Highly Sensitive Analysis of Cervical Mucosal HIV-1 Infection Using Reporter Viruses Expressing Secreted Nanoluciferase.

Ex vivo cervical tissue explant models offer a physiologically relevant approach for studying virus-host interactions that underlie mucosal HIV-1 transmission to women. However, the utility of cervical explant tissue (CET) models has been limited for both practical and technical reasons. These include assay variation, inadequate sensitivity for assessing HIV-1 infection and replication in tissue, and constraints imposed by the requirement for using multiple replica samples of CET to test each experimental variable and assay parameter. Here, we describe an experimental approach that employs secreted nanoluciferase (sNLuc) and current HIV-1 reporter virus technologies to overcome certain limitations of earlier ex vivo CET models. This method augments application of the CET model for investigating important questions involving mucosal HIV-1 transmission.

A Semiquantitative Protein-Fragment Complementation Assay to Study Protein-Protein Interactions of the Polymerase Complex in Cellula.

Protein-fragment complementation assays (PCAs) are powerful tools to investigate protein-protein interactions in a cellular context. These are especially useful to study unstable proteins and weak interactions that may not resist protein isolation or purification. The PCA based on the reconstitution of the Gaussia princeps luciferase (split-luc) is a sensitive approach allowing the mapping of protein-protein interactions and the semiquantitative measurement of binding affinity. Here, we describe the split-luc protocol we used to map the viral interactome of measles virus polymerase complex.

Bioluminescence Imaging of Potassium Ion Using a Sensory Luciferin and an Engineered Luciferase.

Bioluminescent indicators are power tools for studying dynamic biological processes. In this study, we present the generation of novel bioluminescent indicators by modifying the luciferin molecule with an analyte-binding moiety. Specifically, we have successfully developed the first bioluminescent indicator for potassium ions (K+), which are critical electrolytes in biological systems. Our approach involved the design and synthesis of a K+-binding luciferin named potassiorin. Additionally, we engineered a luciferase enzyme called BRIPO (bioluminescent red indicator for potassium) to work synergistically with potassiorin, resulting in optimized K+-dependent bioluminescence responses. Through extensive validation in cell lines, primary neurons, and live mice, we demonstrated the efficacy of this new tool for detecting K+. Our research demonstrates an innovative concept of incorporating sensory moieties into luciferins to modulate luciferase activity. This approach has great potential for developing a wide range of bioluminescent indicators, advancing bioluminescence imaging (BLI), and enabling the study of various analytes in biological systems.

Receptor Targeting Using Copolymer-Modified Gold Nanoparticles for pCMV-Luc Gene Delivery to Liver Cancer Cells In Vitro.

The formulation of novel delivery protocols for the targeted delivery of genes into hepatocytes by receptor mediation is important for the treatment of liver-specific disorders, including cancer. Non-viral delivery methods have been extensively studied for gene therapy. Gold nanoparticles (AuNPs) have gained attention in nanomedicine due to their biocompatibility. In this study, AuNPs were synthesized and coated with polymers: chitosan (CS), and polyethylene glycol (PEG). The targeting moiety, lactobionic acid (LA), was added for hepatocyte-specific delivery. Physicochemical characterization revealed that all nano-formulations were spherical and monodispersed, with hydrodynamic sizes between 70 and 250 nm. Nanocomplexes with pCMV-Luc DNA (pDNA) confirmed that the NPs could bind, compact, and protect the pDNA from nuclease degradation. Cytotoxicity studies revealed that the AuNPs were well tolerated (cell viabilities > 70%) in human hepatocellular carcinoma (HepG2), embryonic kidney (HEK293), and colorectal adenocarcinoma (Caco-2) cells, with enhanced transgene activity in all cells. The inclusion of LA in the NP formulation was notable in the HepG2 cells, which overexpress the asialoglycoprotein receptor on their cell surface. A five-fold increase in luciferase gene expression was evident for the LA-targeted AuNPs compared to the non-targeted AuNPs. These AuNPs have shown potential as safe and suitable targeted delivery vehicles for liver-directed gene therapy.

Dual-Luciferase Reporter Assay for Prescreening CRISPR (d)Cas9-Mediated Epigenetic Editing on a Plant Promoter Using Human Cells.

Epigenetic editing, also known as EpiEdit, offers an exciting way to control gene expression without altering the DNA sequence. In this study, we evaluate the application of EpiEdit to plant promoters, specifically the MLO (mildew locus o) gene promoter. We use a modified CRISPR-(d)Cas9 system, in which the nuclease-deficient Cas9 (dCas9) is fused to an epigenetic modifier, to experimentally demonstrate the utility of this tool for optimizing epigenetic engineering of a plant promoter prior to in vivo plant epigenome editing. Guide RNAs are used to deliver the dCas9-epigenetic modifier fusion protein to the target gene sequence, where it induces modification of MLO gene expression. We perform preliminary experiments using a plant promoter cloned into the luciferase reporter system, which is transfected into a human system and analyzed using the dual-luciferase reporter assay. The results suggest that this approach may be useful in the early stages of plant epigenome editing, as it can aid in the selection of appropriate modifications to the plant promoter prior to conducting in vivo experiments under plant system conditions. Overall, the results demonstrate the potential of CRISPR (d)Cas9-based EpiEdit for precise and controlled regulation of gene expression.

Glycoprotein-glycoprotein Receptor Binding Detection Using Bioluminescence Resonance Energy Transfer.

The glycoprotein receptors, members of the large G protein-coupled receptor family, are characterized by a large extracellular domains responsible for binding their glycoprotein hormones. Hormone-receptor interactions are traditionally analyzed by ligand-binding assays, most often using radiolabeling but also by thermal shift assays. Despite their high sensitivity, these assays require appropriate laboratory conditions and, often, purified plasma cell membranes, which do not provide information on receptor localization or activity because the assays typically focus on measuring binding only. Here, we apply bioluminescence resonance energy transfer in living cells to determine hormone-receptor interactions between a Gaussia luciferase (Gluc)-luteinizing hormone/chorionic gonadotropin receptor (LHCGR) fusion and its ligands (human chorionic gonadotropin or LH) fused to the enhanced green fluorescent protein. The Gluc-LHCGR, as well as other Gluc-G protein-coupled receptors such as the somatostatin and the C-X-C motif chemokine receptors, is expressed on the plasma membrane, where luminescence activity is equal to membrane receptor expression, and is fully functional. The chimeric enhanced green fluorescent protein-ligands are properly secreted from cells and able to bind and activate the wild-type LHCGR as well as the Gluc-LHCGR. Finally, bioluminescence resonance energy transfer was used to determine the interactions between clinically relevant mutations of the hormones and the LHCGR that show that this bioassay provides a fast and effective, safe, and cost-efficient tool to assist the molecular characterization of mutations in either the receptor or ligand and that it is compatible with downstream cellular assays to determine receptor activation/function.

Alfalfa xeno-miR159a regulates bovine mammary epithelial cell proliferation and milk protein synthesis by targeting PTPRF.

Milk protein content is an important index to evaluate the quality and nutrition of milk. Accumulating evidence suggests that microRNAs (miRNAs) play important roles in bovine lactation, but little is known regarding the cross-kingdom regulatory roles of plant-derived exogenous miRNAs (xeno-miRNAs) in milk protein synthesis, particularly the underlying molecular mechanisms. The purpose of this study was to explore the regulatory mechanism of alfalfa-derived xeno-miRNAs on proliferation and milk protein synthesis in bovine mammary epithelial cells (BMECs). Our previous study showed that alfalfa miR159a (mtr-miR159a, xeno-miR159a) was highly expressed in alfalfa, and the abundance of mtr-miR159a was significantly lower in serum and whey from high-protein-milk dairy cows compared with low-protein-milk dairy cows. In this study, mRNA expression was detected by real-time quantitative PCR (qRT-PCR), and casein content was evaluated by enzyme-linked immunosorbent assay (ELISA). Cell proliferation and apoptosis were detected using the cell counting kit 8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, western blot, and flow cytometry. A dual-luciferase reporter assay was used to determine the regulation of Protein Tyrosine Phosphatase Receptor Type F (PTPRF) by xeno-miR159a. We found that xeno-miR159a overexpression inhibited proliferation of BMEC and promoted cell apoptosis. Besides, xeno-miR159a overexpression decreased ß-casein abundance, and increased α-casein and κ-casein abundance in BMECs. Dual-luciferase reporter assay result confirmed that PTPRF is a target gene of xeno-miR159a. These results provide new insights into the mechanism by which alfalfa-derived miRNAs regulate BMECs proliferation and milk protein synthesis.

[Bupleuri Radix-Paeoniae Radix Alba medicated plasma exerts effects on HepG2 hepatoma cells by regulating miR-1297/PTEN signaling axis].

The present study aimed to investigate the effect and mechanism of Bupleuri Radix-Paeoniae Radix Alba medicated plasma on HepG2 hepatoma cells by regulating the microRNA-1297(miR-1297)/phosphatase and tensin homologue deleted on chromosome 10(PTEN) signaling axis. Real-time quantitative PCR(RT-qPCR) was carried out to determine the mRNA levels of miR-1297 and PTEN in different hepatoma cell lines. The dual luciferase reporter assay was employed to verify the targeted interaction between miR-1297 and PTEN. The cell counting kit-8(CCK-8) was used to detect cell proliferation, and the optimal concentration and intervention time of the medicated plasma were determined. The cell invasion and migration were examined by Transwell assay and wound healing assay. Cell cycle distribution was detected by PI staining, and the apoptosis of cells was detected by Annexin V-FITC/PI double staining. The mRNA levels of miR-1297, PTEN, protein kinase B(Akt), and phosphatidylinositol 3-kinase(PI3K) were determined by RT-qPCR. Western blot was employed to determine the protein levels of PTEN, Akt, p-Akt, caspase-3, caspase-9, B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax). The results showed that HepG2 cells were the best cell line for subsequent experiments. The dual luciferase reporter assay confirmed that miR-1297 could bind to the 3'-untranslated region(3'UTR) in the mRNA of PTEN. The medicated plasma inhibited the proliferation of HepG2 cells, and the optimal intervention concentration and time were 20% and 72 h. Compared with the blank plasma, the Bupleuri Radix-Paeoniae Radix Alba medicated plasma, miR-1297 inhibitor, miR-1297 inhibitor + medicated plasma all inhibited the proliferation, invasion, and migration of HepG2 cells, increased the proportion of cells in the G_0/G_1 phase, decreased the proportion of cells in the S phase, and increased the apoptosis rate. The medicated plasma down-regulated the mRNA levels of miR-1297, PI3K, and Akt and up-regulated the mRNA level of PTEN. In addition, it up-regulated the protein levels of PTEN, Bax, caspase-3, and caspsae-9 and down-regulated the protein levels of p-Akt, p-PI3K, and Bcl-2. In conclusion, Bupleuri Radix-Paeoniae Radix Alba medicated plasma can inhibit the expression of miR-1297 in HepG2 hepatoma cells, promote the expression of PTEN, and negatively regulate PI3K/Akt signaling pathway, thereby inhibiting the proliferation and inducing the apoptosis of HepG2 cells.

MicroRNA-542-3p targets Pten to inhibit the myoblasts proliferation but suppresses myogenic differentiation independent of targeted Pten.

BACKGROUND: Non-coding RNA is a key epigenetic regulation factor during skeletal muscle development and postnatal growth, and miR-542-3p was reported to be conserved and highly expressed in the skeletal muscle among different species. However, its exact functions in the proliferation of muscle stem cells and myogenesis remain to be determined. METHODS: Transfection of proliferative and differentiated C2C12 cells used miR-542-3p mimic and inhibitor. RT-qPCR, EdU staining, immunofluorescence staining, cell counting kit 8 (CCK-8), and Western blot were used to evaluate the proliferation and myogenic differentiation caused by miR-542-3p. The dual luciferase reporter analysis and rescued experiment of the target gene were used to reveal the molecular mechanism. RESULTS: The data shows overexpression of miR-542-3p downregulation of mRNA and protein levels of proliferation marker genes, reduction of EdU+ cells, and cellular vitality. Additionally, knocking it down promoted the aforementioned phenotypes. For differentiation, the miR-542-3p gain-of-function reduced both mRNA and protein levels of myogenic genes, including MYOG, MYOD1, et al. Furthermore, immunofluorescence staining immunized by MYHC antibody showed that the myotube number, fluorescence intensity, differentiation index, and myotube fusion index all decreased in the miR-542-3p mimic group, compared with the control group. Conversely, these phenotypes exhibited an increased trend in the miR-542-3p inhibitor group. Mechanistically, phosphatase and tensin homolog (Pten) was identified as the bona fide target gene of miR-542-3p by dual luciferase reporter gene assay, si-Pten combined with miR-542-3p inhibitor treatments totally rescued the promotion of proliferation by loss-function of miR-542-3p. CONCLUSIONS: This study indicates that miR-542-3p inhibits the proliferation and differentiation of myoblast and Pten is a dependent target gene of miR-542-3p in myoblast proliferation, but not in differentiation.

In vivo bioluminescence imaging of the intracerebral fibroin-controlled AAV-α-synuclein diffusion for monitoring the central nervous system and peripheral expression.

Among the several animal models of α-synucleinopathies, the well-known viral vector-mediated delivery of wild-type or mutated (A53T) α-synuclein requires new tools to increase the lesion in mice and follow up in vivo expression. To this end, we developed a bioluminescent expression reporter of the human A53T-α-synuclein gene using the NanoLuc system into an AAV2/9, embedded or not in a fibroin solution to stabilise its expression in space and time. We first verified the expression of the fused protein in vitro on transfected cells by bioluminescence and Western blotting. Next, two groups of C57Bl6Jr mice were unilaterally injected with the AAV-NanoLuc-human-A53T-α-synuclein above the substantia nigra combined (or not) with fibroin. We first show that the in vivo cerebral bioluminescence signal was more intense in the presence of fibroin. Using immunohistochemistry, we find that the human-A53T-α-synuclein protein is more restricted to the ipsilateral side with an overall greater magnitude of the lesion when fibroin was added. However, we also detected a bioluminescence signal in peripheral organs in both conditions, confirmed by the presence of viral DNA corresponding to the injected AAV in the liver using qPCR.

Genome assembly of Genji firefly (Nipponoluciola cruciata) reveals novel luciferase-like luminescent proteins without peroxisome targeting signal.

The Genji firefly, Nipponoluciola cruciata, is an aquatic firefly endemic to Japan, inhabiting a wide area of the Japanese archipelago. The luminescence of fireflies is a scientifically interesting phenomenon, and many studies have evaluated this species in Japan. In this study, we sequenced the whole genome of male N. cruciata and constructed a high-quality genome assembly of 662 Mb with a BUSCO completeness of 99.1% in the genome mode. Using the detected set of 15,169 protein-coding genes, the genomic structures and genetic background of luminescence-related genes were also investigated. We found four new firefly luciferase-like genes in the genome. The highest bioluminescent activity was observed for LLa2, which originated from ancestral PDGY, a mitochondrial acyl-CoA synthetase. A thioesterase candidate, NcruACOT1, which is involved in d-luciferin biosynthesis, was expressed in the lantern. Two opsins were also detected and the absorption wavelength of the UV-type opsin candidate shifted from UV to blue. These findings provide an important resource for unravelling the adaptive evolution of fireflies in terms of luminescence and vision.

Low-Level miR-199 Contribute to Neuropathic Low Back Pain via TRPV1 by Regulating the Production of Pro-Inflammatory Cytokines on Macrophage.

AIM: To explore the post-translational regulation of TRPV1, which plays an important role in neuropathic low back pain (NLBP). MATERIAL AND METHODS: qPCR was used to examine the gene mRNA levels. Western blot was used to examine the protein level. NLBP rat model was established for confirming what we observed in clinical samples. Dual-luciferase assay was used to verify the miR-199 targets on the 3'UTR of TRPV1. Cell coculture was used to explore the interaction between macrophages and nerve cells. RESULTS: We found the mRNA level of TRVP1 decreased in the sinuvertebral nerve biopsy of NLBP. With bioinformatics prediction, miR199 would involve the post-transcription regulation of TRPV1. As the prediction, the miR199 level decreased in the clinical samples. Correlation regression analysis showed a negative correlation between miR-199 and TRPV1. The same phenomenon was confirmed in the rat NLBP model. With dual-luciferase assay, we confirmed that miR199 directly binds to the 3'UTR of TRPV1. Through co-culture of macrophage (THP1) and sNF96.2, we found that up or down-regulates miR-199 in macrophage and sNF96.2 could relieve or aggravate the injury of nerve cells strain. CONCLUSION: These results suggest that the occurrence of NLBP may be caused by the lower expression of miR-199 in macrophages and nerve via TRPV1.

Discovery of Red-Shifting Mutations in Firefly Luciferase Using High-Throughput Biochemistry.

Photinus pyralis luciferase (FLuc) has proven a valuable tool for bioluminescence imaging, but much of the light emitted from the native enzyme is absorbed by endogenous biomolecules. Thus, luciferases displaying red-shifted emission enable higher resolution during deep-tissue imaging. A robust model of how protein structure determines emission color would greatly aid the engineering of red-shifted mutants, but no consensus has been reached to date. In this work, we applied deep mutational scanning to systematically assess 20 functionally important amino acid positions on FLuc for red-shifting mutations, predicting that an unbiased approach would enable novel contributions to this debate. We report dozens of red-shifting mutations as a result, a large majority of which have not been previously identified. Further characterization revealed that mutations N229T and T352M, in particular, bring about unimodal emission with the majority of photons being >600 nm. The red-shifting mutations identified by this high-throughput approach provide strong biochemical evidence for the multiple-emitter mechanism of color determination and point to the importance of a water network in the enzyme binding pocket for altering the emitter ratio. This work provides a broadly applicable mutational data set tying FLuc structure to emission color that contributes to our mechanistic understanding of emission color determination and should facilitate further engineering of improved probes for deep-tissue imaging.

Noxa inhibits oncogenesis through ZNF519 in gastric cancer and is suppressed by hsa-miR-200b-3p.

While Phorbol-12-myristate-13-acetate-induced protein 1 (Noxa/PMAIP1) assumes a pivotal role in numerous tumors, its clinical implications and underlying mechanisms of gastric cancer (GC) are yet enigmatic. In this investigation, our primary objective was to scrutinize the clinical relevance and potential mechanisms of Noxa in gastric cancer. Immunohistochemical analysis was conducted on tissue microarrays comprising samples from a meticulously characterized cohort of 84 gastric cancer patients, accompanied by follow-up data, to assess the expression of Noxa. Additionally, Noxa expression levels in gastric cancer clinical samples and cell lines were measured through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. The effect of Noxa expression on the prognosis of patients with gastric cancer was evaluated using Kaplan-Meier survival. Further insight into the role of Noxa in driving gastric cancer progression was gained through an array of experimental techniques, including cell viability assays (CCK8), plate cloning assays, transwell assays, scratch assays, and real-time cell analysis (RTCA). Potential upstream microRNAs (miRNAs) that might modulate Noxa were identified through rigorous bioinformatics analysis, substantiated by luciferase reporter assays and Western blot experiments. Additionally, we utilized RNA sequencing, qRT-PCR, and Western blot to identify proteins binding to Noxa and potential downstream target. Finally, we utilized BALB/c nude mice to explore the role of Noxa in vivo. Our investigation unveiled a marked downregulation of Noxa expression in gastric cancer and underscored its significance as a pivotal prognostic factor influencing overall survival (OS). Noxa overexpression exerted a substantial inhibitory effect on the proliferation, migration and invasion of GC cells. Bioinformatic analysis and dual luciferase reporter assays unveiled the capacity of hsa-miR-200b-3p to interact with the 3'-UTR of Noxa mRNA, thereby orchestrating a downregulation of Noxa expression in vitro, consequently promoting tumor progression in GC. Our transcriptome analysis, coupled with mechanistic validation, elucidated a role for Noxa in modulating the expression of ZNF519 in the Mitophagy-animal pathway. The depletion of ZNF519 effectively reversed the oncogenic attributes induced by Noxa. Upregulation of Noxa expression suppressed the tumorigenesis of GC in vivo. The current investigation sheds light on the pivotal role of the hsa-miR-200b-3p/Noxa/ZNF519 axis in elucidating the pathogenesis of gastric cancer, offering a promising avenue for targeted therapeutic interventions in the management of this challenging malignancy.

Hepatocyte-derived exosomes deliver the lncRNA CYTOR to hepatic stellate cells and promote liver fibrosis.

Liver fibrosis is characterized by the activation and transformation of hepatic stellate cells (HSCs) induced by various injury factors. The degree of liver fibrosis can be significantly improved, but persistent injury factors present a significant therapeutic challenge. Hepatocytes are the most important parenchymal cell type in the liver. In this study, we explored the molecular mechanisms by which damaged liver cells activate HSCs through extracellular vesicles. We established a coculture model of LO2 and LX2 and validated its exosomal transmission activity. Subsequently, differentially expressed long noncoding RNAs (lncRNAs) were screened through RNA sequencing and their mechanisms of action as competing endogenous RNAs (ceRNAs) further confirmed using biological methods, such as FISH and luciferase assays. Damaged liver cells induced activation of LX2 and upregulation of liver fibrosis-related markers. Exosomes extracted and identified from the supernatant fraction contained differentially expressed lncRNA cytoskeleton regulator RNA (CYTOR) that competed with microRNA-125 (miR-125) for binding to glial cell line-derived neurotrophic factor (GDNF) in HSCs, in turn, promoting LX2 activation. MiR-125 could target and regulate both CYTOR and GDNF and vice versa, as verified using the luciferase assay. In an in vivo model, damaged liver extracellular vesicles induced the formation of liver fibrosis. Notably, downregulation of CYTOR within extracellular vesicles effectively inhibited liver fibrosis. The lncRNA CYTOR in exosomes of damaged liver cells is upregulated and modulates the expression of downstream GDNF through activity as a ceRNA, providing an effective mechanism for activation of HSCs.

miR-215-5p Plays a Key Role in Suppressing Vascular Invasion and Recurrence in Hepatocellular Carcinoma by Blocking Vasculogenic Mimicry.

BACKGROUND: This research explores the significance of miR-215-5p and vasculogenic mimicry (VM) in forecasting the prognosis for hepatocellular carcinoma (HCC). METHODS: We analyzed HCC-associated miRNA expression profiles using data from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). Samples included tissue and blood from 80 early-stage HCC patients and serum from 120 healthy individuals. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was employed to measure miR-215-5p and zinc finger E-box binding homeobox 2 (ZEB2) gene expressions. Hematoxylin and eosin (H&E) and CD34/Periodic Acid-Schiff (PAS) double staining assessed VM presence in HCC tissue sections. Bioinformatics tools predicted interactions between miR-215-5p and ZEB2, confirmed through luciferase reporter assays. We also examined the impact of miR-215-5p or ZEB2 overexpression on HCC cell invasion, migration, and VM formation using scratch, Transwell invasion assays, and Matrigel 3D cultures. RESULTS: Bioinformatics analysis indicated that miR-215-5p was under-expressed in HCC, particularly in cases with vascular invasion, which correlated with worse patient outcomes. In contrast, ZEB2, targeted by miR-215-5p, was overexpressed in HCC. RT-qPCR validated these expression patterns in HCC tissues. Among the HCC patients, 38 were VM positive and 42 VM negative. Logistic regression highlighted a negative correlation between miR-215-5p levels and VM positivity in HCC tissues and a positive correlation for ZEB2 with VM positivity and tumor vascular invasion. Lower miR-215-5p levels were linked to increased HCC recurrence and metastasis. Both bioinformatics analysis and luciferase assays demonstrated a direct interaction between miR-215-5p and ZEB2. Enhancing miR-215-5p levels reduced ZEB2 expression, consequently diminishing invasion, migration, and VM formation of the HCC cells in vitro. CONCLUSIONS: miR-215-5p expression inversely correlates with VM occurrence in HCC tissues, while ZEB2 expression shows a direct correlation. By targeting ZEB2, miR-215-5p may hinder VM in HCC tissues, helping to prevent vascular invasion and HCC recurrence. Thus, miR-215-5p emerges as a vital prognostic indicator for predicting vascular invasion and recurrence in HCC.

Plasma microRNA-320c as a Potential Biomarker for the Severity of Knee Osteoarthritis and Regulates cAMP Responsive Element Binding Protein 5 (CREB5) in Chondrocytes.

Objective: Osteoarthritis (OA) is a commonly known prevalent joint disease, with limited therapeutic methods. This study aimed to investigate the expression of plasma microRNA-320c (miR-320c) in patients with knee OA and to explore the clinical value and potential mechanism of miR-320c in knee OA. Methods: Forty knee OA patients and 20 healthy controls were enrolled. The levels of plasma miR-320c and plasma inflammatory cytokines were measured by real-time PCR or ELISA. Correlations of Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores and cytokine levels with the miR-320c expression level were evaluated by Pearson correlation analysis. Then, a receiver operating characteristic (ROC) curve was drawn to analyse the diagnostic value of miR-320c in OA. Finally, the interaction of miR-320c and cAMP responsive element binding protein 5 (CREB5) was determined using a luciferase reporter assay, and the effect of CREB5 on the cAMP pathway was assessed. Results: The expression level of plasma miR-320c was significantly higher in OA patients than in healthy controls (p < 0.05). The increased plasma miR-320c level was positively correlated with the WOMAC score (r = 0.796, p < 0.001) and the plasma interleukin (IL)-1ß (r = 0.814, p < 0.001) and IL-6 (r = 0.695, p < 0.001) levels in patients with OA. ROC curve analysis demonstrated the relatively high diagnostic accuracy of plasma miR-320c for OA. Furthermore, the luciferase reporter assay results showed that miR-320c regulates CREB5 expression by binding to the CREB5 3'-untranslated region. Moreover, suppression of CREB5 significantly reduced the expression levels of c-fos and c-jun. Conclusion: Our results indicate that plasma miR-320c may serve as a potential novel predictor of the severity of knee OA and that miR-320c may play an important role in the pathogenesis of OA through inhibiting the cAMP pathway by targeting CREB5.

mir-150-5p inhibits the osteogenic differentiation of bone marrow-derived mesenchymal stem cells by targeting irisin to regulate the p38/MAPK signaling pathway.

PURPOSE: To study the effect of miR-150-5p on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), and further explore the relationship between its regulatory mechanism and irisin. METHODS: We isolated mouse BMSCs, and induced osteogenic differentiation by osteogenic induction medium. Using qPCR to detect the expression of osteogenic differentiation-related genes, western blot to detect the expression of osteogenic differentiation-related proteins, and luciferase reporter system to verify that FNDC5 is the target of miR-150-5p. Irisin intraperitoneal injection to treat osteoporosis in mice constructed by subcutaneous injection of dexamethasone. RESULTS: Up-regulation of miR-150-5p inhibited the proliferation of BMSCs, and decreased the content of osteocalcin, ALP activity, calcium deposition, the expression of osteogenic differentiation genes (Runx2, OSX, OCN, OPN, ALP and BMP2) and protein (BMP2, OCN, and Runx2). And down-regulation of miR-150-5p plays the opposite role of up-regulation of miR-150-5p on osteogenic differentiation of BMSCs. Results of luciferase reporter gene assay showed that FNDC5 gene was the target gene of miR-150-5p, and miR-150-5p inhibited the expression of FNDC5 in mouse BMSCs. The expression of osteogenic differentiation genes and protein, the content of osteocalcin, ALP activity and calcium deposition in BMSCs co-overexpressed by miR-150-5p and FNDC5 was significantly higher than that of miR-150-5p overexpressed alone. In addition, the overexpression of FNDC5 reversed the blocked of p38/MAPK pathway by the overexpression of miR-150-5p in BMSCs. Irisin, a protein encoded by FNDC5 gene, improved symptoms in osteoporosis mice through intraperitoneal injection, while the inhibitor of p38/MAPK pathway weakened this function of irisin. CONCLUSION: miR-150-5p inhibits the osteogenic differentiation of BMSCs by targeting irisin to regulate the/p38/MAPK signaling pathway, and miR-150-5p/irisin/p38 pathway is a potential target for treating osteoporosis.

CRISPR-Based Split Luciferase as a Biosensor for Unique DNA Sequences In Situ.

To date, CRISPR-based DNA targeting approaches have typically used fusion proteins between full fluorescent reporters and catalytically inactive Cas9 (dCas9) for imaging rather than detection of endogenous genomic DNA sequences. A promising alternative strategy for DNA targeting is the direct biosensing of user-defined sequences at single copy with single-cell resolution. Our recently described DNA biosensing approach using a dual fusion protein biosensor comprised of two independently optimized fragments of NanoLuc luciferase (NLuc) directionally fused to dCas9 paired with user-defined single-guide RNAs (sgRNAs) could allow users to sensitively detect unique copies of a target sequence in individual living cells using common laboratory equipment such as a microscope or a luminescence-equipped microplate reader. Here we describe a protocol for using such a DNA biosensor noninvasively in situ.

Oxygen imaging of hypoxic pockets in the mouse cerebral cortex.

Consciousness is lost within seconds upon cessation of cerebral blood flow. The brain cannot store oxygen, and interruption of oxidative phosphorylation is fatal within minutes. Yet only rudimentary knowledge exists regarding cortical partial oxygen tension (Po2) dynamics under physiological conditions. Here we introduce Green enhanced Nano-lantern (GeNL), a genetically encoded bioluminescent oxygen indicator for Po2 imaging. In awake behaving mice, we uncover the existence of spontaneous, spatially defined "hypoxic pockets" and demonstrate their linkage to the abrogation of local capillary flow. Exercise reduced the burden of hypoxic pockets by 52% compared with rest. The study provides insight into cortical oxygen dynamics in awake behaving animals and concurrently establishes a tool to delineate the importance of oxygen tension in physiological processes and neurological diseases.