OsWRKY12 negatively regulates the drought-stress tolerance and secondary cell wall biosynthesis by targeting different downstream transcription factor genes in rice.

With the increasing occurrence of global warming, drought is becoming a major constraint for plant growth and crop yield. Plant cell walls experience continuous changes during the growth, development, and in responding to stressful conditions. The plant WRKYs play pivotal roles in regulating the secondary cell wall (SCW) biosynthesis and helping plant defend against abiotic stresses. qRT-PCR evidence showed that OsWRKY12 was affected by drought and ABA treatments. Over-expression of OsWRKY12 decreased the drought tolerance of the rice transgenics at the germination stage and the seedling stage. The transcription levels of drought-stress-associated genes as well as those genes participating in the ABA biosynthesis and signaling were significantly different compared to the wild type (WT). Our results also showed that less lignin and cellulose were deposited in the OsWRKY12-overexpressors, and heterogenous expression of OsWRKY12 in atwrky12 could lower the increased lignin and cellulose contents, as well as the improved PEG-stress tolerance, to a similar level as the WT. qRT-PCR results indicated that the transcription levels of all the genes related to lignin and cellulose biosynthesis were significantly decreased in the rice transgenics than the WT. Further evidence from yeast one-hybrid assay and the dual-luciferase reporter system suggested that OsWRKY12 could bind to promoters of OsABI5 (the critical component of the ABA signaling pathway) and OsSWN3/OsSWN7 (the key positive regulators in the rice SCW thickening), and hence repressing their expression. In conclusion, OsWRKY12 mediates the crosstalk between SCW biosynthesis and plant stress tolerance by binding to the promoters of different downstream genes.

Utilizing a structure-based virtual screening approach to discover potential LSD1 inhibitors.

BACKGROUND: Lysine-specific demethylase 1 (LSD1) is highly expressed in a variety of malignant tumors, rendering it a crucial epigenetic target for anti-tumor therapy. Therefore, the inhibition of LSD1 activity has emerged as a promising innovative therapeutic approach for targeted cancer treatment. METHODS: In our study, we employed innovative structure-based drug design methods to meticulously select compounds from the ZINC15 database. Utilizing virtual docking, we evaluated docking scores and binding modes to identify potential inhibitors. To further validate our findings, we harnessed molecular dynamic simulations and conducted meticulous biochemical experiments to deeply analyze the binding interactions between the protein and compounds. RESULTS: Our results showcased that ZINC10039815 exhibits an exquisite binding mode with LSD1, fitting perfectly into the active pocket and forming robust interactions with multiple critical residues of the protein. CONCLUSIONS: With its significant inhibitory effect on LSD1 activity, ZINC10039815 emerges as a highly promising candidate for the development of novel LSD1 inhibitors.

Phenylboronic acid modification-based novel dumbbell-shaped Au-Ag nanorod SERS substrates for ultrasensitive detection of SO42.

Based on the coordination principle of Lewis acids, a 4-mercaptophenylboronic acid (4-MPBA)-modified novel dumbbell-shaped Au-Ag nanorod (4-MPBA@DS Au-AgNR) substrate was developed, which could be combined with the surface-enhanced Raman scattering (SERS) technique to detect SO42- with high sensitivity and specificity. DS Au-AgNRs synthesized in this study with a dumbbell-shaped structure were verified by finite-difference time domain (FDTD) simulation to be capable of stimulating strong localized electromagnetic enhancement (EM) at nano-edge and gap, generating a large number of "hot spots" exhibiting excellent SERS performance. The 4-MPBA modified on its surface could specifically recognize SO42-, producing a change in the spectral peak at 1382 cm-1, thus realizing highly sensitive and specific sensing of SO42-. Under optimized conditions, this SERS sensor responded rapidly to SO42- within 2 minutes and demonstrated outstanding specificity. Calculation of the ratio of the characteristic peaks at 1382 and 1070 cm-1 (I1382/I1070) enabled the quantitative detection of SO42- in the range of 1 × 10-8-1 × 10-3 M, and the detection threshold was as low as 1 nM, which was superior to those of similar detection methods. Importantly, the utility and reliability of this SERS substrate for the determination of SO42- in actual samples were evaluated using ion chromatography as the gold standard, and there was no significant difference between the two protocols (P > 0.05), and the RSD was less than 6% with a satisfactory recovery rate (97.6-102.3%). Therefore, the present protocol has the advantages of simplicity and rapidity, high sensitivity, specificity, stability, and practicability in the determination of SO42- in aqueous solution, providing a reliable solution for tracing SO42- in the fields of food safety and environmental testing.

LncRNA AGPG Confers Endocrine Resistance in Breast Cancer by Promoting E2F1 Activity.

Resistance to endocrine therapy represents a major concern for patients with estrogen receptor α-positive (ERα+) breast cancer. Endocrine therapy resistance is commonly mediated by activated E2F signaling. A better understanding of the mechanisms governing E2F1 activity in resistant cells could reveal strategies for overcoming resistance. Here, we identified the long noncoding RNA (lncRNA) actin gamma 1 pseudogene 25 (AGPG) as a regulator of E2F1 activity in endocrine-resistant breast cancer. Expression of AGPG was increased in endocrine-resistant breast cancer cells, which was driven by epigenomic activation of an enhancer. AGPG was also abnormally upregulated in patient breast tumors, especially in the luminal B subtype, and high AGPG expression was associated with poor survival of patients with ERα+ breast cancer receiving endocrine therapy. The upregulation of AGPG mediated resistance to endocrine therapy and cyclin-dependent kinase 4/6 inhibition in breast cancer cells. Mechanistically, AGPG physically interacted with PURα, thus releasing E2F1 from PURα and leading to E2F1 signaling activation in ERα+ breast cancer cells. In patients with breast cancer, E2F1 target genes were positively and negatively correlated with expression of AGPG and PURα, respectively. Coadministration of chemically modified AGPG siRNA and tamoxifen strongly suppressed tumor growth in endocrine-resistant cell line-derived xenografts. Together, these results demonstrate that AGPG can drive endocrine therapy resistance and indicate that it is a promising biomarker and potential therapeutic target in breast cancer. SIGNIFICANCE: Blockade of formation of the PURα/E2F1 complex by lncRNA AGPG activates E2F1 and promotes endocrine resistance, providing potential strategies for combatting endocrine-resistant breast cancer.

Modification of lignin composition by ectopic expressing wheat TaF5H1 led to decreased salt tolerance in transgenic Arabidopsis plants.

Lignin is an important cell wall component that provides plants with mechanical support and improved tolerance to pathogen attacks. Previous studies have shown that plants rich in S-lignin content or with a higher S/G ratio always exhibit higher efficiency in the utilization of lignocellulosic biomass. Ferulate 5-hydroxylase, or coniferaldehyde 5-hydroxylase (F5H, or CAld5H), is the critical enzyme in syringyl lignin biosynthesis. Some F5Hs have been characterized in several plant species, e.g., Arabidopsis, rice, and poplar. However, information about F5Hs in wheat remains unclear. In this study, a wheat F5H gene, TaF5H1, together with its native promoter (pTaF5H1), was functionally characterized in transgenic Arabidopsis. Gus staining results showed that TaF5H1 could be expressed predominantly in the highly lignified tissues in transgenic Arabidopsis plants carrying pTaF5H1:Gus. qRT-PCR results showed that TaF5H1 was significantly inhibited by NaCl treatment. Ectopic expression of TaF5H1 driven by pTaF5H1 (i.e., pTaF5H1:TaF5H1) could increase the biomass yield, S-lignin content, and S/G ratio in transgenic Arabidopsis plants, which could also restore the traces of S-lignin in fah1-2, the Arabidopsis F5H mutant, to an even higher level than the wild type (WT), suggesting that TaF5H1 is a critical enzyme in S lignin biosynthesis, and pTaF5H1:TaF5H1 module has potential in the manipulation of S-lignin composition without any compromise on the biomass yield. However, expression of pTaF5H1:TaF5H1 also led to decreased salt tolerance compared with the WT. RNA-seq analysis showed that many stress-responsive genes and genes responsible for the biosynthesis of cell walls were differentially expressed between the seedlings harboring pTaF5H1:TaF5H1 and the WT, hinting that manipulation of the cell wall components targeting F5H may also affect the stress adaptability of the modified plants due to the interference to the cell wall integrity. In summary, this study demonstrated that the wheat pTaF5H1: TaF5H1 cassette has the potential to modulate S-lignin composition without any compromise in biomass yield in future engineering practice. Still, its negative effect on stress adaptability to transgenic plants should also be considered.

Integrative analyses of bulk microarray data to discover genes, pathways, and immune infiltration characteristics associated with targeting of Ewing sarcoma.

PURPOSE: To explore transcriptome and immunological features of patients with Ewing sarcoma (ES) using all publicly available microarray data. METHODS: Data of 479 ES tissues were integrated and normalized. Gene expression, immune infiltration, and cancer-specific pathways were analyzed. Genes of interest were knocked down, followed by cell proliferation and colony formation assays. RESULTS: Consistent with the previous reports of differential expressed genes (DEGs) in ES, our analysis identified CCND1, HMCN1, and NKX2-2 were among the most highly expressed, while TWNC1, MYBPC1, and CKM were among the lowest expressed genes. GO, KEGG, and GSEA enrichment analysis identified that the DEGs related to bone and muscle functioning, those that contributed to crucial cellular, and metabolism pathways such as actin binding, apoptosis, TCA cycle, and cell cycle were also significantly enriched. Immune infiltration analysis discovered that many T cell subsets including CD4T, CD8 T, and Gamma delta T cells were highly infiltrated, while monocytes and B cells were less infiltrated in tumors. A total of 138 genes were both significantly up-regulated in tumors and associated with decreased survival, while 38 significantly down-regulated genes were associated with increased survival, many of which were previously reported as oncogenes and tumor suppressors in ES and other cancers. Silencing of four newly identified top ranked up-regulated genes with decreased survivals in ES inhibited proliferation and colony formation of ES cells. CONCLUSION: This study may provide a clear representative transcriptome profile of ES, providing diagnostic biomarkers, pathways, and immune infiltrative characteristics targets for ES.

Nuclear access of DNlg3 c-terminal fragment and its function in regulating innate immune response genes.

Neuroligins (NLGNs) are one of the autism susceptibility genes, however, the mechanism that how dysfunction of NLGNs leads to Autism remains unclear. More and more studies have shown that the transcriptome alteration may be one of the important factors to generate Autism. Therefore, we are very concerned about whether Neuroligins would affect transcriptional regulation, which may at last lead to Autism. As a single-transmembrane receptor, proteolytic cleavage is one of the most important posttranslational modifications of NLGN proteins. In this study, we demonstrated the existence of DNlg3 C-terminal fragment. Studies in the S2 cells and HEK293T cells showed the evidence for nuclear access of the DNlg3 C-terminal fragment. Then we identified the possible targets of DNlg3 C-terminal fragment after its nuclear access by RNA-seq. The bioinformatics analysis indicated the transcriptome alteration between dnlg3 null flies and wild type flies focused on genes for the innate immune responses. These results were consistent with the infection hypotheses for autism. Our study revealed the nuclear access ability of DNlg3 c-terminal fragment and its possible function in transcriptional regulation of the innate immune response genes. This work provides the new links between synaptic adhesion molecule NLGNs and immune activation, which may help us to get a deeper understanding on the relationship between NLGNs and Autism.

A prominent gene activation role for C-terminal binding protein in mediating PcG/trxG proteins through Hox gene regulation.

The evolutionarily conserved C-terminal binding protein (CtBP) has been well characterized as a transcriptional co-repressor. Herein, we report a previously unreported function for CtBP, showing that lowering CtBP dosage genetically suppresses Polycomb group (PcG) loss-of-function phenotypes while enhancing that of trithorax group (trxG) in Drosophila, suggesting that the role of CtBP in gene activation is more pronounced in fly development than previously thought. In fly cells, we show that CtBP is required for the derepression of the most direct PcG target genes, which are highly enriched by homeobox transcription factors, including Hox genes. Using ChIP and co-IP assays, we demonstrate that CtBP is directly required for the molecular switch between H3K27me3 and H3K27ac in the derepressed Hox loci. In addition, CtBP physically interacts with many proteins, such as UTX, CBP, Fs(1)h and RNA Pol II, that have activation roles, potentially assisting in their recruitment to promoters and Polycomb response elements that control Hox gene expression. Therefore, we reveal a prominent activation function for CtBP that confers a major role for the epigenetic program of fly segmentation and development.

An essential role for PTIP in mediating Hox gene regulation along PcG and trxG pathways.

During Drosophila development, Polycomb-group and Trithorax group proteins function to ensure correct maintenance of transcription patterns by epigenetically repressing or activating target gene expression. To get a deep insight into the PcG and trxG pathways, we investigated a BRCT domain-containing protein called PTIP, which was generally identified as a transcriptional coactivator and belongs to the TRR complex. At the genome scale, we sorted given PTIP-binding peaks into two groups: PTIP/TRR-cobound and PTIP/PC-cobound peaks. In particular, we found that PTIP mediates the molecular switch between H3K4me3/H3K27ac and H3K27me3 histone modifications at TRR or PC occupied regions. Thus, we suggest that PTIP is a mediator rather than a dedicated co-activator along PcG and trxG pathways. Our hypothesis is further supported by the genetic assay: PTIP interacts genetically with either PcG or TrxG in a dosage-dependent manner, suggesting that PTIP functions as a co-factor of PcG/TrxG proteins. In addition, in accordance with the analysis of ChIP-seq, these genetic interactions correlate with modified ectopic HOX protein levels in imaginal discs, which reveals an essential role for PTIP in PcG-mediated Hox gene repression. Hence, we reveal a novel role for PTIP in the epigenetic regulation of gene expression along PcG and trxG pathways.

An F-box protein from wheat, TaFBA-2A, negatively regulates JA biosynthesis and confers improved salt tolerance and increased JA responsiveness to transgenic rice plants.

Soil salinity is a serious problem encountered by agriculture worldwide, which will lead to many harmful effects on plant growth, development, and even crop yield. F-box protein is the core subunit of the Skp1-Cullin-F-box (SCF) complex E3 ligase and plays crucial roles in regulating the growth, development, biotic & abiotic stresses, as well as hormone signaling pathway in plants. In this study, an FBA type F-box gene TaFBA-2A was isolated from wheat (Triticum aestivum L.). This study showed that TaFBA-2A could interact with TaSKP1, and TaOPR2, the crucial enzyme involving in jasmonic acid (JA) biosynthesis. TaFBA-2A negatively regulates JA biosynthesis, probably by mediating the degradation of TaOPR2 via the ubiquitin-26S proteasome pathway. Ectopic expression of TaFBA-2A improved the salt tolerance and increased the JA responsiveness of the transgenic rice lines. In addition, some agronomic traits closely related to crop yield were significantly enhanced in the rice lines ectopic expressing TaFBA-2A. The data obtained in this study shed light on the function and mechanisms of TaFBA-2A in JA biosynthesis and the responses to salt stress and JA treatment; this study also suggested that TaFBA-2A has the potential in improving the salt tolerance and crop yield of transgenic rice plants.

Association between ABCB1 C3435T polymorphism and antiepileptic drug resistance in epilepsy: An updated meta-analysis based on 62 studies.

OBJECTIVE: Previous clinical studies and meta-analyses have shown controversial results on the association between C3435T polymorphism of the ABCB1 gene and anti-epileptic drug (AED) resistance. Based on the fact that sample size and confounding factors could contribute to the inconsistency, we performed an updated meta-analysis by including the most recent studies, and subgroup analysis was conducted to evaluate the effect of confounding factors on the association. MATERIALS AND METHODS: We searched articles in 6 electronic databases including PubMed, Medline, Embase, Web of science, Cochrane Library, CNKI (China National Knowledge Infrastructure) for relevant articles up to June 2020. RESULTS: The current analysis showed that the C allele of C3435T variant was a risk factor for drug resistance in the overall populations (C allele vs. T allele, OR: 1.13; 95% CI: 1.02 - 1.25; p = 0.02) and in the Caucasians (C allele vs. T allele, OR: 1.09; 95% CI: 1.09 - 1.43; p = 0.002), while no association was observed in Asians and Indians. Particularly, our study reported for the first time that the 3435T allele was more common in epilepsy patients with drug resistance in the Tunisian population (C allele vs. T allele, OR: 0.31; 95% CI: 0.15 - 0.65; p = 0.002). In addition, our present analysis suggested an association between C3435T and AED resistance in cryptogenic, symptomatic, but not in idiopathic patients. Subgroup studies based on age and gender showed no association. CONCLUSION: AED resistance in Caucasian and Tunisian populations may benefit from ABCB1 C3435T genotyping. We recommend that more details, such as gender and etiology of epilepsy, should be taken into account to draw a reliable conclusion in future studies.

MAGT1 is required for HeLa cell proliferation through regulating p21 expression, S-phase progress, and ERK/p38 MAPK MYC axis.

Magnesium transporter subtype 1 (MAGT1) is known to participate in animal development and cell differentiation. Thus far, MAGT1 studies have mainly focused on its role in cardiomyocyte regulation and differentiation; only a few studies have demonstrated its role in cell proliferation. To investigate the underlying mechanism of MAGT1 in cell proliferation, HeLa and SiHa cells were transiently knocked down with different siRNAs. We showed that cell proliferation was substantially restricted by S-phase arrest and apoptosis in the MAGT1-knocked down cells, which was further confirmed by the increased expression of p21, cyclin-A1, and cyclin-B1, as well as the decreased expression of MYC, cyclin-D1, cyclin-E1, and CDK2. MAGT1 knockdown also resulted in significant changes in the transcriptional expression of 1,598 genes that were analyzed by RNA sequencing. Bioinformatics analysis showed that MAGT1 was related to the MAPK signaling pathway. Western blot analysis confirmed that the phosphorylation of extracellular signal-related protein kinase 1/2 (ERK1/2) and p38 was remarkably reduced in MAGT1 down-regulated groups. Additionally, MAGT1 was required for the function of viral proteins E6/E7 during cell proliferation and G1/S cell-cycle progression. Therefore, MAGT1 plays a crucial role in the proliferation of HPV-positive cervical cancer cells, S-phase progression, and the ERK/p38 MAPK signaling pathway. These results indicate the potential of MAGT1 as a novel target for anticancer research.Abbreviations: MAGT1: Magnesium transporter subtype 1; MAPK: Mitogen-activated protein kinase; XMEN: X-linked immunodeficiency with Magnesium defect, Epstein-Barr virus infection and Neoplasia; BMMSCs: Bone Marrow Mesenchymal Stem Cells; Dpp: Decapentaplegic; CDKIs: CDK inhibitors; GPCR: G-protein coupled receptor; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; RTK: Receptor Tyrosine Kinase; PTK: Protein Tyrosine Kinase; FGFR: Fibroblast Growth Factor Receptor; BMP: Bone Morphogenetic Protein; HPV18 E6/E7: Human Papillomavirus 18 Early protein 6/ early protein 7; FACS: Fluorescence Activated Cell Sorting; PI: Propidium Iodide.

Naked cuticle inhibits wingless signaling in Drosophila wing development.

Wnt signaling is one of the major signaling pathways that regulate cell differentiation, tissue patterning and stem cell homeostasis and its dysfunction causes many human diseases, such as cancer. It is of tremendous interests to understand how Wnt signaling is regulated in a precise manner both temporally and spatially. Naked cuticle (Nkd) acts as a negative-feedback inhibitor for Wingless (Wg, a fly Wnt) signaling in Drosophila embryonic development. However, the role of Nkd remains controversial in later fly development, particularly on the canonical Wg pathway. In the present study, we show that nkd is essential for wing pattern formation, such that both gain and loss of nkd result in the disruption of Wg target expression in larvae stage and abnormal adult wing morphologies. Furthermore, we demonstrate that a thirty amino acid fragment in Nkd, identified previously in Wharton lab, is critical for the canonical Wg signaling, but is dispensable for Wg/planar cell polarity pathway. Putting aside the pleiotropic nature of nkd function, i.e. its role in the Decapentaplegic signaling, we conclude that Nkd universally inhibits the canonical Wg pathway across a life span of Drosophila development.

Structural studies of a mannoglucan from Cremastra appendiculata (Orchidaceae) by chemical and enzymatic methods.

Pseudobulb of Cremastra appendiculata (Orchidaceae) is a traditionally used medicine in China for treatment of certain cancers. The polysaccharides from this medicinal plant are poorly understood. Therefore, we focused on the isolation and fine structure characterization of C. appendiculata polysaccharides. After isolation by DE-52 and Superdex 200 gel chromatography, the purified polysaccharide (named as CAP) with Mw 557.5 kDa was obtained with a narrow and symmetric peak presented in the HPGPC. The monosaccharide composition results showed in HPAEC that CAP was a heteropolysaccharide composed of glucose and mannose at a molar ratio roughly 0.34:0.66. The methylation results indicated that CAP was a 1,4-ß-mannose and 1,4-ß-glucose linear linkage. The further NMR studies suggested a 0.208 acetylation substitution of CAP and a hexasaccharide repeating unit composed of 1,4-ß-mannose and1, 4-ß-glucose in the CAP structure. The chemical structure of CAP was confirmed further by the specific glucanase and mannanase hydrolysis results.

Risk prediction of drug-drug interaction potential of phenytoin and miconazole topical formulations.

The drug-drug interaction (DDI) risk of phenytoin with several topical formulations of miconazole is still unclear. The present investigation conducted in vitro-in vivo extrapolation to predict the potential risks. Our data indicated that miconazole potently inhibited phenytoin hydroxylation in both pooled human liver microsomes (HLMs) and recombinant cytochrome P450 2C9 (CYP2C9) with the Ki values of 125 ± 7 nM and 30 ± 2 nM, respectively. Quantitative prediction of DDI risk suggests that, beside intravenous administration or swallowed tablet, combination of phenytoin and miconazole high dose oral gel or buccal tablet may also result in a clinically significant increase of phenytoin AUC (>53%) by the inhibition of miconazole against phenytoin hydroxylation, consequently a higher frequency of adverse events, while the coadministration of miconazole vaginal formulation and phenytoin will be safe.

Structure elucidation of arabinogalactoglucan isolated from Sedum sarmentosum Bunge and its inhibition on hepatocellular carcinoma cells in vitro.

Sedum sarmentosum Bunge (SS) is clinically used as Chinese medicine for hepatitis related diseases treatment. The purpose of this study was to explore the chemical structures of polysaccharides from this plant. A neutral polysaccharide (SSWP) was isolated and purified by ion-exchange chromatography and Superdex-75 column. The obtained SSWP was a homogenous one with a molecular weight of 21.5 kDa according to the high-performance gel permeation chromatography. The major monosaccharide composition of SSWP was arabinose, glucose and galactose in a molar ratio of 2.4:1:1.8. The methylation analysis showed that SSWP consists mainly of Araf-(1→, →5)-Araf-(1→, →3,5)-Araf-(1→, →4)-Galp-(1→, →4)-Glcp-(1→. The NMR result and enzymatic digestion data comprehensively indicated that SSWP was a novel arabinogalactoglucan-type structure. The anticancer assay in vitro exhibited that SSWP could effectively inhibit 48.9% of Huh-7 cells growth at 50 µg/mL and arrest cells at S-phase, and induce tumor cells apoptosis. Together, polysaccharide from S. sarmentosum Bunge could be a potential natural antitumor agent.

A novel surface-enhanced Raman scattering probe based on Au nanoboxes for dynamic monitoring of caspase-3 during cervical cancer cell apoptosis.

The highly sensitive and reliable detection, imaging, and monitoring of changes of intracellular caspase-3 are critical for understanding the cell apoptosis and studying the progression of caspase-3-related cervical cancer. Herein, we present a novel surface-enhanced Raman scattering (SERS) probe for the detection of caspase-3 during cervical cancer cell apoptosis, composed of Au nanoboxes modified with Nile blue A as a Raman reporter and a caspase-3-specified peptide as a molecular cross-linker. In the presence of caspase-3, the substrate peptides can be cleaved and the changed surface charge of the Au nanoboxes results in the Au nanoboxes-NBA-peptide assembling to form aggregates and a great enhancement of SERS signal. The finite-difference time-domain simulation showed that hot spots mainly located in the nanogaps of the aggregated Au nanoboxes, which in theory proved the rationality of this signal amplification method. The SERS probes exhibited excellent reproducibility and selectivity toward caspase-3. A detection limit of 0.127 fM was obtained for caspase-3, with a dynamic range from 1 fM to 1 nM. MTT assay demonstrated that the probes had no obvious cytotoxicity within a certain concentration range. HeLa cells were treated with doxorubicin to induce long-term apoptosis. Upon cellular uptake of these probes, the spatiotemporal dynamics of caspase-3 in apoptotic cells could be real-time monitored using SERS. The activity of caspase-3 increased with the prolongation of apoptosis time. The SERS results were in accordance with that of western blotting assay. This kind of probe can offer great potential for the determination of enzymatic activities in the physiological processes of cells.

Gold nanocage-based surface-enhanced Raman scattering probes for long-term monitoring of intracellular microRNA during bone marrow stem cell differentiation.

The ability to monitor the differentiation of living stem cells is essential for understanding stem cell biology and the practical application of stem cell therapies. However, conventional methods of analyzing biomarkers related to differentiation still require a large number of cells or cell lysates. This requirement leads to the unavoidable loss of cell sources and hinders the real-time monitoring of cellular processes. In this study, we report an ultrasensitive surface-enhanced Raman scattering (SERS) method for the long-term detection and imaging of miR-144-3p in osteogenic differentiation of BMSCs, by using target miRNA-induced gold nanocage (GNC)-hairpin DNA1 (hpDNA1)-hpDNA2-GNC assembly in living cells. The finite-difference time domain method demonstrated that the electromagnetic intensities of the dimer and polymer of the GNCs were significantly enhanced compared to that of GNCs only, which theoretically confirmed the rational design of the SERS strategy. The hpDNA-conjugated GNC probes were prepared and used to recognize the target and distinguish from other miRNAs. This method enabled excellent sensitivity and high selectivity toward miR-144-3p with a limit of detection of 13.6 aM and a broad range from 100 aM to 100 pM in cell lysates. Then, we used transmission electron microscopy images, fluorescence microscopy images, and dark-field microscopy images to study the internalization of the probes in BMSCs. A Cell Counting Kit-8 experiment indicated that the probes were not cytotoxic in a certain concentration range. BMSCs were treated with an osteogenic inductor so that they would subsequently differentiate into osteocytes. Upon cellular uptake of these nanoprobes, we observed intense and time-dependent SERS responses from the important osteogenic biomarker miR-144-3p, only in BMSCs undergoing osteogenic differentiation and living undifferentiated BMSCs but not in osteoblasts. Finally, the accuracy of SERS has been proved by a quantitative real-time polymerase chain reaction experiment. The above results demonstrated that our nanoprobes are capable of long-term tracking of the dynamic expression of miR-144-3p (21 days) in the differentiating BMSCs. SERS has broad application prospects in the long-term detection of stem cell differentiation, and identification and isolation of specific cell types as well as in biomedical diagnosis.

Quantitative and specific detection of cancer-related microRNAs in living cells using surface-enhanced Raman scattering imaging based on hairpin DNA-functionalized gold nanocages.

Variations in the intracellular expression level of cancer-related microRNAs (miRNAs) are connected with worsening tumor progression. A simple, accurate, and sensitive analytical method for the imaging and detection of intracellular miRNA is still a great challenge due to the low abundance of miRNAs and the complexity of intracellular environments. In this work, target miRNA (miRNA)-mediated catalytic hairpin assembly (CHA)-induced gold nanocage (GNC)-hairpin DNA1 (hpDNA1)-hpDNA2-GNC nanostructures were designed for surface-enhanced Raman scattering (SERS) detection and imaging of the specific miR-125a-5p in the normal lung epithelial cell line (BEAS-2B cells) and lung cancer cell line (A549 cells). The finite difference time domain (FDTD) simulations showed that the polymer of GNCs possessed a much stronger electromagnetic field in nanogaps than that of single GNC, theoretically confirming the rational design of the CHA assembly strategy. Using this method, miR-125a-5p can be detected in a wide linear range with a detection limit of 43.96 aM and high selectivity over other miRNAs in vitro. Moreover, SERS imaging successfully detected and distinguished the expression levels of intracellular miR-125a-5p in BEAS-2B cells and A549 cells. The results obtained by the SERS assay were consistent with those obtained by the real-time quantitative polymerase chain reaction (qRT-PCR). This method can offer a powerful strategy for the imaging and quantitative detection of various types of biomolecules in vitro as well as in living cells.

MagT1 is essential for Drosophila development through the shaping of Wingless and Decapentaplegic signaling pathways.

Magnesium transporter subtype 1 (MagT1) is a magnesium membrane transporter with channel like properties. We have previously identified MagT1 (CG7830) in Drosophila genome and characterized its protein product by electrophysiological means. Here, we report the generation of fly MagT1 mutants and show that MagT1 is essential for early embryonic development. In wings and primordial wings, by clonal analysis and RNAi knock down of MagT1, we have found that loss of MagT1 results in enhanced/ectopic Wingless (Wg, a fly Wnt) signaling and disrupted Decapentaplegic (Dpp) signaling, indicating the crucial role of MagT1 for fly development at later stages. Finally, we demonstrate directly that magnesium transportations are proportional with the MagT1 expressional levels in Drosophila S2  cells. Taken together, these findings may suggest that MagT1 is a major magnesium transporter/channel profoundly involved in fly development by affecting developmental signaling pathways, such as Wg and Dpp signaling.