Screening substrate-binding positions by rolling circle amplification suggesting a binding model of Nt.BstNBI.

Nicking endonucleases (NEs) become increasingly attractive for their promising applications in isothermal amplification. Unfortunately, in comparison with their applications, their catalytic mechanism studies have relatively lagged behind due to a paucity of crystal structure information. Nt.BstNBI is one of those widely used NEs. However, many aspects of its catalytic mechanism still remained to be explored. Herein, we employed only rolling circle amplification (RCA) assay as a major analytic tool and succeeded in identifying the potential binding positions and regions of the DNA substrate based on locked nucleic acid modification, DNA duplex length of substrate, and substrate mismatch designs. Based on these data, we, for the first time, revealed that Nt.BstNBI was likely to recognize six adjacent positions of the recognition sequence (G1rt, A2rt, G3rt, A2rb, C3rb, and T4rb) in the major groove and hold three positions of the cleavage sequence (N3ct, N4ct, and N7cb) in the minor groove of DNA duplex for nicking. Moreover, this work also demonstrated the unexpected efficiency of RCA to study the macromolecular interaction for certain kind of nucleases in an easy and high-throughput way.

Site-Specific Immuno-PET Tracer to Image PD-L1.

The rapid ascension of immune checkpoint blockade treatments has placed an emphasis on the need for viable, robust, and noninvasive imaging methods for immune checkpoint proteins, which could be of diagnostic value. Immunoconjugate-based positron emission tomography (immuno-PET) allows for sensitive and quantitative imaging of target levels and has promising potential for the noninvasive evaluation of immune checkpoint proteins. However, the advancement of immuno-PET is currently limited by available imaging tools, which heavily rely on full-length IgGs with Fc-mediated effects and are heterogeneous mixtures upon random conjugation with chelators for imaging. Herein, we have developed a site-specific αPD-L1 Fab conjugate with the chelator 1,4,7-triazacyclononane- N, N', N″-triacetic acid (NOTA), enabling radiolabeling for PET imaging, using the amber suppression-mediated genetic incorporation of unnatural amino acid (UAA), p-azidophenylalanine. This Fab conjugate is homogeneous and demonstrated tight binding toward the PD-L1 antigen in vitro. The radiolabeled version, 64Cu-NOTA-αPD-L1, has been employed in PET imaging to allow for effective visualization and mapping of the biodistribution of PD-L1 in two normal mouse models, including the capturing of different PD-L1 expression levels in the spleens of the different mouse types. Follow-up in vivo blocking studies and ex vivo fluorescent staining further validated specific tissue uptakes of the imaging agent. This approach illustrates the utility of UAA-based site-specific Fab conjugation as a general strategy for making sensitive PET imaging probes, which could facilitate the elucidation of the roles of a wide variety of immune checkpoint proteins in immunotherapy.

Graphene Oxide and Lysozyme Ultrathin Films with Strong Antibacterial and Enhanced Osteogenesis.

There is a great demand worldwide for bone-related implant materials. The drawbacks of chronic infections and poor bone healing of current implant materials have limited their clinical applications. Functionalizing the implant surfaces with antibacterial and osteogenic films on implant materials provides new opportunities for fabricating novel implant materials. In the present study, an ultrathin (GO/Lys)8 film of several tens of nanometers was fabricated using a layer-by-layer (LBL) technique with alternative deposition of graphene oxide (GO) and lysozyme (Lys). The deposition of the (GO/Lys) n film exhibited a successive growth as supported by ellipsometry, UV-vis, and Fourier transform infrared data, and the physical properties (morphology, roughness, and stiffness) of this film were characterized with an atomic force microscope. The ultrathin films exhibited a great effect on bacterium sterilization of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli and enhanced osteogenic differentiation efficiency, showing the potential application in bone implant coatings. We believe that this LBL assembling strategy will pave the way for fabricating dual-functional surfaces and guide the design of the implanted surfaces in the future.

Pullulan reduces the non-specific amplification of loop-mediated isothermal amplification (LAMP).

Loop-mediated isothermal amplification (LAMP) has been developed as a versatile method for nucleic acid analysis in many applications. However, non-specific LAMP leading to false-positive outcomes has been observed frequently. To solve this problem, we selected six molecules as the additives for evaluating their effects on the improvement of the LAMP specificity. Experimental results show that bovine serum albumin (BSA) and DL-dithiothreitol (DTT) have negative effects on the LAMP specificity; dimethyl sulfoxide (DMSO), tetramethylene sulfoxide (TMSO), and glycerol could inhibit the non-specific LAMP moderately. Surprisingly, pullulan shows an ability to inhibit the non-specific amplification of LAMP significantly without affecting the sample amplification of LAMP, and this inhibitory effect is concentration dependent. Thus, pullulan could be considered as the most promising additive to improve the amplification specificity in the LAMP-based detection and analysis of nucleic acids.

Astragaloside IV inhibits cell proliferation in vulvar squamous cell carcinoma through the TGF-ß/Smad signaling pathway.

OBJECTIVE: To explore the inhibition of the proliferation of vulvar squamous cell carcinoma (VSCC) by astragaloside IV. METHODS: MTT examined the cell proliferation of VSCC. Flow cytometry analyzed cell cycle and apoptosis. Western blot assay detected the expression of some relevant proteins. RESULTS: AS-IV reduced the proliferation of SW962 cells in a concentration- and time-dependent manner, induced cell-cycle arresting in G0/G1 phase, as demonstrated by the up-regulation of P53 and P21 expression, and the down-regulation of cyclin D1 expression. AS-IV enhanced the expression of Bax and cleaved-caspase 3, and suppressed Bcl-2 and Bcl-xl expression, which resulted in apoptosis increased. Furthermore, the expression of Beclin-1 and LC3-B was upregulated and that of P62 was downregulated, which suggested that AS-IV could increase the incidence of autophagy in SW962 cells. After inhibiting autophagy by 3-methyladenine (3-MA), cell apoptosis decreased upon AS-IV treatment. Similarly, TGF-ß1 stimulated SW962 cells, cell proliferation enhanced, and the expression of TGF-ßRII and Smad4 was decreased. Furthermore, the expression of proteins that promote apoptosis and autophagy decreased. After AS-IV treatment, the expression levels of the above proteins exhibited the opposite effect. CONCLUSION: AS-IV inhibits cell proliferation and induces apoptosis and autophagy through the TGF-ß/Smad signaling pathway in VSCC.

Portable fluorescence-based microRNA detection system based on isothermal signal amplification technology.

MicroRNAs (miRNAs) diagnostics can be useful for diagnosing or confirming miRNA abundance and are used in screening tests and to assess changes in miRNAs in vivo. At present, the use of traditional nucleic acid amplification assays to detect miRNAs has been limited in laboratory environment because of the time, equipment, and technical expertise required to perform these assays. A specialized, rapid affordable miRNA detection system is necessary when there are limited resources or point-of-care testing needs. We designed a portable and affordable fluorescence-based miRNA detection system based on isothermal signal amplification technology, using SYBR Green II as a fluorescent dye. To reduce costs, we chose LED as a light source and designed the corresponding optical path for LED. The portable detection system shows results consistent with those by real-time PCR, and can be used to detect miR-183 with a limit of detection of approximately 2 fmol. We used the system to detect miR-183 in tissues and blood from patients with hepatocellular carcinoma (HCC). The results from the portable detection device were compared with those from clinical trials and indicated that the miR-183 fluorescence signal could successfully identify HCC and provide information related to cancer progression.

Effects of Central Loop Length and Metal Ions on the Thermal Stability of G-Quadruplexes.

The central loop of G-quadruplex molecular beacons is a key element to sense target DNA or RNA sequences. In this study, circular dichroism spectroscopy (CD), thermal difference spectrum (TDS), non-denatured non-denaturing gel electrophoresis, and thermal stability analysis were used to investigate the effect of the central loop length on G-quadruplex features. Two series of G-quadruplexes, AG3TTAG3-(TTA)n-G3TTAG3T (n = 1-8) (named TTA series) and AG3TTTG3-(TTA)n-G3TTTG3T (n = 1-8) (named TTT series) were examined in K+ and Na+ solutions, respectively. CD and TDS spectral data indicated that TTA series adopted an antiparallel G-quadruplex structure in Na+ solution and a hybrid G-quadruplex structure in K+ solution respectively. TTT series exhibited a hybrid G-quadruplex structure in both Na+ and K+ solutions. UV melting curves indicated that the stability of G-quadruplex in both series was reduced by the elongation of central loop. Thermal stability analysis concluded that the G-quadruplex destabilization with long central loop is an entropy-driven process due to more flexible and longer central loops.

Screening oligonucleotide sequences for silver staining and d-galactose visual detection using RCA silver staining in a tube.

Oligonucleotides were screened for strongly silver-stained repetitive sequences. An 'AG'-clustered purine sequence showed strong staining, and the staining density can be compromised by disrupting the continuity of the 'AG'-clustered sequence. The staining-favored sequence was then employed in rolling circle amplification (RCA) for its product detection. A tube-staining method was developed for convenient and visual RCA assay. Moreover, by introducing GalR into RCA, d-galactose was detected by RCA tube-staining with naked eyes without any equipment. About 10 mM d-galactose can be easily identified, and the detection of d-galactose was specific in comparison with that of several other monosaccharides.

A Novel Design Combining Isothermal Exponential Amplification and Gold-Nanoparticles Visualization for Rapid Detection of miRNAs.

MicroRNAs (miRNAs) play important roles in a wide range of biological processes, and their aberrant expressions are associated with various diseases. The levels of miRNAs can be useful biomarkers for cellular events or disease diagnosis; thus, sensitive and selective detection of microRNAs is of great significance in understanding biological functions of miRNAs, early-phase diagnosis of cancers, and discovery of new targets for drugs. However, traditional approaches for the detection of miRNAs are usually laborious and time-consuming, with a low sensitivity. Here, we develop a simple, rapid, ultrasensitive colorimetric assay based on the combination of isothermal Exponential Amplification Reaction (EXPAR) and AuNP-labeled DNA probes for the detection of miRNAs (taking let-7a as a model analyte). In this assay, the presence of let-7a is converted to the reporter Y through EXPAR under isothermal conditions. The subsequent sandwich hybridization of the reporter Y with the AuNP-labeled DNA probes generates a red-to-purple color change. In other words, if the reporter Y is complementary to the AuNP-labeled DNA probes, the DNA-functionalized AuNPs will be aggregated, resulting in the change of solution color from red to purple/blue, while when the AuNP-labeled DNA probes are mismatched to the reporter Y, the solution remains red. This assay represents a simple, time-saving technique, and its results can be visually detected with the naked eye due to the colorimetric change. The method provides superior sensitivity, with a detection limit of 4.176 aM over a wide range from 1 nM to 1 aM under optimal conditions. The method also shows high selectivity for discriminating even single-nucleotide differences between let-7 miRNA family members. Notably, it is comparable to the most sensitive method reported to date, thus providing a promising alternative to standard approaches for the direct detection of let-7a miRNA. Importantly, through combination with specific templates, different miRNAs can be converted to the same reporter Y, which can hybridize with the same set of AuNP-labeled DNA probes to form sandwich hybrids. The color change of the solution can be observed in the presence of the target miRNA. This technique has potential as a routine method for assessing the levels of miRNAs, not only for let-7, but also for various miRNAs in the early phase of cancers. In addition, it can be a useful tool in biomedical research and clinical diagnosis, as well as diagnosis or surveillance programs in field conditions.

Phytochemical and Biological Characteristics of Mexican Chia Seed Oil.

The purpose of this research was to investigate the chemical profile, nutritional quality, antioxidant and hypolipidemic effects of Mexican chia seed oil (CSO) in vitro. Chemical characterization of CSO indicated the content of α-linolenic acid (63.64% of total fatty acids) to be the highest, followed by linoleic acid (19.84%), and saturated fatty acid (less than 11%). Trilinolenin content (53.44% of total triacylglycerols (TAGs)) was found to be the highest among seven TAGs in CSO. The antioxidant capacity of CSO, evaluated with ABTS•+ and DPPH• methods, showed mild antioxidant capacity when compared with Tocopherol and Catechin. In addition, CSO was found to lower triglyceride (TG) and low-density lipoprotein-cholesterol (LDL-C) levels by 25.8% and 72.9%respectively in a HepG2 lipid accumulation model. As CSO exhibits these chemical and biological characteristics, it is a potential resource of essential fatty acids for human use.

A sensitive colorimetric assay system for nucleic acid detection based on isothermal signal amplification technology.

Rapid and accurate detection of microRNAs in biological systems is of great importance. Here, we report the development of a visual colorimetric assay which possesses the high amplification capabilities and high selectivity of the rolling circle amplification (RCA) method and the simplicity and convenience of gold nanoparticles used as a signal indicator. The designed padlock probe recognizes the target miRNA and is circularized, and then acts as the template to extend the target miRNA into a long single-stranded nucleotide chain of many tandem repeats of nucleotide sequences. Next, the RCA product is hybridized with oligonucleotides tagged onto gold nanoparticles. This interaction leads to the aggregation of gold nanoparticles, and the color of the system changes from wine red to dark blue according to the abundance of miRNA. A linear correlation between fluorescence and target oligonucleotide content was obtained in the range 0.3-300 pM, along with a detection limit of 0.13 pM (n = 7) and a RSD of 3.9% (30 pM, n = 9). The present approach provides a simple, rapid, and accurate visual colorimetric assay that allows sensitive biodetection and bioanalysis of DNA and RNA nucleotides of interest in biologically important samples. Graphical abstract The colorimetric assay system for analyzing target oligonucleotides.

Potent Inhibitor of Drug-Resistant HIV-1 Strains Identified from the Medicinal Plant Justicia gendarussa.

Justicia gendarussa, a medicinal plant collected in Vietnam, was identified as a potent anti-HIV-1 active lead from the evaluation of over 4500 plant extracts. Bioassay-guided separation of the extracts of the stems and roots of this plant led to the isolation of an anti-HIV arylnaphthalene lignan (ANL) glycoside, patentiflorin A (1). Evaluation of the compound against both the M- and T-tropic HIV-1 isolates showed it to possess a significantly higher inhibition effect than the clinically used anti-HIV drug AZT. Patentiflorin A and two congeners were synthesized, de novo, as an efficient strategy for resupply as well as for further structural modification of the anti-HIV ANL glycosides in the search for drug leads. Subsequently, it was determined that the presence of a quinovopyranosyloxy group in the structure is likely essential to retain the high degree of anti-HIV activity of this type of compounds. Patentiflorin A was further investigated against the HIV-1 gene expression of the R/U5 and U5/gag transcripts, and the data showed that the compound acts as a potential inhibitor of HIV-1 reverse transcription. Importantly, the compound displayed potent inhibitory activity against drug-resistant HIV-1 isolates of both the nucleotide analogue (AZT) and non-nucleotide analogue (nevaripine). Thus, the ANL glycosides have the potential to be developed as novel anti-HIV drugs.

An intramolecular G-quadruplex structure formed in the human MET promoter region and its biological relevance.

Previous studies have shown that promoter regions of many proto-oncogenes can fold into G-quadruplexes, which are potentially involved in the regulation of genes. Bioinformatics analysis suggested that there was a G-rich sequence within -48 to -26 region of the human MET promoter (named Pu23WT). In this study, we proved that Pu23WT adopted an intramolecular parallel G-quadruplex structure under physiological conditions in vitro, and the cationic porphyrin TMPyP4 enhanced the stability of the Pu23WT G-quadruplex. To better understand the functions of Pu23WT in the MET expression, we performed a series of analysis on several cancer cells. Experimental data revealed that TMPyP4 treatment attenuated the expression of MET in HepG2, BGC823, and U87MG cells, resulting in the cellular proliferation inhibition, G1 phase cell cycle arrest and cell migration retardation. ChIP assay results indicated that TMPyP4 probably prohibited the Pu23WT G-quadruplex from binding to the activator Sp1, which could be one of the mechanisms that led to the transcription inhibition of MET gene. It is the first study on the G-quadruplex structure in the human MET promoter and its functions in cancer cells. We believe that this structure is a potential target for anticancer treatment.

Suppression of rolling circle amplification by nucleotide analogs in circular template for three DNA polymerases.

Among wide applications of nucleotide analogs, their roles in enzyme catalytic reactions are significant in both fundamental and medical researches. By introducing analogs into circular templates, we succeeded in determining effects of four analogs on RCA efficiency for three different DNA polymerases. Results showed an obvious suppression effect for 2'-OMeRNA modification, which might be due to the size of the C2'-modified moieties. 2'-F RNA, LNA and PS had little interference, suggesting good analog candidates for application in RCA. Different polymerases and nucleobases made a little difference according to analogs we used. These results are useful for understanding polymerase catalytic mechanism and analogs applications in RCA reaction.

Dihydro-Resveratrol Ameliorates Lung Injury in Rats with Cerulein-Induced Acute Pancreatitis.

Acute pancreatitis is an inflammatory process originated in the pancreas; however, it often leads to systemic complications that affect distant organs. Acute respiratory distress syndrome is indeed the predominant cause of death in patients with severe acute pancreatitis. In this study, we aimed to delineate the ameliorative effect of dihydro-resveratrol, a prominent analog of trans-resveratrol, against acute pancreatitis-associated lung injury and the underlying molecular actions. Acute pancreatitis was induced in rats with repetitive injections of cerulein (50 µg/kg/h) and a shot of lipopolysaccharide (7.5 mg/kg). By means of histological examination and biochemical assays, the severity of lung injury was assessed in the aspects of tissue damages, myeloperoxidase activity, and levels of pro-inflammatory cytokines. When treated with dihydro-resveratrol, pulmonary architectural distortion, hemorrhage, interstitial edema, and alveolar thickening were significantly reduced in rats with acute pancreatitis. In addition, the production of pro-inflammatory cytokines and the activity of myeloperoxidase in pulmonary tissues were notably repressed. Importantly, nuclear factor-kappaB (NF-κB) activation was attenuated. This study is the first to report the oral administration of dihydro-resveratrol ameliorated acute pancreatitis-associated lung injury via an inhibitory modulation of pro-inflammatory response, which was associated with a suppression of the NF-κB signaling pathway.

Inhibition of mdr1 by G-quadruplex oligonucleotides and reversal of paclitaxel resistance in human ovarian cancer cells.

The expression of ATP-dependent efflux pump P-glycoprotein (P-gp) in cancer cells generally results in multidrug resistance (MDR) to chemotherapeutic drugs, which is the main cause of chemotherapy failure in cancer treatment. The intracellular drug levels could be increased by some MDR reversal agents that inhibited the drug efflux activity of P-gp. The synthesized DNA nucleic acids of G-quadruplex represent a novel and unique class of anti-cancer agents. While there was no report on the roles of DNA G-quadruplex oligonucleotides (GQ-ODNs) in the MDR reversal, the present study was performed to investigate the ability of synthesized GQ-ODNs to reverse P-gp-mediated MDR and its mechanism in paclitaxel (PTX)-resistant SKOV3 (SKOV3/PTX) cells and their sensitive cell lines SKOV3. The ability of GQ-ODNs to reverse drug resistance was evaluated by MTS assay. The results showed that GQ-ODNs can reverse PTX resistance effectively. The potential of GQ-ODNs as reversal agents was evaluated with the nude mice tumor xenograft model and showed that the co-administration of the GQ-ODNs and PTX had better effects and was also more evident than treatment with only PTX. The P-gp expression was assessed by the Western blot; it showed that SKOV3/PTX cells showed highly expressed P-gp protein, while their sensitive cells scarcely showed P-gp. The presence of GQ-ODNs efficiently decreased the P-gp expression, showing that GQ-ODNs could reverse P-gp-mediated MDR by decreasing the expression of P-gp. This study indicated that GQ-ODNs could effectively reverse P-gp-mediated PTX resistance by inhibiting the expression of P-gp and by the co-administration of GQ-ODNs and PTX that could increase the apoptosis of SKOV3/PTX cells. Thus, the synthesized GQ-ODNs may be a potential inhibitor to overcome drug resistance.

Discriminative identification of miRNA let-7 family members with high specificity and sensitivity using rolling circle amplification.

Rolling circle amplification (RCA) is a new method based on virus DNA reproduction, which has been widely used in the field of miRNA detection. However, discrimination of highly homologous miRNAs is a bottleneck in the research of miRNA. In this study, the RCA process was creatively used to conduct the discrimination of miRNAs. Results showed that T4 RNA ligase 2 could reach the highest circularization efficiency during the RCA process with higher specificity. By using RCA technology, a member of highly homologous miRNAs, let-7, could be discriminated at the amount of 2.5 fmol. This sensitivity could not be achieved by using traditional reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. In addition, detection of miRNAs by using RCA could reach the amount limit of fmol with a good linearity. Optimal RCA technology used in this study is better than RT-qPCR in discriminating highly homologous family miRNAs. Results from this study can promote the applications of RCA in clinical diagnosis, environment protection, health care, disease inspection and prevention, and national security.

BAG3 is upregulated by c-Jun and stabilizes JunD.

BAG3 plays a regulatory role in a number of cellular processes, including cell proliferation, apoptosis, adhesion and migration, epithelial-mesenchymal transition (EMT), autophagy activation, and virus infection. The AP-1 transcription factors are implicated in a variety of important biological processes including cell differentiation, proliferation, apoptosis and oncogenesis. Recently, it has been reported that AP-1 protein c-Jun inhibits autophagy and enhances apoptotic cell death mediated by starvation. However, the molecular mechanisms remain unclear. For the first time, the current study demonstrated that serum starvation downregulated BAG3 at the transcriptional level via c-Jun. In addition, the current study reported that BAG3 stabilized JunD mRNA, which was, at least in part, responsible for the promotion of serum starvation mediated-growth inhibition by BAG3.

BAG3 sensitizes cancer cells exposed to DNA damaging agents via direct interaction with GRP78.

Bcl-2 associated athanogene 3 (BAG3) has a modular structure that contains a BAG domain, a WW domain, a proline-rich (PxxP) domain to mediate potential interactions with chaperons and other proteins that participate in more than one signal transduction. In search for novel interacting partners, the current study identified that 78kDa glucose-regulated protein (GRP78) was a novel partner interacting with BAG3. Interaction between GRP78 and BAG3 was confirmed by coimmunoprecipitation and glutathione S-transferase (GST) pulldown. We also identified that the ATPase domain of GRP78 and BAG domain of BAG3 mediated their interaction. Counterintuitive for a prosurvival protein, BAG3 was found to promote the cytotoxicity of breast cancer MCF7, thyroid cancer FRO and glioma U87 cells subjected to genotoxic stress. In addition, the current study demonstrated that BAG3 interfered with the formation of the antiapoptotic GRP78-procaspase-7 complex, which resulted in an increased genotoxic stress-induced cytotoxicity in cancer cells. Furthermore, overexpression of GRP78 significantly blocked the enhancing effects of BAG3 on activation of caspase-7 and induction of apoptosis by genotoxic stress. Overall, these results suggested that through direct interaction BAG3 could prevent the antiapoptotic effect of GRP78 upon genotoxic stress.

Bioactive compounds from Vitex leptobotrys.

A new lignan, vitexkarinol (1), as well as a known lignan, neopaulownin (2), a known chalcone, 3-(4-hydroxyphenyl)-1-(2,4,6-trimethoxyphenyl)-2-propen-1-one (3), two known dehydroflavones, tsugafolin (4) and alpinetin (5), two known dipeptides, aurantiamide and aurantiamide acetate, a known sesquiterpene, vemopolyanthofuran, and five known carotenoid metabolites, vomifoliol, dihydrovomifoliol, dehydrovomifoliol, loliolide, and isololiolide, were isolated from the leaves and twigs of Vitex leptobotrys through bioassay-guided fractionation. The chalcone (3) was found to inhibit HIV-1 replication by 77% at 15.9 µM, and the two dehydroflavones (4 and 5) showed weak anti-HIV activity with IC50 values of 118 and 130 µM, respectively, while being devoid of cytotoxicity at 150 µM. A chlorophyll-enriched fraction of V. leptobotrys, containing pheophorbide a, was found to inhibit the replication of HIV-1 by 80% at a concentration of 10 µg/mL. Compounds 1 and 3 were further selected to be evaluated against 21 viral targets available at NIAID (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA).