ResearchPad - molecular-biosciences https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Cellular Plasticity in Breast Cancer Progression and Therapy]]> https://www.researchpad.co/article/elastic_article_12895 With the exception of non-melanoma skin cancer, breast cancer is the most frequently diagnosed malignant disease among women, with the majority of mortality being attributable to metastatic disease. Thus, even with improved early screening and more targeted treatments which may enable better detection and control of early disease progression, metastatic disease remains a significant problem. While targeted therapies exist for breast cancer patients with particular subtypes of the disease (Her2+ and ER/PR+), even in these subtypes the therapies are often not efficacious once the patient's tumor metastasizes. Increases in stemness or epithelial-to-mesenchymal transition (EMT) in primary breast cancer cells lead to enhanced plasticity, enabling tumor progression, therapeutic resistance, and distant metastatic spread. Numerous signaling pathways, including MAPK, PI3K, STAT3, Wnt, Hedgehog, and Notch, amongst others, play a critical role in maintaining cell plasticity in breast cancer. Understanding the cellular and molecular mechanisms that regulate breast cancer cell plasticity is essential for understanding the biology of breast cancer progression and for developing novel and more effective therapeutic strategies for targeting metastatic disease. In this review we summarize relevant literature on mechanisms associated with breast cancer plasticity, tumor progression, and drug resistance.

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<![CDATA[Cancer Stem Cell Plasticity – A Deadly Deal]]> https://www.researchpad.co/article/elastic_article_7630 Intratumoral heterogeneity is a major ongoing challenge in the effective therapeutic targeting of cancer. Accumulating evidence suggests that a fraction of cells within a tumor termed Cancer Stem Cells (CSCs) are primarily responsible for this diversity resulting in therapeutic resistance and metastasis. Adding to this complexity, recent studies have shown that there can be different subpopulations of CSCs with varying biochemical and biophysical traits resulting in varied dissemination and drug-resistance potential. Moreover, cancer cells can exhibit a high level of plasticity or the ability to dynamically switch between CSC and non-CSC states or among different subsets of CSCs. In addition, CSCs also display extensive metabolic plasticity. The molecular mechanisms underlying these different interconnected axes of plasticity has been under extensive investigation and the trans-differentiation process of Epithelial to Mesenchymal transition (EMT) has been identified as a major contributing factor. Besides genetic and epigenetic factors, CSC plasticity is also shaped by non-cell-autonomous effects such as the tumor microenvironment (TME). In this review, we discuss the latest developments in decoding mechanisms and implications of CSC plasticity in tumor progression at biochemical and biophysical levels, and the latest in silico approaches being taken for characterizing cancer cell plasticity. These efforts can help improve existing therapeutic approaches by taking into consideration the contribution of cellular plasticity/heterogeneity in enabling drug resistance.

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<![CDATA[Short Residence Times of DNA-Bound Transcription Factors Can Reduce Gene Expression Noise and Increase the Transmission of Information in a Gene Regulation System]]> https://www.researchpad.co/article/elastic_article_7536 Gene expression noise is not just ubiquitous but also variable, and we still do not understand some of the most elementary factors that affect it. Among them is the residence time of a transcription factor (TF) on DNA, the mean time that a DNA-bound TF remains bound. Here, we use a stochastic model of transcriptional regulation to study how residence time affects the gene expression noise that arises when a TF induces gene expression. We find that the effect of residence time on gene expression noise depends on the TF’s concentration and its affinity to DNA, which determine the level of induction of the gene. At high levels of induction, residence time has no effect on gene expression noise. However, as the level of induction decreases, short residence times reduce gene expression noise. The reason is that fast on-off TF binding dynamics prevent long periods where proteins are predominantly synthesized or degraded, which can cause excessive fluctuations in gene expression. As a consequence, short residence times can help a gene regulation system acquire information about the cellular environment it operates in. Our predictions are consistent with the observation that experimentally measured residence times are usually modest and lie between seconds to minutes.

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<![CDATA[Structural and Functional Analysis of Gly212 Mutants Reveals the Importance of Intersubunit Interactions in ASIC1a Channel Function]]> https://www.researchpad.co/article/elastic_article_7390 Acid-sensing ion channels (ASICs) act as pH sensors in neurons. ASICs contribute to pain sensation, learning, fear behavior and to neuronal death after ischemic stroke. Extracellular acidification induces a transient activation and subsequent desensitization of these Na+-selective channels. ASICs are trimeric channels made of identical or homologous subunits. We have previously shown that mutation of the highly conserved Gly212 residue of human ASIC1a to Asp affects the channel function. Gly212 is located in the proximity of a predicted Cl binding site at a subunit interface. Here, we have measured the function of a series of Gly212 mutants. We show that substitution of Gly212 affects the ASIC1a pH dependence and current decay kinetics. Intriguingly, the mutations to the acidic residues Asp and Glu have opposing effects on the pH dependence and the current decay kinetics. Analysis of molecular dynamics simulation trajectories started with the coordinates of the closed conformation indicates that the immediate environment of residue 212 in G212E, which shifts the pH dependence to more alkaline values, adopts a conformation closer to the open state. The G212D and G212E mutants have a different pattern of intersubunit salt bridges, that, in the case of G212E, leads to an approaching of neighboring subunits. Based on the comparison of crystal structures, the conformational changes in this zone appear to be smaller during the open-desensitized transition. Nevertheless, MD simulations highlight differences between mutants, suggesting that the changed function upon substitution of residue 212 is due to differences in intra- and intersubunit interactions in its proximity.

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<![CDATA[Modeling Early Stages of Bone and Joint Infections Dynamics in Humans: A Multi-Agent, Multi-System Based Model]]> https://www.researchpad.co/article/N8fbc090b-d0e3-4fac-aba0-42bdecf39dae

Diagnosis and management of bone and joint infections (BJI) is a challenging task. The high intra and inter patient’s variability in terms of clinical presentation makes it impossible to rely on a systematic description or classical statistical analysis for its diagnosis. Advances can be achieved through a better understanding of the system behavior that results from the interactions between the components at a micro-scale level, which is difficult to mastered using traditional methods. Multiple studies from the literature report factors and interactions that affect the dynamics of the BJI system. The objectives of this study were (i) to perform a systematic review to identify relevant interactions between agents (cells, pathogens) and parameters values that characterize agents and interactions, and (ii) to develop a two dimensional computational model of the BJI system based on the results of the systematic review. The model would simulate the behavior resulting from the interactions on the cellular and molecular levels to explore the BJI dynamics, using an agent-based modeling approach. The BJI system’s response to different microbial inoculum levels was simulated. The model succeeded in mimicking the dynamics of bacteria, the innate immune cells, and the bone mass during the first stage of infection and for different inoculum levels in a consistent manner. The simulation displayed the destruction in bone tissue as a result of the alteration in bone remodeling process during the infection. The model was used to generate different patterns of system behaviors that could be analyzed in further steps. Simulations results suggested evidence for the existence of latent infections. Finally, we presented a way to analyze and synthesize massive simulated data in a concise and comprehensive manner based on the semi-supervised identification of ordinary differential equations (ODE) systems. It allows to use the known framework for temporal and structural ODE analyses and therefore summarize the whole simulated system dynamical behavior. This first model is intended to be validated by in vivo or in vitro data and expected to generate hypotheses to be challenged by real data. Step by step, it can be modified and complexified based on the test/validation iteration cycles.

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<![CDATA[In silico, in vitro, and in vivo Approaches to Identify Molecular Players in Fragile X Tremor and Ataxia Syndrome]]> https://www.researchpad.co/article/Nd16aaea3-5641-45ad-ae52-767d074e1bb6

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative monogenetic disorder affecting carriers of premutation (PM) forms of the FMR1 gene, resulting in a progressive development of tremors, ataxia, and neuropsychological problems. This highly disabling disease is quite common in the general population with an estimation of about 20 million PM carriers worldwide. The chances of developing FXTAS increase dramatically with age, with about 45% of male carriers over the age of 50 being affected. Both the gene and pathogenic trigger, a mutant expansion of CGG RNA, causing FXTAS are known. This makes it an interesting disease to develop targeted therapeutic interventions for. Yet, no such interventions are available at this moment. Here we discuss in silico, in vitro, and in vivo approaches and how they have been used to identify the molecular determinants of FXTAS pathology. These approaches have yielded substantial information about FXTAS pathology and, consequently, many markers have emerged to play a key role in understanding the disease mechanism. Integration of the different approaches is expected to provide crucial information about the value of these markers as either therapeutic target or biomarker, essential to monitor therapeutic interventions in the future.

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<![CDATA[Selective Inhibition of HDAC1 by Macrocyclic Polypeptide for the Treatment of Glioblastoma: A Binding Mechanistic Analysis Based on Molecular Dynamics]]> https://www.researchpad.co/article/Ndcc4a273-ece4-4d77-a258-8c9760545e84

Glioblastoma (GBM) is the most common and aggressive intracranial malignant brain tumor, and the abnormal expression of HDAC1 is closely correlated to the progression, recurrence and metastasis of GBM cells, making selective inhibition of HDAC1 a promising strategy for GBM treatments. Among all available selective HDAC1 inhibitors, the macrocyclic peptides have gained great attention due to their remarkable inhibitory selectivity on HDAC1. However, the binding mechanism underlying this selectivity is still elusive, which increases the difficulty of designing and synthesizing the macrocyclic peptide-based anti-GBM drug. Herein, multiple computational approaches were employed to explore the binding behaviors of a typical macrocyclic peptide FK228 in both HDAC1 and HDAC6. Starting from the docking conformations of FK228 in the binding pockets of HDAC1&6, relatively long MD simulation (500 ns) shown that the hydrophobic interaction and hydrogen bonding of E91 and D92 in the Loop2 of HDAC1 with the Cap had a certain traction effect on FK228, and the sub-pocket formed by Loop1 and Loop2 in HDAC1 could better accommodate the Cap group, which had a positive effect on maintaining the active conformation of FK228. While the weakening of the interactions between FK228 and the residues in the Loop2 of HDAC6 during the MD simulation led to the large deflection of FK228 in the binding site, which also resulted in the decrease in the interactions between the Linker region of FK228 and the previously identified key amino acids (H134, F143, H174, and F203). Therefore, the residues located in Loop1 and Loop2 contributed in maintaining the active conformation of FK228, which would provide valuable hints for the discovery and design of novel macrocyclic polypeptide HDAC inhibitors.

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<![CDATA[The Small-Molecule Flunarizine in Spinal Muscular Atrophy Patient Fibroblasts Impacts on the Gemin Components of the SMN Complex and TDP43, an RNA-Binding Protein Relevant to Motor Neuron Diseases]]> https://www.researchpad.co/article/N5f33db38-5430-4add-9f84-b663b0bcb2b5

The motor neurodegenerative disease spinal muscular atrophy (SMA) is caused by alterations of the survival motor neuron 1 (SMN1) gene involved in RNA metabolism. Although the disease mechanisms are not completely elucidated, SMN protein deficiency leads to abnormal small nuclear ribonucleoproteins (snRNPs) assembly responsible for widespread splicing defects. SMN protein localizes in nuclear bodies that are lost in SMA and adult onset amyotrophic lateral sclerosis (ALS) patient cells harboring TDP-43 or FUS/TLS mutations. We previously reported that flunarizine recruits SMN into nuclear bodies and improves the phenotype of an SMA mouse model. However, the precise mode of action remains elusive. Here, a marked reduction of the integral components of the SMN complex is observed in severe SMA patient fibroblast cells. We show that flunarizine increases the protein levels of a subset of components of the SMN-Gemins complex, Gemins2-4, and markedly reduces the RNA and protein levels of the pro-oxydant thioredoxin-interacting protein (TXNIP) encoded by an mRNA target of Gemin5. We further show that SMN deficiency causes a dissociation of the localization of the SMN complex components from the same nuclear bodies. The accumulation of TDP-43 in SMN-positive nuclear bodies is also perturbed in SMA cells. Notably, TDP-43 is found to co-localize with SMN in nuclear bodies of flunarizine-treated SMA cells. Our findings indicate that flunarizine reverses cellular changes caused by SMN deficiency in SMA cells and further support the view of a common pathway in RNA metabolism underlying infantile and adult motor neuron diseases.

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<![CDATA[Spontaneous Embedding of DNA Mismatches Within the RNA:DNA Hybrid of CRISPR-Cas9]]> https://www.researchpad.co/article/Nfad48beb-417e-4db6-a5e3-6b7d588817e1

CRISPR-Cas9 is the forefront technology for editing the genome. In this system, the Cas9 protein is programmed with guide RNAs to process DNA sequences that match the guide RNA forming an RNA:DNA hybrid structure. However, the binding of DNA sequences that do not fully match the guide RNA can limit the applicability of CRISPR-Cas9 for genome editing, resulting in the so-called off-target effects. Here, molecular dynamics is used to probe the effect of DNA base pair mismatches within the RNA:DNA hybrid in CRISPR-Cas9. Molecular simulations revealed that the presence of mismatched pairs in the DNA at distal sites with respect to the Protospacer Adjacent Motif (PAM) recognition sequence induces an extended opening of the RNA:DNA hybrid, leading to novel interactions established by the unwound nucleic acids and the protein counterpart. On the contrary, mismatched pairs upstream of the RNA:DNA hybrid are rapidly incorporated within the heteroduplex, with minor effect on the protein-nucleic acid interactions. As a result, mismatched pairs at PAM distal ends interfere with the activation of the catalytic HNH domain, while mismatches fully embedded in the RNA:DNA do not affect the HNH dynamics and enable its activation to cleave the DNA. These findings provide a mechanistic understanding to the intriguing experimental evidence that PAM distal mismatches hamper a proper function of HNH, explaining also why mismatches within the heteroduplex are much more tolerated. This constitutes a step forward in understanding off-target effects in CRISPR-Cas9, which encourages novel structure-based engineering efforts aimed at preventing the onset of off-target effects.

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<![CDATA[Studying DNA Double-Strand Break Repair: An Ever-Growing Toolbox]]> https://www.researchpad.co/article/Nb78421e7-225e-4ed3-907b-e8aaf4b9cd46

To ward off against the catastrophic consequences of persistent DNA double-strand breaks (DSBs), eukaryotic cells have developed a set of complex signaling networks that detect these DNA lesions, orchestrate cell cycle checkpoints and ultimately lead to their repair. Collectively, these signaling networks comprise the DNA damage response (DDR). The current knowledge of the molecular determinants and mechanistic details of the DDR owes greatly to the continuous development of ground-breaking experimental tools that couple the controlled induction of DSBs at distinct genomic positions with assays and reporters to investigate DNA repair pathways, their impact on other DNA-templated processes and the specific contribution of the chromatin environment. In this review, we present these tools, discuss their pros and cons and illustrate their contribution to our current understanding of the DDR.

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<![CDATA[Routine Optical Clearing of 3D-Cell Cultures: Simplicity Forward]]> https://www.researchpad.co/article/Ne2d11b6b-5c1e-4e35-9c3d-0964b3a618bb

Three-dimensional cell cultures, such as spheroids and organoids, serve as increasingly important models in fundamental and applied research and start to be used for drug screening purposes. Optical tissue clearing procedures are employed to enhance visualization of fluorescence-stained organs, tissues, and three-dimensional cell cultures. To get a more systematic overview about the effects and applicability of optical tissue clearing on three-dimensional cell cultures, we compared six different clearing/embedding protocols on seven types of spheroid- and chip-based three-dimensional cell cultures of approximately 300 μm in size that were stained with nuclear dyes, immunofluorescence, cell trackers, and cyan fluorescent protein. Subsequent whole mount confocal microscopy and semi-automated image analysis were performed to quantify the effects. Quantitative analysis included fluorescence signal intensity and signal-to-noise ratio as a function of z-depth as well as segmentation and counting of nuclei and immunopositive cells. In general, these analyses revealed five key points, which largely confirmed current knowledge and were quantified in this study. First, there was a massive variability of effects of different clearing protocols on sample transparency and shrinkage as well as on dye quenching. Second, all tested clearing protocols worked more efficiently on samples prepared with one cell type than on co-cultures. Third, z-compensation was imperative to minimize variations in signal-to-noise ratio. Fourth, a combination of sample-inherent cell density, sample shrinkage, uniformity of signal-to-noise ratio, and image resolution had a strong impact on data segmentation, cell counts, and relative numbers of immunofluorescence-positive cells. Finally, considering all mentioned aspects and including a wish for simplicity and speed of protocols – in particular, for screening purposes – clearing with 88% Glycerol appeared to be the most promising option amongst the ones tested.

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<![CDATA[Drought Stress Causes Specific Changes to the Spliceosome and Stress Granule Components]]> https://www.researchpad.co/article/Nb8d79b96-002d-4774-8621-f27d675ee31e

The spliceosome processes RNAs from a pre-RNA state to a mature mRNA thereby influencing RNA availability for translation, localization, and turnover. It consists of complex structures containing RNA-binding proteins (RBPs) essential for post-transcriptional gene expression control. Here we investigate the dynamic modifications of spliceosomal RBPs under stress and in particular drought stress. We do so by mRNA interactome capture in Arabidopsis thaliana using label free quantitation. This approach identified 44 proteins associated with the spliceosome and further 32 proteins associated with stress granules. We noted a high enrichment in the motifs RDRR and RSRSRS that are characteristic of RNA interacting proteins. Identification of splicing factors reflect direct and/or indirect stress induced splicing events that have a direct effect on transcriptome and proteome changes under stress. Furthermore, detection of stress granule components is consistent with transcriptional arrest. Identification of drought induced stress granule components is critical in determining common abiotic stress-induced foci that can have biotechnological applications. This study may therefore open ways to modify plant stress responses at a systems level through the modification of key spliceosome components.

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<![CDATA[Why Selenocysteine Is Unique?]]> https://www.researchpad.co/article/N073a7fa8-88f2-4b9a-9938-6b26380382a4 ]]> <![CDATA[Characterizing the Binding Sites for GK Domain of DLG1 and DLG4 via Molecular Dynamics Simulation]]> https://www.researchpad.co/article/Ncd2c0e35-955f-40db-b142-48f20542f642

Discs-large (DLG) is a member that belongs to the membrane-associated guanylate kinase (MAGUK) family. The GK domain of DLGs has evolved into a protein–protein interaction module that could bind with kinds of proteins to regulate diverse cellular functions. Previous reports have demonstrated the GK domain of DLGs functioned as a phosphor-peptide-binding module by resolving the crystal structures. Here we investigated into the interactions of DLG1 and DLG4 with their reported phosphor-peptides by molecular dynamics simulations. Post-dynamics analysis showed that DLG1/4 formed extensive interactions with phosphorylated ligands, including hydrophobic and hydrogen bonding interactions. Among them, the highly conserved residues among the DLGs in phosphor-site and β5 sheet were crucial for the binding according to the energy decomposition calculations. Additionally, the binding interactions between DLG4 and reported unphosphorylated peptides including MAP1A and designed GK inhibitory (GKI-QSF) peptides were analyzed. We found the key residues that played important roles in DLG4/unphosphorylated peptide systems were very similar as in DLG4/phosphor-peptide systems. Moreover, the molecular dynamic simulation for the complex of DLG1 and GKI-QSF was carried out and predicted that the GKI-QSF could bind with DLG1 with similar Kd value compared to DLG4/GKI-QSF, which was verified by using ITC assay (Kd = 1.20 ± 0.29 μM). Our study might be helpful for the better understanding of the structural and biological function of DLGs GK domain and encourage the discovery of new binders.

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<![CDATA[Dynamics Insights Into the Gain of Flexibility by Helix-12 in ESR1 as a Mechanism of Resistance to Drugs in Breast Cancer Cell Lines]]> https://www.researchpad.co/article/N7e16a1fc-a3d4-446a-98af-697f835276ba

Incidents of breast cancer (BC) are on the rise on a daily basis and have proven to be the most prevelant cause of death for women in both developed and developing countries. Among total BC cases diagnosed after menopause, 70% of cases are Estrogen Receptor (ER) positive (ER-positive or ER+). Mutations in the LBD (ligand-binding domain) of the ER have recently been reported to be the major cause of resistance to potent antagonists. In this study, the experimentally reported mutations K303R, E380Q, V392I, S463P, V524E, P535H, P536H, Y537C, Y537N, Y537S, and D538G were analyzed, and the most significant mutations were shortlisted based on multiple analyses. Initial analyses, such as mCSM stability, occluded depth analysis, mCSM-binding affinity, and FoldX energy changes shortlisted only six mutations as being highly resistant. Finally, simulations of force field-based molecular dynamics (MD on wild type (WT) ERα) on six mERα variants (E380Q, S463P, Y537S, Y537C, Y537N, and D538G) were carried out to justify mechanism of the resistance. It was observed that these mutations increased the flexibility of the H12. A bonding analysis suggested that previously reported important residue His524 lost bonding upon mutation. Other parameters, such as PCA (principal component analysis), DCCM (dynamics cross-correlation), and FEL (free energy landscape), verified that the shortlisted mutations affect the H12 helix, which opens up the co-activator binding conformation. These results provide deep insight into the mechanism of relative resistance posed to fulvestrant due to mutations in breast cancer. This study will facilitate further understanding of the important aspects of designing specific and more effective drugs.

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<![CDATA[The Role of RNA Binding Proteins for Local mRNA Translation: Implications in Neurological Disorders]]> https://www.researchpad.co/article/N6e6ed449-895c-4c7f-b7be-fc655a8ec0f0

As neurons are one of the most highly polarized cells in our body, they require sophisticated cellular mechanisms to maintain protein homeostasis in their subcellular compartments such as axons and dendrites. When neuronal protein homeostasis is disturbed due to genetic mutations or deletions, this often results in degeneration of neurons leading to devastating outcome such as spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), and fragile X syndrome (FXS). Ribonucleoprotein (RNP) complexes are macromolecular complexes composed of RNA binding proteins (RBPs) and their target RNAs. RBPs contain RNA binding domains and bind to RNA molecules via specific sequence motifs. RNP complexes have various functions in gene expression including messenger RNA (mRNA) trafficking, RNA processing and silencing. In neurons, RBPs deliver specific sets of mRNAs to subcellular compartments such as axons and dendrites to be locally translated. Mutations or deletions in genes coding for RNPs have been reported as causes for neurological disorders such as SMA, ALS, and FXS. As RBPs determine axonal or dendritic mRNA repertoires as well as proteomes by trafficking selective mRNAs and regulating local protein synthesis, they play a crucial role for neuronal function. In this review, we summarize the role of well-known RBPs, SMN, TDP-43, FUS, and FMRP, and review their function for local protein synthesis in neurons. Furthermore, we discuss their pathological contribution to the neurological disorders.

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<![CDATA[Circular RNAs—The Road Less Traveled]]> https://www.researchpad.co/article/Nfc26f478-21f2-41de-a7b2-8e5395fca063

Circular RNAs are the most recent addition in the non-coding RNA family, which has started to gain recognition after a decade of obscurity. The first couple of reports that emerged at the beginning of this decade and the amount of evidence that has accumulated thereafter has, however, encouraged RNA researchers to navigate further in the quest for the exploration of circular RNAs. The joining of 5′ and 3′ ends of RNA molecules through backsplicing forms circular RNAs during co-transcriptional or post-transcriptional processes. These molecules are capable of effectively sponging microRNAs, thereby regulating the cellular processes, as evidenced by numerous animal and plant systems. Preliminary studies have shown that circular RNA has an imperative role in transcriptional regulation and protein translation, and it also has significant therapeutic potential. The high stability of circular RNA is rendered by its closed ends; they are nevertheless prone to degradation by circulating endonucleases in serum or exosomes or by microRNA-mediated cleavage due to their high complementarity. However, the identification of circular RNAs involves diverse methodologies and the delineation of its possible role and mechanism in the regulation of cellular and molecular architecture has provided a new direction for the continuous research into circular RNA. In this review, we discuss the possible mechanism of circular RNA biogenesis, its structure, properties, degradation, and the growing amount of evidence regarding the detection methods and its role in animal and plant systems.

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<![CDATA[“An End to a Means”: How DNA-End Structure Shapes the Double-Strand Break Repair Process]]> https://www.researchpad.co/article/N9a48f8fb-7616-4c38-a517-1dde0e6fc387

Endogenously-arising DNA double-strand breaks (DSBs) rarely harbor canonical 5′-phosphate, 3′-hydroxyl moieties at the ends, which are, regardless of the pathway used, ultimately required for their repair. Cells are therefore endowed with a wide variety of enzymes that can deal with these chemical and structural variations and guarantee the formation of ligatable termini. An important distinction is whether the ends are directly “unblocked” by specific enzymatic activities without affecting the integrity of the DNA molecule and its sequence, or whether they are “processed” by unspecific nucleases that remove nucleotides from the termini. DNA end structure and configuration, therefore, shape the repair process, its requirements, and, importantly, its final outcome. Thus, the molecular mechanisms that coordinate and integrate the cellular response to blocked DSBs, although still largely unexplored, can be particularly relevant for maintaining genome integrity and avoiding malignant transformation and cancer.

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<![CDATA[To Be or Not To Be…Toxic—Is RNA Association With TDP-43 Complexes Deleterious or Protective in Neurodegeneration?]]> https://www.researchpad.co/article/N771401a6-baca-4025-bded-8d043eb615f6

TAR DNA binding protein (TDP-43) is a nucleic acid binding protein associated with insoluble cytoplasmic aggregates in several neurodegenerative disorders, including 97% of the ALS cases. In healthy individuals, TDP-43 is primarily localized to the nucleus; it can shuttle between the nucleus and the cytoplasm, and is involved in several aspects of RNA processing including transcription, splicing, RNA stability, transport, localization, stress granule (SG) formation, and translation. Upon stress, TDP-43 aggregates in the cytoplasm and associates with several types of RNA and protein assemblies, resulting in nuclear depletion of TDP-43. Under conditions of prolonged stress, cytoplasmic TDP-43 undergoes liquid-liquid phase separation (LLPS) and becomes less mobile. Evidence exists to support a scenario in which insoluble TDP-43 complexes sequester RNA and/or proteins causing disturbances in both ribostasis and proteostasis, which in turn contribute to neurodegeneration. However, the relationship between RNA binding and TDP-43 toxicity remains unclear. Recent studies provide conflicting views on the role of RNA in TDP-43 toxicity, with some finding RNA as a toxic factor whereby RNA binding contributes to TDP-43 toxicity, while others find RNA to be a protective factor that inhibits TDP-43 aggregation. Here we review and discuss these recent reports, which ultimately highlight the importance of understanding the heterogeneity of TDP-43 assemblies and collectively point to solubilizing TDP-43 as a potential therapeutic strategy.

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<![CDATA[Dissecting the Extracellular Complexity of Neuromuscular Junction Organizers]]> https://www.researchpad.co/article/N0e7edde6-060c-4a9b-b987-6efc455fc4f6

Synapse formation is a very elaborate process dependent upon accurate coordination of pre and post-synaptic specialization, requiring multiple steps and a variety of receptors and signaling molecules. Due to its relative structural simplicity and the ease in manipulation and observation, the neuromuscular synapse or neuromuscular junction (NMJ)—the connection between motor neurons and skeletal muscle—represents the archetype junction system for studying synapse formation and conservation. This junction is essential for survival, as it controls our ability to move and breath. NMJ formation requires coordinated interactions between motor neurons and muscle fibers, which ultimately result in the formation of a highly specialized post-synaptic architecture and a highly differentiated nerve terminal. Furthermore, to ensure a fast and reliable synaptic transmission following neurotransmitter release, ligand-gated channels (acetylcholine receptors, AChRs) are clustered on the post-synaptic muscle cell at high concentrations in sites opposite the presynaptic active zone, supporting a direct role for nerves in the organization of the post-synaptic membrane architecture. This organized clustering process, essential for NMJ formation and for life, relies on key signaling molecules and receptors and is regulated by soluble extracellular molecules localized within the synaptic cleft. Notably, several mutations as well as auto-antibodies against components of these signaling complexes have been related to neuromuscular disorders. The recent years have witnessed strong progress in the understanding of molecular identities, architectures, and functions of NMJ macromolecules. Among these, prominent roles have been proposed for neural variants of the proteoglycan agrin, its receptor at NMJs composed of the lipoprotein receptor-related protein 4 (LRP4) and the muscle-specific kinase (MuSK), as well as the regulatory soluble synapse-specific protease Neurotrypsin. In this review we summarize the current state of the art regarding molecular structures and (agrin-dependent) canonical, as well as (agrin-independent) non-canonical, MuSK signaling mechanisms that underscore the formation of neuromuscular junctions, with the aim of providing a broad perspective to further stimulate molecular, cellular and tissue biology investigations on this fundamental intercellular contact.

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