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Human papillomavirus (HPV) is one of the most common sexually transmitted infections which causes cervical cancer in women. In order to avoid the development of cancer and the onset of the disease, a reliable and on-site diagnosis for early screening of HPV is still required. To achieve this goal, a smartphone-assisted paper-based colorimetric assay using pyrrolidinyl peptide nucleic acid (acpcPNA) as a probe for the detection of HPV DNA was developed. Dextrin-stabilized gold nanoparticles (d-AuNPs) were used as the colorimetric reagent. The aggregation of the d-AuNPs can be induced by the outstanding properties of the positively charged acpcPNA, generating a distinctive color change. After the duplex formation between the acpcPNA probe and the HPV DNA target, the acpcPNA probe was depleted and the residual probe can cause different degrees of d-AuNPs aggregation, resulting in a detectable color change. The different color change before and after the introduction of the DNA target as a function of the DNA concentration was quantified by analyzing the color intensity through a smartphone application. Under the optimal conditions, the linearity in the range from 1 to 1000 nM with a correlation coefficient of 0.9996 and an experimental limit of detection of 1 nM was obtained. In addition, the acpcPNA probe exhibited high selectivity for the DNA target over single-base-mismatch, two-base-mismatch, and non-complementary DNA. Moreover, this DNA sensor was successfully applied for the detection of HPV DNA from cell line samples. The proposed smartphone-based colorimetric DNA sensor offers a simple, sensitive, and selective way of detecting HPV DNA. Therefore, this DNA sensing device can be utilized as an alternative tool for the point-of-care (POC) screening of HPV. [ABSTRACT FROM AUTHOR]

A novel fluorescent paper-based DNA sensor employing a highly specific pyrrolidinyl peptide nucleic acid (acpcPNA) probe was developed for the sensitive and selective detection of hepatitis C virus (HCV). The acpcPNA was covalently immobilized onto partially oxidized cellulose paper via reductive alkylation between the amine and the aldehyde groups. The fluorescence-based detection was performed by monitoring the fluorescence signal response of a fluorescent dye that selectively binds to the single-strand region of the DNA target over the PNA probe employing a custom-made portable fluorescent camera gadget in combination with a smartphone camera. Under the optimal conditions, a linear relationship between the fluorescence change in the green channel and the amount of HCV DNA from 5 to 100 pmol with a correlation coefficient of 0.9956, and the limit of detection of 5 pmol were obtained for short synthetic oligonucleotides. The acpcPNA probe exhibited very high selectivity for the complementary oligonucleotides over the single-base-mismatched, two-base-mismatched, and non-complementary DNA targets. Benefitting from the signal amplification achieved through the numerous binding sites for the dye provided by the overhanging tail of long ssDNA target sequences, this system was successfully applied to detect the HCV complementary DNA (cDNA) obtained from clinical samples with satisfactory results. The proposed fluorescent paper-based sensor demonstrated a great potential to be used as a low-cost, simple, label-free, sensitive, and selective DNA sensor for point-of-care applications. [Display omitted] • A paper-based fluorogenic DNA sensor using acpcPNA probe was developed for detecting HCV. • The ssDNA-specific fluorescent dye allows straightforward label-free visualization. • The signal amplification was achieved through the overhang region of the ssDNA target. • The sensor was successfully applied to detect HCV cDNA from clinical samples without requiring any DNA amplification. [ABSTRACT FROM AUTHOR]

To explore the signal transduction pathway that TaRHA2b gene is involved in regulating, yeast two-hybrid system was used to screen and identify proteins interacting with TaRHA2b. The homogenized cDNA library was constructed from the embryo of mixed barley. The decoy protein vector pGBKT7- TaRHA2b was constructed and transformed into yeast AH109 cells for self-activation detection. Candidate positive clones were screened by one to one yeast co-transfection, SD/-Leu/-Trp and SD/-Leu/- Trp/-Ade/-His medium screening and β-galactosidase chromogenic reaction. The in vitro co-transfection and GST-pull down experiments were used for further verification. The homogenized cDNA library of the barley embryos with titer of 1.2 106 cfu/ml and storage capacity of 1.1 106 was successfully constructed. The inserted fragment size was between 1000-3000 bp. The plasmids of the cDNA library were extracted and sequenced on a large scale, and 5000 plasmids were obtained. The decoy vector without self-activation function was successfully constructed. The x-gal filter paper of eighty-four hybrid clones showed strong blue color and forty-nine pieces of effective sequence information were obtained. Eight of them were selected for co-transfection verification. YTH2450 was selected for GST-pull down verification. The results showed that they were positive hybrid clones. TaRHA2b interacted with YTH2450 and other proteins. The information of above genes could be used for genetic improvement of crops, further improving the adaptability of crops to the environment. [ABSTRACT FROM AUTHOR]

The present research is focused on the development of a biofunctionalized hydrogel with a surface diffractive micropattern as a label-free biosensing platform. The biosensors described in this paper were fabricated with a holographic recording of polyethylene terephthalate (PET) surface micro-structures, which were then transferred into a hydrogel material. Acrylamide-based hydrogels were obtained with free radical polymerization, and propargyl acrylate was added as a comonomer, which allowed for covalent immobilization of thiolated oligonucleotide probes into the hydrogel network, via thiol-yne photoclick chemistry. The comonomer was shown to significantly contribute to the immobilization of the probes based on fluorescence imaging. Two different immobilization approaches were demonstrated: during or after hydrogel synthesis. The second approach showed better loading capacity of the bioreceptor groups. Diffraction efficiency measurements of hydrogel gratings at 532 nm showed a selective response reaching a limit of detection in the complementary DNA strand of 2.47 µM. The label-free biosensor as designed could significantly contribute to direct and accurate analysis in medical diagnosis as it is cheap, easy to fabricate, and works without the need for further reagents. [ABSTRACT FROM AUTHOR]

Purpose: The present study aimed to explore the binding ability of acyl-CoA binding protein 2 to fatty acid acyl-CoA esters and its effect on Monascus pigment production in M. ruber CICC41233. Methods: The Mracbp2 gene from M. ruber CICC41233 was cloned with a total DNA and cDNA as the templates through the polymerase chain reaction. The cDNA of the Mracbp2 gene fragment was ligated to expression vector pGEX-6P-1 to construct pGEX-MrACBP2, which was expressed in Escherichia coli BL21 to obtain the fusion protein GST-MrACBP2 and then measure the binding ability of fatty acid acyl-CoA esters. Additionally, the DNA of the Mracbp2 gene fragment was ligated to expression vector pNeo0380 to construct pNeo0380-MrACBP2, which was homologously over-expressed in M. ruber CICC41233 to evaluate Monascus pigment production and fatty acid. Results: The cloned Mracbp2 gene of the DNA and cDNA sequence was 1525 bp and 1329 bp in length, respectively. The microscale thermophoresis binding assay revealed that the purified GST-MrACBP2 had the highest affinity for palmitoyl-CoA (Kd =70.57 nM). Further, the Mracbp2 gene was homologously overexpressed in M. ruber CICC41233, and a positive transformant M. ruber ACBP-E was isolated. In the Monascus pigments fermentation, the expression level of the Mracbp2 gene was increased by 1.74-fold after 2 days and 2.38-fold after 6 days. The palmitic acid content and biomass in M. ruber ACBP2-E were significantly lower than that in M. ruber CICC41233 on 2 days and 6 days. However, compared with M. ruber CICC41233, the yields of total pigment, ethanol-soluble pigment, and water-soluble pigment in M. ruber ACBP2-E increased by 63.61%, 71.61%, and 29.70%, respectively. Conclusions: The purified fusion protein GST-MrACBP2 exhibited the highest affinity for palmitoyl-CoA. The Mracbp2 gene was overexpressed in M. ruber CICC41233, which resulted in a decrease in palmitic acid and an increase in Monascus pigments. Overall, the effect of MrACBP2 on the synthesis of fatty acid and Monascus pigment was explored. This paper explored the effect of MrACBP2 on the fatty acid synthesis and the synthesis of Monascus pigment. The results indicated the regulation of fatty acid synthesis could affect Monascus pigment synthesis, providing a novel strategy for improving the yield of Monascus pigment. [ABSTRACT FROM AUTHOR]

NIK (NF-κB inducing kinase) belongs to the mitogen-activated protein kinase family, which activates NF-κB and plays a vital role in immunology, inflammation, apoptosis, and a series of pathological responses. In NF-κB noncanonical pathway, NIK and IKKα have been often studied in mammals and zebrafish. However, few have explored the relationship between NIK and other subunits of the IKK complex. As a classic kinase in the NF-κB canonical pathway, IKKβ has never been researched with NIK in fish. In this paper, the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) NIK (CiNIK) was first cloned and identified. The expression level of CiNIK in grass carp cells was increased under GCRV stimuli. Under the stimulation of GCRV, poly (I:C), and LPS, the expression of NIK in various tissues of grass carp was also increased. This suggests that CiNIK responds to viral stimuli. To study the relationship between CiNIK and CiIKKβ , we co-transfected CiNIK -FLAG and CiIKKB -GFP into grass carp cells in coimmunoprecipitation and immunofluorescence experiments. The results revealed that CiNIK interacts with CiIKKβ. Besides, the degree of autophosphorylation of CiNIK was enhanced under poly (I:C) stimulation. CiIKKβ was phosphorylated by CiNIK and then activated the activity of p65. The activity change of p65 indicates that NF-κB downstream inflammatory genes will be functioning. CiNIK or CiIKKβ up-regulated the expression of IL-8. It got higher when CiNIK and CiIKKβ coexisted. This paper revealed that NF-κB canonical pathway and noncanonical pathway are not completely separated in generating benefits. • The expression level of CiNIK is increased under GCRV stimuli. • CiNIK interacts with CiIKKβ and promotes the phosphorylation of CiIKKβ. • CiNIK up-regulates the expression of IL-8 by activating NF-κB canonical pathway. [ABSTRACT FROM AUTHOR]

We previously reported that fully complementary DNA duplexes formed on gold nanoparticle (GNP) surfaces aggregate at high salt concentrations. We previously reported that DNA-functionalized gold nanoparticles (GNPs) aggregate by hybridization with fully complementary DNA at high salt concentrations. Although this behavior has been applied to some precise naked-eye colorimetric analyses of DNA-related molecules, the aggregation mechanism is still unclear and comprehensive studies are needed. In this paper, we reveal the key factors that influence GNP aggregation. The effects of temperature, electrolyte concentration, probe length, and particle size, which control the stabilities of double-stranded DNAs and GNPs, were investigated. Larger GNPs aggregated more easily, and GNP aggregates were easily formed with ∼15-mer-long probes, while longer probes prevented aggregation, perhaps by preventing the formation of rigid double-stranded DNA layers, compared to shorter probes. Furthermore, GNPs with purine bases at their 5′ ends aggregated more easily than those with these bases at their 3′ ends. This phenomenon is different from that based on the melting-temperature trend calculated using the nearest-neighbor method. [ABSTRACT FROM AUTHOR]

Patulin (PAT) is a kind of mycotoxin which must be monitored for the sake of quality and safety in traditional Chinese medicine (TCM) owing to its harm to human health. On this account, a rationally designed ratiometric fluorescent aptasensor was developed based on the studies of the interaction mechanism between PAT and its aptamer (PAT-APT). First, CD spectroscopy, molecular docking, and molecular dynamic simulation were applied to investigate the details on how PAT-APT binds with its target molecule. The results indicated that the structure of PAT-APT changed to a certain extent and was stabilized after binding with PAT. C-11, C-37 and C-38 were the key sites for the recognition and interaction between PAT-APT and its target. Second, based on these results, a ratiometric aptasensor was designed using fluorescence resonance energy transfer (FRET) and synchronous fluorescence spectroscopy. A complementary sequence (cDNA) to the aptamer with an appropriate length and hybridization position was obtained through rational design and optimization. Both PAT-APT and cDNA were labeled using a pair of fluorophores, which could generate FRET when the two single-stranded oligonucleotides hybridized. The accurate detection of PAT could be realized according to the change ratio of the fluorescence intensity at the corresponding wavelengths of the two fluorophores before and after the assay. The aptasensor achieved an ultralow limit of detection of 0.16 nM, perfect selectivity, and satisfactory practicability in complex TCM samples. To our knowledge, this is the first aptasensor for PAT designed through the interaction mechanism between its aptamer and the target molecule. Moreover, the assay for PAT is cost-effective, does not need complicated pretreatment and only takes less than an hour. In summary, this study makes a contribution to the safety control of TCM and provides a thinking mode from mechanism to rational design to conquer the problem of sensitive aptasensing of one component in a complex system. [ABSTRACT FROM AUTHOR]

Caspase-3 is generally considered to be the most important terminal shear enzyme in the process of apoptosis, as well as an important part of cytotoxic T lymphocytes (CTL) killing mechanism, which is confirmed to play an important role in vertebrate cell apoptosis and immune system, and is poorly reported in invertebrates. In this paper, we used bioinformatics to perform amino acid multiple sequence alignment and protein structural domain analysis, and constructed a phylogenetic tree to identify the full-length cDNA of the cloned caspase-3 of Cristaria plicata (Named CpCaspase-3). The expression of caspase-1, caspase-7, caspase-8, and caspase-9 was found to be down-regulated by double-stranded RNA interference of CpCaspase-3 in C. plicata. Some degree of disruption of the caspase signaling pathway occurs. The expression of CpCaspase-3 was affected after injection of Lipopolysaccharide (LPS), Peptidoglycan (PGN), polyinosinic-polycytidylic acid (poly(I:C)), and Aeromonas hydrophila. These results were suggested that CpCaspase-3 was involved in the immune response of C. plicata. The wound recovery process of C. plicata was simulated and CpCaspase-3 was found to promote wound recovery. An autophagy inhibition and autophagy activation model of mussels was constructed, where apoptosis and autophagy undergo crosstalk, and inhibition of autophagy induces the onset of apoptosis, and similarly autophagy activation inhibits the process of apoptosis instead. In addition, a recombinant CpCaspase-3-pEGFP-C1 plasmid was constructed for subcellular localization experiments and found that CpCaspase-3 was distributed in both the nucleus and the cytoplasm. This paper aims to unveil the immune mechanism of C. plicata and provide a theoretical basis for the healthy culture of shellfish. • It was demonstrated that CpCaspase-3 plays a key role in the wound healing process of Cristaria plicata. • A negative correlation between autophagy and apoptosis in the Cristaria plicata was revealed. • It was revealed that microcystin increased the complexity of the crosstalk between autophagy and apoptosis. [ABSTRACT FROM AUTHOR]

Oxford Nanopore (ONT) is a leading long-read technology which has been revolutionizing transcriptome analysis through its capacity to sequence the majority of transcripts from end-to-end. This has greatly increased our ability to study the diversity of transcription mechanisms such as transcription initiation, termination, and alternative splicing. However, ONT still suffers from high error rates which have thus far limited its scope to reference-based analyses. When a reference is not available or is not a viable option due to reference-bias, error correction is a crucial step towards the reconstruction of the sequenced transcripts and downstream sequence analysis of transcripts. In this paper, we present a novel computational method to error correct ONT cDNA sequencing data, called isONcorrect. IsONcorrect is able to jointly use all isoforms from a gene during error correction, thereby allowing it to correct reads at low sequencing depths. We are able to obtain a median accuracy of 98.9–99.6%, demonstrating the feasibility of applying cost-effective cDNA full transcript length sequencing for reference-free transcriptome analysis. Nanopore sequencing technologies applied to transcriptome analysis suffer from high error rates, limiting them largely to reference-based analyses. Here, the authors develop a computational error correction method for transcriptome analysis that reduces the median error rate from ~7% to ~1%. [ABSTRACT FROM AUTHOR]

Clostridium difficile can cause antibiotic‐associated diarrhoea or pseudo‐membranous colitis in humans and animals. Currently, the various methods such as microbiological culture, cytotoxic assay, ELISA and polymerase chain reaction have been used to detect Clostridium difficile infection (CDI). These conventional methods, however, require long detection time and professional staff. The paper is to describe a simple strategy which employs immunomagnetic separation and aptamer‐mediated colorimetric assay for the detection of toxin B of C. difficile (TcdB) in the stool samples. HRP‐labelled aptamer against TcdB selected by SELEX was firstly captured on the surface of magnetic beads (MB) by DNA hybridization with a complementary strand. In the presence of TcdB, aptamer specifically recognized and bound TcdB, disturbing the DNA hybridization and causing the release of HRP‐aptamer from MB. This reduced the catalytic capacity of HRP and consequently the absorption intensity. As there was a relationship between the decrease in the absorption intensity and target concentration, a quantitative analysis of TcdB can be accomplished by the measurement of the absorption intensity. Under the optimal conditions, the assay system is able to detect TcdB at a concentration down to 5 ng ml−1. Moreover the method had specificity of 97% and sensitivity of 66% and the system remained excellent stability within 4 weeks. The proposed method is a valuable screening procedure for CDI and can be extended readily to detection of other clinically important pathogens. [ABSTRACT FROM AUTHOR]

We recently developed a piggyback knockdown method that was used to knockdown genes in adult zebrafish. In this method, a vivo morpholino (VMO) piggybacks an antisense deoxyoligonucleotide (dO) into the somatic cells and reduces the cognate mRNA levels. In this paper, we tested whether we can piggyback more than one dO with one VMO. We designed various hybrids that had more than one dO that could be piggybacked with one VMO. We chose f7, f8, and αIIb genes and tested their knockdown by the appropriate assays. The knockdown with piggybacking either two or three dOs by one VMO yielded > 85% knockdown efficiency. We also performed knockdown of argonautes and rnaseh separately along with f7. We found the knockdown of f7 occurs when knockdown of argonautes happens and not when rnaseh knockdown was performed, suggesting that RNaseH is involved in mRNA degradation. In conclusion, we developed a method where we could knockdown three genes at one time, and by increasing the concentration of VMO by twofold, we could knockdown six genes simultaneously. These multiple gene knockdowns will not only increase the efficiency of the method in whole genome-wide knockdowns but will also be useful to study multifactorial disorders. [ABSTRACT FROM AUTHOR]

A sulfadiazine (SDZ) aptamer sensor based on carboxylated tea residue carbon quantum dots (C-TCDs) was studied to detect SDZ residues in honey. C-TCDs were synthesized by a hydrothermal method. The C-TCD labeled SDZ aptamer was used as a recognition probe, and the C-TCD labeled cDNA was used as a complementary chain to construct a fluorescent aptamer sensor. The fluorescence intensity of the constructed sensor has a good linear relationship with the concentration of SDZ in the detection range of 0–80 ng mL−1 (y = 12.857x + 828.721, R2 = 0.99768), and the detection limit is 0.95 ng mL−1. The sensor constructed using this method has good specificity and excellent anti-interference ability for ions that often appear in honey. The accuracy of the fluorescent sensor is high, the spiked recovery is 93.2–102.4%, and the standard deviation is 4.5–9.7%. These results show that this fluorescent sensor based on the C-TCD modified SDZ aptamer can detect SDZ residues in honey sensitively, accurately and selectively. [ABSTRACT FROM AUTHOR]

The enzyme strictosidine synthase (STR, EC: 4.3.3.2) plays a key role in the biosynthetic pathway of terpenoid indole alkaloid (TIA). It catalyzes the condensation of the tryptamine and secologanin to form 3α(S)-strictosidine, which is the common precursor of all TIAs. In this paper, a STR gene designated as DoSTR (GenBank: KX068707) was first cloned and characterized from Dendrobium officinale with rapid amplified cDNA ends method (RACE). DoSTR has a length of 1380bp with 1179bp open reading frame encoding 392 amino acids. BlastP analyses showed that its amino acid sequence was classified into Str_synth superfamily. qRT-PCR showed that DoSTR was expressed in all tissues tested, with a significantly higher level in flower and the lowest in stem. Four different treatments with MeJA, SA, ABA and AgNO3, respectively, could induce the DoSTR expression to a different extent. And the effect of MeJA was the most obvious and transcript level of DoSTR induced by MeJA was 20.7 times greater than that of control at 48 hours after treatment. Furthermore, it was found that DoSTR was localized in vacuole through transient expression in tobacco. The characterization and expression of DoSTR can help in further studying the role of DoSTR in the biosynthesis of TIAs in D. officinale. This study may throw light on the alkaloid biosynthesis pathway of D. officinale. [ABSTRACT FROM AUTHOR]

[Objective] Human microplasminogen is a peptide chain consisted of 261 amino acids, located in 549 —790 of human plasminogen and containing plasminogen active region. Alter being activated, it has fibrinolytic activity to degrade fibrin. This paper aims to optimize the expression of human microplasminogen cDNA in Pichia past oris. [Method] The amino acid sequence of human plasminogen was obtained from NCBI according to the principle of codon preference of Pichia pastoris, the corresponding DNA sequence was designed and then synthesized, and it was cloned into the expression vector pPICZαA to obtain one copy expression unit in pPICZαA-mPlg. The expression unit of human plasminogen was obtained by double enzyme digestion of positive expression plasmid, pPICZαA-mPlg, and final a 4-copy (expression u-nit) pPICZ a-mplg was constructed in pPICZαA. The 4 copys expression plasmid was linearized by single enzyme digestion, and was transformed into Pichia pastor'is SMD1168 by electroporation. The positive colony was screened bv phenotype and PCR. The screened genetic engineering Pichia past oris was induced with methyl alcohol, the molecular weight and activity was detected by SDS-PAGE and ehromogenie substrate assay, respectively. The yield of human microplasminogen was increased by optimizing fermentation and Sp-sepharose fast flow gel chromatography was used to purify the target protein. [Result] The human microplasminogen band was observed from SDS-PAGE gel and the plasmin activity was 90 11/mb. [Conclusion]This paper paves the way for subsequent large scale expression of human microplasminogen. [ABSTRACT FROM AUTHOR]

Licorice is one of the most-frequently-used Chinese herbs. Glycyrrhizic acid (GA), as the most important marker component in licorice, is biosynthesized by mevalonic acid (MVA) pathway and regulated by many key enzymes. Among them, β -amyrin synthase (β -AS) is responsible for the triterpene skeleton formation. In this paper, we tried to find out the influence of β - AS gene polymorphism on the GA biosynthesis. The contents of 18 α -glycyrrhizic acid (18 α -GA) and 18 β -glycyrrhizic acid (18 β -GA) in 60 licorice samples were assayed by HPLC. For the β-AS gene polymorphism analysis, six samples with high GA content and seven samples with low GA content were selected. Thirteen β-AS cDNA sequences were obtained and eleven haplotypes were determined. The full length of these β-AS cDNA sequences is 2289 bp, with a complete open reading frame (ORF), encoding 762 amino acid residues. Haplotype-3 was the major haplotype in the group with high GA content, while haplotype-8 was the major haplotype in the group with low GA content. The missense mutation sites between haplotype-3 and haplotype-8 were G/C at 104 bp and 106 bp, resulting in the mutations of serine/threonine at the 35th amino acid residue site and alanine/proline at the 36th amino acid residue site. The yeast expression vectors, GS115-BAS-3 and GS115-BAS-8, containing haplotype-3 or haplotype-8 were constructed, and later transformed into Pichia pastoris GS115 by electrotransformation, respectively. The catalytic efficiency of protein BAS-3 and BAS-8 expressed in the different recombinant P. pastoris GS115 strains were analyzed by GC–MS. It was showed that compared with BAS-8, BAS-3 had a higher catalytic activity. The relative expression analysis showed that there was a positive correlation between the expression level of β-AS gene and the content of GA. We hope this paper will provide a basis for functional gene studies involved in the biosynthetic pathway of GA and high-quality cultivated licorice selection in the future. • Two special β -AS haplotypes, haplotype-3 and haplotype-8 were the major haplotypes in the group with high/low content of glycyrrhizic acid, respectively. • Comparatively, haplotype-3 has a higher catalytic activity in Pichia pastoris GS115. • There was a positive correlation between the expression level of β-AS gene and the content of glycyrrhizic acid. • The β - AS gene polymorphism influenced the biosynthesis of glycyrrhizic acid. [ABSTRACT FROM AUTHOR]

This study investigated the expression levels of FAD2 genes important for the conversion of oleic acid to linoleic acid at cotyledon, root and leaf tissues of four different safflower cultivars (Olas, Remzibey, Dincer and Gokturk) subjected to B by qRT-PCR. Safflower species were grown in a controlled environmental growth chamber for 21 days and then exposed to different B concentrations ranging from 20 to 1 280 µmol for 24 h. RNA isolation, cDNA synthesis and RT-PCR analysis were performed on root, cotyledon and leaf tissues exposed to B stress for 24 h. It was determined that the expression levels of FAD2 genes decreased in the safflower cultivars exposed to increasing B concentrations. Under B stress conditions, the expression levels of FAD2 genes revealed an overall pattern of increase and reduction up to 160 µmol relative to the control group, and they reached the highest expression level. After 320 µmol, the activity of FAD2 genes was almost absent at increasing concentrations. All results show that the application of B causes significant changes in the expression of FAD2 genes and plays an important role in the defence mechanism against increased B toxicity. [ABSTRACT FROM AUTHOR]

Carrier-free delivery of therapeutically relevant small molecules and functional oligonucleotides is extremely challenging and is one of the major hurdles in cancer therapy. Herein, we report a non-covalent approach for the design of self-deliverable nanoparticles of camptothecin and antisense DNA. Our strategy relies on the electrostatic attraction between the cationic ammonium group of the camptothecin prodrug and the negatively charged backbone of antisense DNA as the key interaction. Self-assembly between the camptothecin prodrug and antisense DNA results in the formation of a supramolecular amphiphile, wherein the hydrophobic camptothecin is electrostatically tethered along the phosphate backbone of DNA. The subsequent amphiphilicity-driven self-assembly of the supramolecular amphiphile leads to the formation of stimuli-responsive prodrug nanoparticles. The most notable structural feature of prodrug nanoparticles is the presence of redox-responsive disulfide bonds, which permits thiol-triggered disassembly of the nanoparticles and the release of active drugs. By exploring the high concentration of GSH inside the cancerous cells, we in vitro demonstrate the carrier-free self-delivery of two anticancer drugs simultaneously. Excellent anticancer activity is observed due to the combined therapeutic effect of camptothecin and antisense DNA. [ABSTRACT FROM AUTHOR]

Successful construction of a detection method for Salmonella typhimurium (S. typhimurium) based on the synergy of hybridization chain reaction (HCR) and fluorescence was realized in this paper. First, the aptamer modified with the quenching group Black Hole Quencher-1 acid (BHQ1) was immobilized on the magnetic beads in combination with the complementary chain of the aptamer modified with 6-carboxyfluorescein (6-FAM). Second, S. typhimurium and cDNA-6-FAM immobilized on magnetic beads competitively bound to the aptamer. Finally, the cDNA-6-FAM was released after magnetic separation acted as a promoter to trigger HCR amplification when the target presented. The fluorescence signal could be significantly improved by the combination of green SYBR Green I (SGI) and HCR long double-stranded DNA and the fluorescent synergy of 6-FAM and SGI. Because of the separation of target and its aptamer, the trigger strand was abstracted by magnetic separation. There was no HCR to generate long double-stranded DNA, and the fluorescence of excess hairpin/SGI could be adsorbed through UIO66 so that only a very low background signal was detected. This fluorescent sensor was capable of monitoring S. typhimurium in the range of 10–3.2 × 107 CFU mL−1 with a limit of detection as low as 1.5 CFU mL−1. Because of the excellent properties of the aptasensor and the validity of SGI fluorescence synergy, this HCR enzyme-free amplification strategy could be generalized to other areas. [Display omitted] • Enzyme-free isothermal fluorescence signal amplification was realized by HCR. • Background signal was reduced by magnetic separation and UIO66 quenching effect. • SGI and 6-FAM were used as signaling probes for fluorescence synergy. [ABSTRACT FROM AUTHOR]

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The expansion of long non-coding RNAs (lncRNAs) in organismal genomes has been associated with the emergence of sophisticated regulatory networks that may have contributed to more complex neuronal processes, such as higher-order cognition. In line with the important roles of lncRNAs in the normal functioning of the human brain, dysregulation of lncRNA expression has been implicated in aging and age-related neurodegenerative disorders. In this paper, we discuss the function and expression of known neuronal-associated lncRNAs, their impact on epigenetic changes, the contribution of transposable elements to lncRNA expression, and the implication of lncRNAs in maintaining the 3D nuclear architecture in neurons. Moreover, we discuss how the complex molecular processes that are orchestrated by lncRNAs in the aged brain may contribute to neuronal pathogenesis by promoting protein aggregation and neurodegeneration. Finally, this review explores the possibility that age-related disturbances of lncRNA expression change the genomic and epigenetic regulatory landscape of neurons, which may affect neuronal processes such as neurogenesis and synaptic plasticity. [ABSTRACT FROM AUTHOR]

Dinoflagellates are the large group of marine phytoplankton with primary studies interest regarding their symbiosis with coral reef and the abilities to form harmful algae blooms (HABs). Toxin produced by dinoflagellates during events of HABs cause severe negative impact both in the economy and health sector. However, attempts to understand the dinoflagellates genomic features are hindered by their complex genome organization. Transcriptomics have been employed to understand dinoflagellates genome structure, profile genes and gene expression. RNA-seq is one of the latest methods for transcriptomics study. This method is capable of profiling the dinoflagellates transcriptomes and has several advantages, including highly sensitive, cost effective and deeper sequence coverage. Thus, in this review paper, the current workflow of dinoflagellates RNA-seq starts with the extraction of high quality RNA and is followed by cDNA sequencing using the next-generation sequencing platform, dinoflagellates transcriptome assembly and computational analysis will be discussed. Certain consideration needs will be highlighted such as difficulty in dinoflagellates sequence annotation, post-transcriptional activity and the effect of RNA pooling when using RNA-seq. [ABSTRACT FROM AUTHOR]

Computational analysis of promoters is hindered by the complexity of their architecture. In less studied genomes with complex organization, false positive promoter predictions are common. Accurate identification of transcription start sites and core promoter regions remains an unsolved problem. In this paper, we present a comprehensive analysis of genomic features associated with promoters and show that probabilistic integrative algorithms-driven models allow accurate classification of DNA sequence into “promoters” and “non-promoters” even in absence of the full-length cDNA sequences. These models may be built upon the maps of the distributions of sequence polymorphisms, RNA sequencing reads on genomic DNA, methylated nucleotides, transcription factor binding sites, as well as relative frequencies of nucleotides and their combinations. Positional clustering of binding sites shows that the cells of Oryza sativa utilize three distinct classes of transcription factors: those that bind preferentially to the [-500,0] region (188 “promoter-specific” transcription factors), those that bind preferentially to the [0,500] region (282 “5′ UTR-specific” TFs), and 207 of the “promiscuous” transcription factors with little or no location preference with respect to TSS. For the most informative motifs, their positional preferences are conserved between dicots and monocots. [ABSTRACT FROM AUTHOR]

Abiotic stresses particularly heat stress constitutes a serious threat to the world food production in bread wheat. In the present study, candidate gene expression analysis of HDR77 genotype (which is a susceptible genotype based on electrolyte leakage experiment) was carried out and only those candidate genes which were showing two fold change differences were selected from previous research papers These genes were run over cDNA of HDR 77genotype used as template and intensity of bands was checked by semi-quantative RT PCR. Few genes like HSP 80 gene, MADSWP12 gene, HSP 26.6B gene, WRKY53b gene, WRKY72-a gene, HSP26 gene and WRKY72b gene were upregulated whereas TaVRTMADS gene, NAC gene and WRKY10 gene were down-regulated. The heat stress responsive genes will increase our understanding of molecular basis for heat tolerance in different wheat genotypes and future improvement of heat tolerance in wheat and other crops. [ABSTRACT FROM AUTHOR]

A simple and rapid electrochemical sensing method with high sensitivity and specificity of aptamers was developed for the detection of methylamphetamine (MAMP). A short anti-MAMP thiolated aptamer (Apt) with a methylene blue (MB) probe at 3ʹ-end was immobilized on the surface of a gold electrode (MB-Apt-S/GE). The electrochemical signal appeared when MAMP presenting in the sample solution competed with cDNA for binding with MB-Apt-S. Under optimized conditions, the liner range of this signal-on electrochemical aptasensor for the detection of MAMP achieved from 1.0 to 10.0 nmol/L and 10.0–400 nmol/L. LOD 0.88 nmol/L were obtained. Satisfactory spiked recoveries of saliva and urine were also obtained. In this method, only 5 min were needed to incubate before the square wave voltammetry (SWV) analysis, which was much more rapid than other electrochemical sensors, leading to a bright and broad prospect for the detection of MAMP in biological sample. This method can be used for on-site rapid detection on special occasions, such as drug driving scenes, entertainment venues suspected of drug use, etc. [ABSTRACT FROM AUTHOR]

Alu elements in the human genome are present in more than one million copies, accounting for 10% of the genome. However, the biological functions of most Alu repeats are unknown. In this present study, we detected the effects of Alu elements on EGFP gene expression using a plasmid system to find the roles of Alu elements in human genome. We inserted 5′-4TMI-Alus-CMV promoter-4TMI-Alus (or antisense Alus)-3′ sequences into the pEGFP-C1 vector to construct expression vectors. We altered the copy number of Alus, the orientation of the Alus, and the presence of an enhancer (4TMI) in the inserted 5′-4TMI-Alus-CMV promoter-4TMI-Alus (or antisense Alus)-3′ sequences. These expression vectors were stably transfected into HeLa cells, and EGFP reporter gene expression was determined. Our results showed that combined sense-antisense Alu elements activated the EGFP reporter gene in the presence of enhancers and stable transfection. The combined sense-antisense Alu vectors carrying four copies of Alus downstream of inserted CMV induced much stronger EGFP gene expression than two copies. Alus downstream of inserted CMV were replaced to AluJBs (having 76% homology with Alu) to construct expression vectors. We found that combined sense-antisense Alu (or antisense AluJB) vectors induced strong EGFP gene expression after stable transfection and heat shock. To further explore combined sense-antisense Alus activating EGFP gene expression, we constructed Tet-on system vectors, mini-C1-Alu-sense-sense and mini-C1-Alu-sense-antisense ( EGFP gene was driven by mini-CMV). We found that combined sense-antisense Alus activated EGFP gene in the presence of reverse tetracycline repressor (rTetR) and doxycycline ( Dox). Clone experiments showed that Mini-C1-Alu-sense-antisense vector had more positive cells than that of Mini-C1-Alu-sense-sense vector. The results in this paper proved that Alu repetitive sequences inhibited gene expression and combined sense-antisense Alus activated EGFP reporter gene when Alu transcribes, which suggests that Alus play roles in maintaining gene expression (silencing genes or activating genes) in human genome. [ABSTRACT FROM AUTHOR]

The article discusses a study on the cloning of complementary DNA (cDNA) encoding the murine immunoglobulin G1 (IgG1) induction factor. It references the study "Cloning of cDNA Encoding the Murine IgG1 Induction Factor by a Novel Strategy Using SP6 Promoter"by Y. Noma et al. published in Volume 319 of "Nature." The author's involvement in the molecular cloning of interleukin-4 (IL-4) is also described.

Oysters contain significant amounts of the zinc element, which may also be found in their proteins. In this study, a novel zinc-binding protein was purified from the mantle of the oyster Magallana hongkongensis using two kinds of gel filtration chromatograms. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) showed that its molecular weight was approximately 36 kDa. The protein identified by the Q-Exactive mass spectrometer shared the highest sequence identity with carbonic anhydrase derived from Crassostrea gigas concerning amino acid sequence similarity. Based on homologous cloning and RACE PCR, the full-length cDNA of carbonic anhydrase from Magallana hongkongensis (designated as MhCA) was cloned and sequenced. The cDNA of MhCA encodes a 315-amino-acid protein with 89.74% homology to carbonic anhydrase derived from Crassostrea gigas. Molecular docking revealed that the two zinc ions primarily form coordination bonds with histidine residues in the MhCA protein. These results strongly suggest that MhCA is a novel zinc-binding protein in Magallana hongkongensis. [ABSTRACT FROM AUTHOR]

Plant growth and development are inhibited by the high levels of ions and pH due to soda saline-alkali soil, and the cell wall serves as a crucial barrier against external stresses in plant cells. Proteins in the cell wall play important roles in plant cell growth, morphogenesis, pathogen infection and environmental response. In the current study, the full-length coding sequence of the vegetative cell wall protein gene OsGP1 was characterized from Lj11 (Oryza sativa longjing11), it contained 660 bp nucleotides encoding 219 amino acids. Protein-protein interaction network analysis revealed possible interaction between CESA1, TUBB8, and OsJ_01535 proteins, which are related to plant growth and cell wall synthesis. OsGP1 was found to be localized in the cell membrane and cell wall. Furthermore, overexpression of OsGP1 leads to increase in plant height and fresh weight, showing enhanced resistance to saline-alkali stress. The ROS (reactive oxygen species) scavengers were regulated by OsGP1 protein, peroxidase and superoxide dismutase activities were significantly higher, while malondialdehyde was lower in the overexpression line under stress. These results suggest that OsGP1 improves saline-alkali stress tolerance of rice possibly through cell wall-mediated intracellular environmental homeostasis. [ABSTRACT FROM AUTHOR]

Tomato black ring virus (TBRV) is a member of the Nepovirus genus in the Secoviridae family, which infects a wide range of important crop species worldwide. In this work, we constructed four cDNA infectious clones of the TBRV tagged with the green fluorescent protein (TBRV-GFP), which varied in (i) the length of the sequences flanking the GFP insert, (ii) the position of the GFP insert within the RNA2 polyprotein, and (iii) the addition of a self-cutting 2A protein. The presence of the GFP coding sequence in infected plants was verified by RT-PCR, while the infectivity and stability of the constructs were verified by mechanical inoculation of the host plants. The systemic spread of TBRV-GFP within plants was observed under UV light at a macroscopic level, monitoring GFP-derived fluorescence in leaves, and at a microscopic level using confocal microscopy. The obtained clones are a valuable tool for future studies of TBRV-host interactions, virus biology, and the long-term monitoring of its distribution in infected plants. [ABSTRACT FROM AUTHOR]

Pectate lyases are known to play a key role in pectin degradation by catalyzing the random cleavage of internal polymer linkages (endo-pectinases). In this paper, four novel cDNAs, designated Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7, that encode pectate lyases were cloned and characterized from the soybean cyst nematode, Heterodera glycines. The predicted protein sequences of HG-PEL-3, HG-PEL-4 and HG-PEL-6 differed significantly in both their amino acid sequences and their genomic structures from other pectate lyases of H. glycines (HG-PEL-1, HG-PEL-2 and HG-PEL-7). A phylogenetic study revealed that the pectate lyase proteins of H. glycines are clustered into distinct clades and have distinct numbers and positioning of introns, which suggests that the pectate lyase genes of H. glycines may have evolved from at least two ancestral genes. A Southern blot analysis revealed that multiple Hg-pel-6-like genes were present in the H. glycines genome. In situ hybridization showed that four novel pectate lyases (Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7) were actively transcribed in the subventral esophageal gland cells. A semi-quantitative RT-PCR assay supported the finding that the expression of these genes was strong in the egg, pre-parasitic second-stage juvenile (J2) and early parasitic J2 stages and that it declined in further developmental stages of the nematode. This expression pattern suggests that these proteins play a role in the migratory phase of the nematode life cycle. Knocking down Hg-pel-6 using in vitro RNA interference resulted in a 46.9% reduction of the number of nematodes that invaded the plants and a 61.5% suppression of the development of H. glycines females within roots compared to the GFP-dsRNA control. Plant host-derived RNAi induced the silencing of the Hg-pel-6gene, which significantly reduced the nematode infection levels at 7 Days post inoculation (dpi). Similarly, this procedure reduced the number of female adults at 40 dpi, which suggests the important roles of this gene in the early stages of parasitism. Our combined data suggest that two types of pectate lyases are present in the H. glycines genome and may have different roles during infection. [ABSTRACT FROM AUTHOR]

Japanese red pine (Pinus densiflora) is extensively cultivated in Japan, Korea, China, and Russia and is harvested for timber, pulpwood, garden, and paper markets. However, genetic information and molecular markers were very scarce for this species. In this study, over 51 million sequencing clean reads from P. densiflora mRNA were produced using Illumina paired-end sequencing technology. It yielded 83,913 unigenes with a mean length of 751 bp, of which 54,530 (64.98%) unigenes showed similarity to sequences in the NCBI database. Among which the best matches in the NCBI Nr database were Picea sitchensis (41.60%), Amborella trichopoda (9.83%), and Pinus taeda (4.15%). A total of 1953 putative microsatellites were identified in 1784 unigenes using MISA (MicroSAtellite) software, of which the tri-nucleotide repeats were most abundant (50.18%) and 629 EST-SSR (expressed sequence tag- simple sequence repeats) primer pairs were successfully designed. Among 20 EST-SSR primer pairs randomly chosen, 17 markers yielded amplification products of the expected size in P. densiflora. Our results will provide a valuable resource for gene-function analysis, germplasm identification, molecular marker-assisted breeding and resistance-related gene(s) mapping for pine for P. densiflora. [ABSTRACT FROM AUTHOR]

Two double-stranded RNA (dsRNA) mycoviruses were found in isolate QSP5 of the rice blast fungus Magnaporthe oryzae. Sequence analysis of the two dsRNA mycoviruses revealed that one is closely related to Magnaporthe oryzae virus 2 (MoV2), and the other one is related to Magnaporthe oryzae chrysovirus 1-A (MoCV1-A). Therefore, they were named Magnaporthe oryzae virus 3 (MoV3) and Magnaporthe oryzae chrysovirus 1-C (MoCV1-C), respectively. In this paper, the molecular and structural characteristics of MoV3 were analyzed in detail. The full genome sequence (5181 bp) of MoV3 was obtained by cDNA cloning. Sequence analysis indicated that MoV3 has two overlapping open reading frames (ORF1 and ORF2). The 5′-proximal ORF1 encodes a putative coat protein (CP) with a molecular weight of 80,939 Da; the 3′-proximal ORF2 encodes a putative RNA-dependent RNA polymerase (RdRp) with a molecular weight of 90,506 Da. The stop codon of ORF1 overlaps the start codon of ORF2, with the tetranucleotide sequence AUGA, which is characteristic of members of the genus Victorivirus of the family Totiviridae. Phylogenetic analysis of RdRp and CP further supported the view that MoV3, a novel mycovirus, belongs to the genus Victorivirus of the family Totiviridae. [ABSTRACT FROM AUTHOR]

In vitro detection of low abundance biomolecules including microRNAs (miRNAs) is essential to biological research and early clinical diagnosis. In this work, a versatile magnetic bead (MB)-based flow cytometric assay was developed for the detection of hsa-miR-221-3p, which is strongly associated with papillary thyroid carcinoma (PTC). In the presence of hsa-miR-221-3p, the complementary DNA probe attached to the surface of MBs is hybridized with the target to form DNA/RNA heteroduplexes. After the recognition of the DNA/RNA heteroduplexes by PicoGreen, the fluorescence signals of each MB were readily detected using a flow cytometer. This assay can selectively detect hsa-miR-221-3p with a detection limit of 2.1 pM. The practicality of the assay is demonstrated by the discrimination of thyroid cancer tissues from normal tissues, and a satisfactory result is obtained. Moreover, this assay can be rapidly carried out in one step at room temperature, providing a generic method for the sensitive detection of miRNAs in molecular diagnosis. [ABSTRACT FROM AUTHOR]

Resonance energy transfer donor (MIL-53-apt) and acceptor (RuSiO 2 -cDNA) fluorescence probes were synthesized by simple methods. Base pairing between 17β-Estradiol (E2) aptamer and its complementary DNA shortens the distance between the donor and the acceptor, leading to the transfer of energy from the donor to the acceptor. The presence of E2 hinder the process of FRET and make the fluorescence of donor recover and the fluorescence of acceptor quench, so as to realize the ratiometric analysis for E2. The detection of E2 in serum samples shows that the sensor has a practical application prospect, and the method is also expected to be applied in the detection of other biomarkers. • In situ enhancement of fluorescence emission of RuSiO 2 was achieved by encapsulating Ru(bpy) 3 2+ with silica. • The opening of the FRET is completed by base pairing, which realize the sensitive detection of E2 in serum, showing the practical application prospect of this sensor. 17β-Estradiol (E2), an important endocrine disrupting compound, could be quantitatively detected by fluorescence resonance energy transfer (FRET) aptasensor, designed in this paper. Metal organic frameworks have large specific surface area and easily modifiable groups, which are helpful for the construction of aptasensor. Specifically, streptavidin was immobilized on the synthesized MIL-53-NH 2 by covalent bonding, and further linked with the biotin modified E2 aptamer (apt) through specific bonding between avidin and biotin to obtain the FRET donor probe (MIL-53-apt). Meanwhile, complementary DNA (cDNA) modified Ru(bpy) 3 2+-doped silica nanoparticles (RuSiO 2 -cDNA) were prepared through covalent bonding. They acted as the FRET acceptor probe, since its absorption spectrum showed large overlap with the emission spectrum of MIL-53-apt. In the presence of E2, aptamer modified donor probes tended to bind with E2, owing to their higher selectivity and affinity. Therefore, the optimal distance between FRET pairs was broken, resulting in the fluorescence emission recovery of donor and the fluorescence emission of acceptor decreased. Under optimal conditions, this proposed aptasensor displayed sensitive detection of E2 ranging from 0.5 to 1000 nM with a detection limit of 0.2 nM. Furthermore, the sensor provides a promising method for rapid and sensitive detection of other small biological molecules. [ABSTRACT FROM AUTHOR]

Hexaammineruthenium(III), RuHex3+, is a DNA-binding metal complex that is widely used as a redox marker for the indirect determination of DNA coverage at the electrode surface. The conversion of electrochemically quantifiable surface excess of RuHex3+ into DNA surface coverage requires the knowledge of the binding site size of RuHex3+ (s). Traditionally, s on surface-immobilized DNA has been assumed to be equivalent to that on solution-phase DNA, which was experimentally determined in previous studies. Nevertheless, the different local microenvironments existing inside DNA monolayers in comparison to that in bulk solutions cast doubt on the validity of this assumption. In this report, we used electrochemical techniques to investigate s on surface-immobilized DNA. The values of s inside the DNA monolayers were found to be significantly smaller than that reported on solution-phase DNA. Besides, s was found to depend on the DNA packing density and became larger by increasing the DNA surface coverage or hybridizing the surface-tethered DNAs with complementary strands. Our data indicate that the RuHex3+ method, in which an s value of 3 nucleotides is used for the conversion of RuHex3+ to DNA surface coverage, does not always give reliable results. [ABSTRACT FROM AUTHOR]

Gold-sputtered microelectrodes with built-in gold reference and counter electrodes represent a promising platform for the development of disposable DNA sensors. Pretreating gold electrode surfaces and immobilization of DNA thereon is commonly employed in biosensing applications. However, with no scientific or practical guidelines to prepare a DNA sensor using these miniature gold-sputtered microelectrodes, cleaning and immobilization steps need to be systematically optimized and updated. In this work, we present efficient cleaning and modification of miniaturized gold-sputtered microelectrodes with thiolated DNA probes for DNA detection. Additional discussions on subtleties and nuances involved at each stage of pretreating and modifying gold-sputtered microelectrodes are included to present a robust, well-founded protocol. It was evident that the insights on cleaning polycrystalline gold disk electrodes with a benchmark electrode surface for DNA sensors, cannot be transferred to clean these miniature gold-sputtered microelectrodes. Therefore, a comparison between five different cleaning protocols was made to find the optimal one for gold-sputtered microelectrodes. Additionally, two principally different immobilization techniques for gold-sputtered microelectrode modification with thiolated ssDNA were compared i.e., immobilization through passive chemisorption and potential perturbation were compared in terms of thiol-specific attachment and thiol-unspecific adsorption through nitrogenous bases. The hybridization performance of these prepared electrodes was characterized by their sensitive complementary DNA capturing ability, detected by a standard alkaline phosphatase assay. Immobilization through passive chemisorption proved to be efficient in capturing the complementary target DNA with a detection limit of 0.14 nM and sensitivity of 9.38 A M−1 cm2. In general, this work presents a comprehensive understanding of cleaning, modification and performance of gold-sputtered microelectrodes with built-in gold reference and counter electrodes for both fundamental investigations and practical DNA sensing applications. [ABSTRACT FROM AUTHOR]

From its discovery in the 1980s, Polymerase chain reaction was further developed and is nowadays used as the foundation for the various PCR-based techniques used in molecular diagnosis across different species, and numerous types of samples. Real-Time PCR enables the user to monitor the amplification of a deoxyribonucleic acid (DNA) or complementary DNA (cDNA) target during the PCR run, in real-time, and not at the end, as it is the case in conventional PCR. The most frequent types of applications include gene expression analysis, gene silencing, variant analysis, and fusion temperature analysis. Given its vast field of application, a key question remains, and it is related to the controls (negative controls, positive controls, internal exogenous and endogenous controls) and their purpose in a Real-Time PCR experiment. In this paper, we set out to find how and when to use them, and which type of controls are suitable for certain experiment types, since the use of appropriate controls during Real-Time PCR experiments will reduce the effects of variables aside from the independent variable within the sample, therefore yielding accurate results, be it in research or diagnostic purposes. [ABSTRACT FROM AUTHOR]

Molecular studies that involved cDNA and genomic DNA sequencing as well as multiplex PCR of the HPRT gene were performed to determine the molecular mutational spectrum for 1,377 HPRT mutant isolates obtained from 61 Veterans of the 1991 Gulf War, most of whom were exposed to depleted uranium (DU). Mutant colonies were isolated from one to four times from each Veteran (in 2003, 2005, 2007, and/or 2009). The relative frequencies of the various types of mutations (point mutations, deletions, insertions, etc.) were compared between high versus low DU exposed groups, (based on their urine U concentration levels), with HPRT mutant frequency (as determined in the companion paper) and with a database of historic controls. The mutational spectrum includes all classes of gene mutations with no significant differences observed in Veterans related to their DU exposures. Environ. Mol. Mutagen. 56:594-608, 2015. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Jatropha curcasis an important non feed crop, increasingly important as a biofuel crop. It is hardy and resistant to different stress conditions in the field. In the wastelands of Gujarat (India), it is being grown for land reclamation and for socio-economic benefits. The long coastline in this state also promotes the growth of a large number of halophytes. Exploiting the genetic resource of Jatropha and halophytes for drought and salt-induced gene is an important area of research. For the isolation of genes and to study the molecular mechanism a good qualitative and quantitative RNA is a prerequisite.Jatrophaleaves have latex, and therefore isolating RNA using guanidine thiocyanate or cetyltrimethylammonium bromide did not yield desirable quality of RNA. This paper reports a very simple and economical protocol for the isolation of good quality RNA fromJatrophaand a few halophytes. The sodium dodecyl sulphate was used as a detergent for lysis of plant cells in the extraction buffer along with bentonite, which inhibits the ribonuclease’s activity. The addition of water saturated phenol in mortar-pestle, during grinding, facilitated better homogenisation of the tissues. Absolute RNA precipitation was obtained with the help of 2-butoxyethanol. Further this RNA was used successfully in preparation of complementary DNA and subsequently used for gene isolation. [ABSTRACT FROM PUBLISHER]

A new block cipher for gray images is proposed in this paper which fully utilizes whole set of DNA complementary rules dynamically for encoding and decoding each pixel of a block. The most significant (MSB) part of each block is added under DNA addition operation with least significant (LSB) while LSB part itself get encrypted by chaotically selecting different DNA rules for each pixel. The initial condition is calculated from 128-bits external input key and then the said key is modified for each subsequent block of an image. An image is permuted by Piecewise Linear Chaotic Map (PWLCM) while logistic sequence is used for the selection of encoding and decoding rules for each pixel of a block. The simulated experimental results and the security analysis in terms of quantitative and qualitative way show that our cipher can not only achieve good encryption effect to resist the exhaustive and statistical attacks but also is a good candidate for encrypting large sized uncompressed gray images. [ABSTRACT FROM AUTHOR]

Amphibian skin secretion has great potential for drug discovery and contributes hundreds of bioactive peptides including bradykinin-related peptides (BRPs). More than 50 BRPs have been reported in the last two decades arising from the skin secretion of amphibian species. They belong to the families Ascaphidae (1 species), Bombinatoridae (3 species), Hylidae (9 speices) and Ranidae (25 species). This paper presents the diversity of structural characteristics of BRPs with N-terminal, C-terminal extension and amino acid substitution. The further comparison of cDNA-encoded prepropeptides between the different species and families demonstrated that there are various forms of kininogen precursors to release BRPs and they constitute important evidence in amphibian evolution. The pharmacological activities of isolated BRPs exhibited unclear structure-function relationships, and therefore the scope for drug discovery and development is limited. However, their diversity shows new insights into biotechnological applications and, as a result, comprehensive and systematic studies of the physiological and pharmacological activities of BRPs from amphibian skin secretion are needed in the future. [ABSTRACT FROM AUTHOR]

Data on glucose dehydrogenases (GDHs) are scarce and availability of these enzymes for application purposes is limited. This paper describes a new GDH from the fungus Pycnoporus cinnabarinus CIRM BRFM 137 that is the first reported GDH from a white-rot fungus belonging to the Basidiomycota. The enzyme was recombinantly produced in Aspergillus niger, a well-known fungal host producing an array of homologous or heterologous enzymes for industrial applications. The full-length gene that encodes GDH from P. cinnabarinus ( PcGDH) consists of 2,425 bp and codes for a deduced protein of 620 amino acids with a calculated molecular mass of 62.5 kDa. The corresponding complementary DNA was cloned and placed under the control of the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. The signal peptide of the glucoamylase prepro sequence of A. niger was used to target PcGDH secretion into the culture medium, achieving a yield of 640 mg L, which is tenfold higher than any other reported value. The recombinant PcGDH was purified twofold to homogeneity in a one-step procedure with a 41 % recovery using a Ni Sepharose column. The identity of the recombinant protein was further confirmed by immunodetection using western blot analysis and N-terminal sequencing. The molecular mass of the native PcGDH was 130 kDa, suggesting a homodimeric form. Optimal pH and temperature were found to be similar (5.5 and 60 °C, respectively) to those determined for the previously characterized GDH, i.e., from Glomerella cingulata. However PcGDH exhibits a lower catalytic efficiency of 67 M s toward glucose. This substrate is by far the preferred substrate, which constitutes an advantage over other sugar oxidases in the case of blood glucose monitoring. The substrate-binding domain of PcGDH turns out to be conserved as compared to other glucose-methanol-choline (GMCs) oxidoreductases. In addition, the ability of PcGDH to reduce oxidized quinones or radical intermediates was clearly demonstrated, which raises prospects for applying this enzyme to detoxify toxic compounds formed during the degradation of lignin. [ABSTRACT FROM AUTHOR]

This is a perspective based on the paper "Cloning of complementary DNA encoding T cell-replacing factor and identity with B cell growth factor II," by Kinashi et al. (1). We have been interested in understanding the molecular basis ofT-B cell cooperation for antibody formation. Although many investigators had described a number of different soluble factors that appeared to have biological relevance to T-B cell interactions, molecular basis of such active substances remained unknown for a long period of time. In this perspective, I will briefly summarize the history of the initial discovery of T cell-replacing factor/B cell growth factor II that appeared to be involved in B cell growth and differentiation, and outline the discovery and characterization of interleukin-5. Studies of interleukin-5 have provided strong evidence that a single cytokine exerts a variety of activities on diverse target cells. [ABSTRACT FROM AUTHOR]

Trehalose is a non-reducing disaccharide that accumulates to large quantities in microbial cells, but in plants it is generally present in very low, barely-detectible levels. A notable exception is the desiccation-tolerant plant Selaginella lepidophylla, which accumulates very high levels of trehalose in both the hydrated and dehydrated state. As trehalose is known to protect membranes, proteins, and whole cells against dehydration stress, we have been interested in the characterization of the trehalose biosynthesis enzymes of S. lepidophylla; they could assist in engineering crop plants towards better stress tolerance. We previously isolated and characterized trehalose-6-phosphate synthases from Arabidopsis thaliana (desiccation sensitive) and S. lepidophylla (desiccation tolerant) and found that they had similar enzymatic characteristics. In this paper, we describe the isolation and characterization of trehalose-6-phosphate phosphatase from S. lepidophylla and show that its catalytic activities are also similar to those of its homolog in A. thaliana. Screening of an S. lepidophylla cDNA library using yeast trehalose biosynthesis mutants resulted in the isolation of a large number of trehalose biosynthesis genes that were of microbial rather than plant origin. Thus, we suggest that the high trehalose levels observed in S. lepidophylla are not the product of the plant but that of endophytes, which are known to be present in this plant. Additionally, the high trehalose levels in S. lepidophylla are unlikely to account for its desiccation tolerance, because its drought-stress-sensitive relative, S. moellendorffii, also accumulated high levels of trehalose. [ABSTRACT FROM AUTHOR]

DNA microarray technology has enabled biologists to study all the genes within an entire organism to obtain a global view of genes? interaction and regulation. This technology has a great potential in obtaining a deep understanding of the functional organization of cells. This paper is concerned with improving the processes involved in the analysis of microarray image data. The main focus is to clarify an image?s feature space in an unsupervised manner. Rather than using the raw microarray image, it proposes to produce filtered versions of the image data by applying nonlinear anisotropic diffusion so that the dynamic range of the image could be increased and, therefore, a better ability of signal extraction could be achieved. In this paper, a novel segmentation algorithm is developed. This algorithm is based on the Cellular Neural Network computational paradigm integrated with median and anisotropic diffusion filters. The AnaLogic CNN Simulation Toolbox for MATLAB (InstantVision Toolboxes for MATLAB) is used during the segmentation process. Quantitative comparisons among the proposed methods and GenePix® are carried out from both the objective and subjective points of view. It is shown that analogic algorithm integrated with Complex Diffusion filter is the best one to be applied to accomplish the segmentation. [ABSTRACT FROM AUTHOR]

Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on secondstrand complementary DNA (cDNA) synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without secondstrand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods. [ABSTRACT FROM AUTHOR]

【Objective】This paper aimed to establish the 293 T and HT 1080 cell lines with CRISPR/Cpf1 gene by lentiviral vector and further to realize the high-efficiency gene editing. 【Method】AsCpf1 and LbCpf1 gene was amplified by PCR technique, which was inserted into the pLent-EF1 a-MF-CMV-GFP-P2 A-Puro vector. Monolayer of 293 FT cells were cotransfected with three plasmids psPAX2, pMD2.G and Cpf1 in pLent-EF1 a-MF-CMV-GFP-P2 A-Puro. The recombinant lentivirus expressing LbCpf1 and AsCpf1 was harvested, and the titer of virus was calculated by fluorescence microscope 48 hours later. The purified lentivirus was used to infect 293 T cell and the transduced cells were selected with puromycin and single cell colonies were isolated and verified for DNA, RNA and cDNA. Single cell colonies were transfected by sgRNA targeting the exons ABCG1 CDS regions to the functional domains of ABCG1 genes, and gene knockout efficiency was measured by DNA specificity and T7 EΙ digestion. 【Result】DNA sequnencing verified that Cpf1-pLent was constructed successfully, and the titer of recombinant lentivirus was greater than 10~6 TU/mL. Three cell lines expressing Cpf1 stably were obtained, two of which(AsCpf1-293 T-7 D and AsCpf1-HT1080 02-3 B) the targeted gene was knocked out successfully by T7 EΙ digestion. 【Conclusion】 293 T and HT 1080 cells stably expressing Cpf1 was successfully constructed, which could be a valuable tool for gene editing. [ABSTRACT FROM AUTHOR]