ca2+ ) activity and MK maturation. We noticed that TGF-ß1 increased intracellular free Ca2+ ([Ca2+ ]i) via reactive oxygen species-mediated activation of transient receptor potential (TRP) ion channels. Moreover, we observed that decreased cytosolic Ca2+ activity in the siRUNX1-transfected cells was associated with down-regulation of TRP ion channel expression. Finally, we demonstrated that TGF-ß/SMAD signaling augments the development of MKs derived from CB-CD34+ . Present data suggest that RUNX1/TGF-ß pathway cross talk is crucial for MK maturation.

Beta-Glycerophosphate-Induced ORAI1 Expression and Store Operated Ca2+ Entry in Megakaryocytes.

Impairment of renal phosphate elimination in chronic kidney disease (CKD) leads to enhanced plasma and tissue phosphate concentration, which in turn up-regulates transcription factor NFAT5 and serum & glucocorticoid-inducible kinase SGK1. The kinase upregulates ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE). ORAI1 is stimulated following intracellular store depletion by Ca2+-sensors STIM1 and/or STIM2. In megakaryocytes and blood platelets SOCE and thus ORAI1 are powerful regulators of activity. The present study explored whether the phosphate-donor ß-glycerophosphate augments NFAT5, ORAI1,2,3 and/or STIM1,2 expressions and thus SOCE in megakaryocytes. Human megakaryocytic Meg01cells were exposed to 2 mM of phosphate-donor ß-glycerophosphate for 24 hours. Platelets were isolated from blood samples of patients with impaired kidney function or control volunteers. Transcript levels were estimated utilizing q-RT-PCR, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). NFAT5 and ORAI1 protein abundance was estimated with Western blots. As a result, ß-glycerophosphate increased NFAT5, ORAI1/2/3, STIM1/2 transcript levels, as well as SOCE. Transcript levels of NFAT5, SGK1, ORAI1/2/3, and STIM1/2 as well as NFAT5 and ORAI1 protein abundance were significantly higher in platelets isolated from patients with impaired kidney function than in platelets from control volunteers. In conclusion, phosphate-donor ß-glycerophosphate triggers a signaling cascade of NFAT5/SGK1/ORAI/STIM, thus up-regulating store-operated Ca2+-entry.

Endoplasmic reticulum stress induced apoptosis and caspase activation is mediated through mitochondria during megakaryocyte differentiation.

Megakaryocytopoiesis involves the process of the development of hematopoietic stem cells into megakaryocytes (MKs), which are the specialized cells responsible for the production of blood platelets. Platelets are one of the crucial factors for hemostasis and thrombosis. In terminally differentiated MKs, many molecular process such as caspase activation and a massive cytoskeletal rearrangement drive the formation of cytoplasmic extensions called proplatelets. These cytoplasmic extensions packed with granules and organelles are then released from the bone marrow into the blood circulation as platelets. Classically, caspase activation is associated with apoptosis and recent reports suggest their involvement in cell differentiation and maturation. There is no clear evidence about the stimulus for caspase activation during megakaryocyte development. In the current study, we attempted to understand the importance of endoplasmic reticulum stress in the caspase activation during megakaryocyte maturation. We used human megakaryoblstic cell line (Dami cells) as an experimental model. We used PMA (Phorbol 12-myristate 13 acetate) to induce megakaryocytic differentiation to understand the involvement of ER stress and caspase activation during MK maturation. Further, we used Thapsigargin, a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) as a positive control to induce ER stress. We observed larger and adherent cells with the increased expression of megakaryocytic markers (CD41 and CD61) and UPR markers in PMA or Thapsigargin treated cells as compared to control. Also, Thapsigargin treatment induced increased caspase activity and PARP cleavage. The increased expression of megakaryocyte maturation markers alongside with ER stress and caspase activation suggests the importance of ER stress in caspase activation during MK maturation.

Long non-coding RNA: Classification, biogenesis and functions in blood cells.

While there exist some long non-coding RNAs (lncRNAs) that are structurally similar to mRNAs (capped, spliced, poly a tail), not all of the lncRNAs exhibit these features. Structurally, lncRNAs are classified under the regulatory non-coding RNAs category these lncRNA molecules operate as signals, decoys, guides, and scaffolds. In eukaryotes, lncRNAs are transcribed by RNA Polymerase II and RNA Polymerase III at several loci of the genome. Unlike other protein-coding mRNAs, lncRNAs exhibit functional uniqueness by participating in and modulating the various cellular processes such as, histone modification, DNA methylation, and cellular transcription (Wei et al., 2017). LncRNA alters chromatin structure and DNA accessibility, thereby regulating patterns of gene expression (Wang et al., 2011b). Disordered lncRNA with quantitative or qualitative alterations lead to the progression of numerous diseases including blood associated diseases. LncRNAs not only regulate lineage commitment such as cardiovascular lineage but also contribute for the hematopoietic stem cell development with a significant role in myeloid and lymphoid lineage commitment. However, the key molecular functions of lncRNAs in hematopoiesis are still unclear, particularly, their functional role during megakaryocyte development from hematopoietic stem cells (HSCs) is largely unexplored. This review summarizes the current status of knowledge on lncRNAs classification, biogenesis and its role in blood cells.

Exosome mediated differentiation of megakaryocytes: a study on TLR mediated effects.

Megakaryocytes are large polyploid bone marrow cells whose function is to produce circulatory platelets. Megakaryocytes are also known to release extracellular vesicles (EVs) of varying sizes. Toll like receptors (TLRs), present on the sentinel cells are essential components of the innate immune response, these receptors are also expressed by platelets and megakaryocytes. Our data provide the evidence that TLR-2 induced MKEVs are able to recapitulate TLR-2 signalling in megakaryocytic cell line (Dami cells) and that likely induces megakaryocytic maturation by increasing the production of cytokines involved in MK maturation. TLR-2 induced MKEVs may be involved in replenishment of the immune effector platelets in circulation and its progenitor megakaryocyte in bone marrow for the physiological need of the platelets by inducing the maturation of megakaryocyte.