CRISPR/Cas9-based gene therapy has gotten a lot of interest during last decade. This treatment protocol tries to fix disease-causing traits by changing the DNA code in the exact spot on the chromosome. Therefore, we have tried to elaborate the most current information that shows how well CRISPR/Cas9-based gene therapy works. In vivo methods, like the ones described in the next two lines, are used to study cells in live creatures, like babies that are growing or animals that are already grown up. It has also been suggested to use ex-vivo methods to change somatic stem cells or progenitor cells in a culture outside of the body before putting them back into the patient. CRISPR/Cas is a good way to change specific genes in the DNA. Clustered Regularly Interspaced Short Palindromic Repeats is the full name for CRISPR. Biotechnologists can use them to fix DNA instead of the body's natural ways of doing so. This helps physicians in treatment diseases that run in families more successfully. Cas9 is an enzyme that, along with a guide RNA, is part of the CRISPR/Cas system. As this new technology grows quickly, it is replacing normal medical processes with treatments that are more cutting-edge and can change people's lives. CRISPR-Cas technology has changed biology by making it possible to change genes quickly. CRISPR molecular tools (Cas9 or Cas12a) have a lot of potential, but they are not very useful right now because they depend on the target cell's own DNA repair systems. With or without a template, the body's natural DNA repair processes can fix DNA breaks caused by Cas9 and Cas12a-based technologies. People use these methods a lot, but their effectiveness ranges from cell type to cell type. HDR-mediated DNA repair is a part of cell division, so tools that target it don't work on cells that don't divide, like neurons. CRISPR-associated transposase (CAST) has recently been studied, which suggests that it may offer new ways to change genes with CRISPR. The CRISPR activator of type V-K that is part of CAST has the same structure as the transposase. This is because CRISPR systems can put DNA in the right place without the help of the cell's own DNA repair systems. But a lot of work is being done right now to improve Cas9 and Cas12a so that DNA can be put into a target gene more precisely. Experts are still working hard to come up with better and more reliable ways to change genes, so even though there may be a problem, both methods have useful uses.

The production and secretion of very-low density lipoproteins (VLDL) by hepatocytes has a direct impact on liver fat content as well as the concentration of cholesterol and triglycerides in the circulation and thus affects liver and cardiovascular health, respectively. Importantly, excess caloric intake and lack of physical activity are associated with overproduction of VLDL, hepatic steatosis, and increased levels of atherogenic lipoproteins. Cholesterol as well as triglycerides in remnant particles after VLDL lipolysis are risk factors for atherosclerotic cardiovascular disease (ASCVD) and have garnered increasing attention over the last few decades. To date, however, increased risk of ASCVD is not the only concern when considering today’s cardiometabolic patients, as they often also suffer from hepatic steatosis. This notion highlights the importance of understanding the molecular regulation of VLDL biogenesis. Fortunately, there has been a resurgence of interest in the intracellular assembly, trafficking, degradation, and secretion of VLDL by hepatocytes, that has led to many exciting new molecular insights, the topic of this review. We think that increasing our understanding of the biology of this pathway will help improve the health of the cardiometabolic patient in the long term.

Following ischemic stroke, the degradation of myelin and other cellular membranes surpasses the lipid-processing capabilities of resident microglia and infiltrating macrophages. This imbalance leads to foam cell formation in the infarct and areas of secondary neurodegeneration, instigating sustained inflammation and furthering neurological damage. Given that mitochondria are the primary sites of fatty acid metabolism, augmenting mitochondrial biogenesis (MB) may enhance lipid processing, curtailing foam cell formation and post-stroke chronic inflammation. Previous studies have shown that the pharmacological activation of the β2-adrenergic receptor (β2-AR) stimulates MB. Consequently, our study sought to discern the effects of intensified β2-AR signal-ing on MB, the processing of brain lipid debris, and neurological outcome using a mouse stroke model. To achieve this goal, aged mice were treated with formoterol, a long-acting β2-AR ago-nist, daily for two and eight weeks following stroke. Formoterol increased MB in the infarct re-gion, modified fatty acid metabolism, and reduced foam cell formation. However, it did not re-duce markers of post-stroke neurodegeneration or improve recovery. Our findings suggest that boosting MB in myeloid cells is one component of a therapeutic avenue for improving brain lipid debris processing after stroke.

Circular RNAs (circRNAs), which are a class of non-coding RNA with covalently closed loops, play important roles in epigenetics regulation of gene expression at both the transcriptional and post-transcriptional level. Accumulating evidence demonstrated that numerous circRNAs were abnormally expressed in tumors and their dysregulation was involved in the tumorigenesis and metastasis of cancer. Although the functional mechanisms of many circRNAs have been revealed, why circRNAs are dysregulated in cancer remains elusive. CircRNAs are generated by a “backsplicing” process, which is regulated by different cis-regulatory elements and trans-acting proteins. Therefore, how these cis- and trans-elements change during tumorigenesis and how they regulate the biogenesis of circRNAs in cancer are two questions that interest us. In this review, we summarized the pathways for the biogenesis of circRNAs; and then illustrated why circRNAs dysregulated in cancer by discussing the changes of cis-regulatory elements and trans-acting proteins that related to circRNA splicing and maturation in cancer.

Processing of the RNA polymerase I pre-rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs requires removing the “spacer” sequences. The canonical pathway for the removal of the ITS1 spacer, located between 18S and 5.8S rRNAs in the primary transcript, involves cleavages at the 3’ end of 18S rRNA and at two sites inside ITS1. The process generates a long and a short 5.8S rRNA that differ in the number of ITS1 nucleotides retained at the 5.8S 5’ end. Here we document a novel pathway that generates the long 5.8S for ITS1 while bypassing cleavage within ITS1. It entails a single endonuclease cut at the 3’-end of 18S rRNA followed by exonuclease Xrn1 degradation of ITS1. Mutations in RNase MRP increase the accumulation of long relative to short 5.8S rRNA; traditionally this is attributed to a decreased rate of RNase MRP cleavage at its target in ITS1, called A3. In contrast, we report here that the MRP induced switch between long and short 5.8S rRNA formation occurs even when the A3 site is deleted. Based on this and our published data, we propose that the switch may depend on RNase MRP processing RNA molecules other than pre-rRNA.

The early generative phase of cauliflower (Brassica oleracea var. botrytis) curd ripening is sensitive to the water deficit. Mitochondrial responses under drought within Brassica genus are poorly understood. The main goal of this study was to investigate the mitochondrial biogenesis of three cauliflower cultivars varying with drought tolerance. Diverse quantitative changes (down-regulations mostly) in the mitochondrial proteome were assayed by 2D PAGE coupled with LC-MS/MS. Respiratory (e.g. CII, CIV and ATP synthase subunits), transporter (including diverse porin isoforms) and matrix multifunctional proteins (e.g. components of RNA editing machinery) appeared diversely affected in their abundance under two drought levels. Western immunoassays showed also cultivar-specific responses of selected mitochondrial proteins. Dehydrin-related tryptic peptides found in few 2D spots that appeared immunopositive with dehydrin-specific antisera highlighted the relevance of mitochondrial dehydrin-like proteins for the drought response. The level of selected messengers participating in drought response was also determined. We conclude that the mitochondrial biogenesis was strongly, but diversely affected in various cauliflower cultivars and associated with drought tolerance on the proteomic and functional levels. However, transcriptomic and proteomic regulations were largely uncoordinated due to the suggested altered availability of messengers for translation, mRNA/ribosome interactions and/or miRNA impact on transcript abundance and translation.

Ribosomal proteins (RPs) mediate protein synthesis and stability of the ribosomal complex. RPs and rRNA form ribosomal subunits and ri-bosomes, the cellular machinery for protein synthesis, a fundamental biological process related to cell growth and proliferation. The differ-ences between paralogs within the same RP family have led them to ac-quire extraribosomal functions involved in plants' growth, develop-ment, and biotic and abiotic stresses. From transcriptomic analysis, it has been revealed that ribosomal proteins have a crucial role during MTs development. The RPLs; RPL11, RPL29 and RPL40 interact with PEBP family members (Phosphatidylethanolamine-binding proteins), poten-tial activators in the MTs development. In potatoes, genome editing is a promising technology to introduce crop breeding traits. Recently, the edition of the genome through the CRISPR/Cas9 system has made it the most efficient and powerful genetic modification method. Based on this, the gene modulation by overexpression and silencing in RPL11, RPL29 and RPL40 will guide us to understand the effect on the microtuberiza-tion process and produce improved potato plants with the capability of growing under adverse environments (biotic and abiotic stresses). By understanding the molecular biology mechanisms that RPs govern, we can improve crops under today's changing climatic conditions.

Osteosarcoma (OSA) is the most common primary malignant bone tumor. More than 40% of patients with OSA have poor prognoses. We aimed to discover a biomarker for patient stratification and therapeutic targets for these high-risk patients. Using Single Sample Gene Set Enrichment Analysis (ssGSEA) and univariate Cox analysis, six hallmarks were identified as significant prognostic factors for overall survival (OS). Three were selected to construct a multivariate Cox model. Then, WGCNA, univariate Cox regression, Kaplan-Meier (KM) survival analyses, and multivariate Cox analyses were combined to filter promising candidates and establish a seven-gene signature to predict OS, whose prognostic value was validated internally and externally. Subsequently, Differential Expression Analysis was conducted between high- and low-risk patients, and the Robust Rank Aggregation algorithm was used to determine the robust DEGs. Metascape was used to perform pathway and process enrichment analyses as well as construct protein-protein interaction (PPI) networks. Finally, RPS28 was identified as an independent risk factor by using univariate and multivariate Cox regression, which was preliminarily validated as a promising therapeutic target by using RNA interference. In conclusion, we might contribute to optimizing risk stratification and an excellent therapeutic target for high-risk patients with OSA.

In "genome editing," a type of genetic engineering, DNA is added, taken away, or changed in an organism's genome. Biomedicine, biotechnology, and synthetic biology, just to name a few, have all gained a lot from how often this method is used. Before the editing process can start, a Double-Strand Breaks (DSB) must be made in the DNA at a specific gene. Researchers have made nucleases, which are sometimes called "molecular scissors," to fix this DSB. Transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs), and homing endonucleases are all examples of protein targets that have been studied and changed. The last part gave an overview of these study projects and explained how to make Cas12a for specific uses. The applications of Cas12a have been extensively studied in recent years, but the protein still holds a lot of promise as a treatment and screening tool. In order to give a quick review of CRISPR-Cas12a and its uses, we will briefly talk about the structure and function of the different parts of the reaction pathway that lead up to the catalysis of the target DNA. Cas12a uses a multistep process to make sure that it is selecting the right DNA. This is a good trait for a device that changes the DNA because it makes it less likely that something bad will happen. Even though data shows that a new CRISPR RNAs (crRNA) molecule can stop Cas12a from randomly destroying ssDNA, it may still hurt the host cell while trying to change the genome. The action of Cas12a catalysis, which is a powerful tool for changing the genome, needs to be changed so that it can be more easily controlled and managed. Using what we know about how Cas12a works, we have made mutants that are less active on ssDNA and more active on dsDNA. So far, only Cas9 and Cas12a, which are both part of the CRISPR family, have been used to change the genome. Because of how similar and different these two endonucleases are, CRISPR can now be used for multiple scientific purposes. Cas12a is better than Cas9 because it makes double-strand breaks (DSBs) that favor Homology-Directed Repair (HDR) over Non-homologous end joining (NHEJ) and can change more than one copy of the genome at the same time. Both Cas9 and Cas12a are being changed to make them better at detecting Protospacer Adjacent Motif (PAM) than they are in their natural state. This will make it possible to start focusing on more genes.

The present study was conducted to investigate the effects of rhizome extract of Dioscorea batatas (Dioscoreae Rhizoma, Chinese Yam) and its bioactive compound, allantoin, on myoblast differentiation and mitochondrial biogenesis in skeletal muscle cells. Yams were extracted in water and the extract was analyzed by HPLC. The expression of C2C12 myotubes differentiation and mitochondrial biogenesis regulators were determined by reverse transcriptase (RT)-PCR or Western blot. The glucose levels and total ATP contents were determined by glucose consumption, glucose uptake and ATP assays, respectively. Treatment with yam extract (1 mg/mL) and allantoin (0.2 and 0.5 mM) significantly increased of MyHC expression compared with non-treated myotubes. Yam extract and allantoin significantly increased the expression of mitochondrial biogenesis regulating proteins, PGC1?, Sirt-1, NRF-1, and TFAM, as well as the phosphorylation of AMPK and ACC in C2C12 myotubes. Furthermore, yam extract and allantoin significantly increased the glucose uptake levels and the ATP contents. Finally, HPLC analysis revealed that the yam extract contained 1.53% of allantoin. Yam extract and allantoin, stimulated myoblast differentiation into myotubes and increased energy production through upregulation of mitochondrial biogenesis regulators. These findings indicate that yam extract and allantoin can help to prevent the skeletal muscle dysfunction through stimulation of energy metabolism.

Age-related declines in vitamin D receptor (VDR) expression have been implicated in the onset of sarcopenia. The increased expression of VDR in muscle is known to increase muscle mass, whereas VDR knockout in the muscle decreases muscle mass in rats. Similar to VDR, the age-related decline in protein deacetylase sirtuin (SIRT) expression is linked to the development of sarcopenia and age-related muscle dysfunction. This study aimed to investigate whether the VDR agonist 1,25-dihydroxyvitamin D3 (1,25VD3) exerts beneficial effects on muscles through interactions with sirtuins and, if so, the underlying molecular mechanisms. 1,25VD3 treatment increases the expression of VDR, SIRT1, and SIRT3 in C2C12 myotubes. Additionally, it stimulates the differentiation of C2C12 myoblasts into myotubes. Furthermore, 1,25VD3 significantly enhanced the expression of key myogenic markers, including myosin heavy chain (MyHC) proteins (MyHC I and II), MyoD, and MyoG, and increased the phosphorylation of AMPK and AKT. Conversely, VDR knockdown in myoblasts induces myotube atrophy by downregulating SIRT1 and SIRT3 expression. Furthermore, 1,25VD3 ameliorated muscle atrophy and apoptosis induced by IFN-γ/TNF-α co-treatment to C2C12 myotube. Moreover, 1,25VD3 downregulated the increased expression of muscle atrophy-associated proteins, including FoxO3a, MAFbx, and MuRF1 in IFN-γ/TNF-α induced atrophy model. Importantly, IFN-γ/TNF-α significantly reduced the mtDNA copy number in C2C12 myotube, whereas the presence of 1,25VD3 effectively prevented this decrease. These results indirectly suggest that 1,25VD3 has the potential to develop as a therapeutic or preventive agent for age-mediated muscle atrophy by enhancing the VDR/SIRT1/SIRT3 axis and inhibiting the FoxO3-mediated atrophy pathway.

Polycystic ovary syndrome (PCOS) is a complex, but relatively common endocrine disorder associated with chronic anovulation, hyperandrogenism, and micro-polycystic ovaries [1]. In addition to reduced fertility, people with PCOS have a higher risk of obesity, insulin resistance, and metabolic disease [1], all comorbidities that are associated with mitochondrial dysfunction. This review summarizes human and animal data that report mitochondrial dysfunction and metabolic dysregulation in PCOS to better understand how mitochondria impact reproductive organ pathophysiology. This in-depth review considers all the elements regulating mitochondrial quantity and quality, from mitochondrial biogenesis under transcriptional regulation of both the nuclear and the mitochondrial genome, to the ultrastructural and functional complexes that regulate cellular metabolism and reactive oxygen species production, to dynamics which regulate subcellular interactions that are key to mitochondrial quality control. When any of these mitochondrial functions are disrupted the energetic equilibrium within the cell changes, cell processes can fail, and cell death can occur. If this process is ongoing, it affects tissue and organ function to cause disease. The objective of this review is to consolidate and classify a broad number of PCOS studies to understand how various mitochondrial processes impact reproductive organs including the ovary (oocyte and granulosa cell), uterus, placenta, and circulation to cause reproductive pathophysiology. A secondary objective is to uncover the potential role of mitochondria in transgenerational transmission of PCOS and metabolic disorders.

Mitochondrial dysregulation is implicated in numerous neurological disorders. Mitochondrial dynamics, including biogenesis, fusion and fission, are essential components of mitostasis which is modulated by complex regulatory mechanisms. Although expression studies are often used to investigate mitochondrial dynamics, these studies may be limited by the interdependent and temporal nature of mitostasis. Transmission electron microscopy (TEM) and cryogenic preparation methods provide a direct approach to examine mitochondrial ultrastructure in neurons. We investigated the utility of TEM to visualize mitochondrial morphological changes in SH-SY5Y cells treated with propionic acid (PPA). We examined whether morphological alterations were associated with differences in membrane potential or expression of biogenesis, fusion and fission genes. PPA induced a significant decrease in mitochondrial area (p<0.01 5mM), Feret's diameter and perimeter (p<0.05 5mM), and in area2 (p<0.05 3mM, p<0.01 5mM) – consistent with a shift towards fission. Morphological changes were not associated with significant differences in mitochondrial membrane potential. However, we observed decreased gene expression of NRF1 (p<0.01), TFAM (p<0.05), and STOML2 (p<0.0001). These data support a disruption of the balance in dynamics to preserve function under stress. This demonstrates the utility of TEM to provide insight into mitochondrial dynamics and function which can inform targeted mechanistic investigations into neuropathology.

It is standard to identify and compare predicted protein sequence of the Drosha and Pasha genes subsidiary to detection and identification of novel microRNAs in newly sequenced taxa or review of previous deep sequencing data. Drosha and Pasha proteins are the key, conserved members of the ‘microprocessor’ protein complex which facilitates nuclear nuclear localized, pri to pre miRNA processing miRNAs of the canonical eumetazoan complement. Because of the necessity of the microprocessor for production of c anonical eumetazoan miRNA, the detection of both (1) bona fide microRNAs and (2) presence of Drosha/Pasha orthologs (or homologs) is often presented as sufficient to represent a functional canonical eumetazoan microRNA biogenesis pathway. However, the fun ctional role of the Drosha and Pasha homologs sometimes, though not always experimentally validated in non model taxa. Differentiation of ‘bona fide miRNAs’, opposed to ‘non bona fide’ small RNAs of similar size, are also necessary for miRNA identificatio n projects. Recent rubrics are based on structural and sequence elements of the miRNAs themselves, however these inclusion criteria include paraphyletic groupings of miRNAs, for example eumetazoan miRNAs and S treptophyte (green plant) miRNAs which are not produced by the Drosha/Pasha microprocessor mechanism. Therefore, a dichotomy exists between the structural definitions for miRNAs and understanding of the evolutionarily conserved function of the microprocessor and its components. In this article, I re view literature in the context of this topic and discuss philosophical significance for understanding the importance of the microprocessor in understanding the evolutionary and molecular origins of miRNA.

Theaflavins, the characteristic and bioactive polyphenols in black tea, possess the potential improvement effects on insulin resistance-associated metabolic abnormalities including obesity and type 2 diebetes. However, the molecular mechanisms of theaflavins improving insulin sensitivity are still not clear. In this study, we investigated the protective effects and mechanisms of theaflavins on palmitic acid-induced insulin resistance in HepG2 cells. Theaflavins could significantly increase glucose uptake of insulin-resistant cells at noncytotoxic doses. This activity was mediated by upregulating the glucose transporter 4 protein expression, increasing the phosphorylation of IRS-1 at Ser307, and reduced the phosphor-Akt (Ser473) level. Moreover, theaflavins were found to enhance mitochondrial DNA copy number through down-regulate the PGC-1β mRNA level and up-regulate PRC mRNA expression in insulin-resistant HepG2 cells. These results indicated that theaflavins could improve free fatty acid-induced hepatic insulin resistance by promoting mitochondrial biogenesis, and were promising functional food and medicines for insulin resistance-related disorders.

The advent of RNA-sequencing (RNA-Seq) technologies has markedly improved our knowledge and expanded the compendium of small non-coding RNAs, most of which derive from the processing of longer RNA precursors. In this review article, we will discuss about the biogenesis and function of small non-coding RNAs derived from eukaryotic ribosomal RNA (rRNA), called rRNA fragments (rRFs), and their potential role(s) as regulator of gene expression. This relatively new class of ncRNAs remained poorly investigated and underappreciated until recently, due mainly to the a priori exclusion of rRNA sequences—because of their overabundance—from RNA-Seq datasets. The situation surrounding rRFs resembles that of microRNAs (miRNAs), which used to be readily discarded from further analyses, for more than five decades, because we could not believe that RNA of such a short length could bear biological significance. As if we had not yet learned our lesson not to restrain our investigative, scientific mind from challenging widely accepted beliefs or dogmas, and from looking for the hidden treasures in the most unexpected places.

Mitochondrial dysfunction is relevant mechanism in cardiac aging. Here, we investigated the effects of late-life enalapril administration at non-antihypertensive dose on mitochondrial genomic stability, oxidative damage, and mitochondrial quality control (MQC) signaling in the heart of aged rats. The protein expression of selected mediators (i.e., mitochondrial antioxidant enzymes, energy metabolism, mitochondrial biogenesis, dynamics, and autophagy) was measured in old rats randomly assigned to receive enalapril (n=8) or placebo (n=8) from 24 to 27 months of age. We also assessed mitochondrial DNA (mtDNA) content, citrate synthase activity, oxidative lesions to protein and mtDNA (i.e., carbonyls and abundance of mtDNA4834 deletion), and mitochondrial transcription factor A (TFAM) binding to specific mtDNA regions. Enalapril attenuated cardiac hypertrophy and oxidative stress-derived damage (mtDNA oxidation, mtDNA4834 deletion, and protein carbonylation), while increasing mitochondrial antioxidant defenses. TFAM binding to mtDNA regions involved in replication and deletion generation was increased following enalapril administration. Increased mitochondrial mass as well as mitochondriogenesis and autophagy signaling was found in enalapril-treated rats. Late-life enalapril administration mitigates age-dependent cardiac hypertrophy and oxidative damage, while increasing mitochondrial mass and modulating MQC signaling. Further analyses are needed to conclusively establish whether enalapril may offer cardioprotection during aging.

Cisplatin (CDDP), carboplatin (CP), and oxaliplatin (OXP) are three platinating agents clinically approved worldwide for use against a variety of cancers. They are canonically known as DNA damage inducers; however, that is only one of their mechanisms of cytotoxicity. CDDP mediates its effects through DNA damage-induced transcription inhibition and apoptotic signalling. In addition, CDDP targets the endoplasmic reticulum (ER) to induce ER-stress, the mitochondria via mitochondrial DNA damage leading to ROS production, and the plasma membrane and cytoskeletal components. CP acts in a similar fashion to CDDP by inducing DNA damage, mitochondrial damage, and ER stress. Additionally, CP is also able to upregulate micro-RNA activity, enhancing intrinsic apoptosis. OXP, on the other hand, at first induces damage to all the same targets as CDDP and CP, yet it is also capable of inducing immunogenic cell death via ER stress and can decrease ribosome biogenesis through its nucleolar effects. In this comprehensive review, we provide detailed mechanisms of action for the three platinating agents, going beyond their nuclear effects to include their cytoplasmic impact within cancer cells. In addition, we cover their current clinical use and limitations, including side effects and mechanisms of resistance.