cDNA Sequence Research Articles

Characterization, Recombinant Production, and Bioactivity of a Novel Immunomodulatory Protein from Hypsizygus marmoreus.

A novel fungal immunomodulatory protein (FIP), identified as FIP-hma, was discovered in the genome of an edible mushroom Hypsizygus marmoreus. Bioinformatics analysis suggested FIP-hma contained the cerato-platanin (CP) conserved domain and was categorized into Cerato-type FIP. In phylogenetic analysis, FIP-hma was clustered into a new branch of the FIP family, displaying large system divergence from most of the other FIPs. The higher gene expression of FIP-hma was observed during the vegetative growth stages than that during the reproductive growth stages. In addition, the cDNA sequence of FIP-hma was cloned and successfully expressed in Escherichia coli (E. coli) BL21(DE3). The recombinant protein of FIP-hma (rFIP-hma) was neatly purified and isolated by Ni-NTA and SUMO-Protease. The iNOS, IL-6, IL-1β, and TNF-α levels of RAW 264.7 macrophages were upregulated by rFIP-hma, indicating its activation of an immune response by regulating central cytokines. No cytotoxic effects were observed in an MTT test. The findings of this work discovered a novel immunoregulatory protein from H. marmoreus, provided a systematic bioinformatic profile, suggested an effective approach for its heterologous recombinant production, and reported its potent immunoregulatory activity in macrophages. This study sheds light on the physiological function research of FIPs and their further industrial utilization.

Analysis of LncRNA43234-Associated ceRNA Network Reveals Oil Metabolism in Soybean.

Soybean [Glycine max (Linn.) Merr.] is an important oil crop. Long noncoding RNAs (lncRNAs) play a variety of functions in plants. However, their function in the soybean oil synthesis pathway is yet to be uncovered. Here, the lncRNA43234 gene related to soybean oil synthesis was screened, and the full-length cDNA sequence of the lncRNA was obtained using rapid amplification of cDNA ends. Overexpression of lncRNA43234 increased the content of crude protein in seeds, decreased the content of oleic acid, and affected the content of alanine and arginine in free amino acids. RNA interference of the lncRNA43234 gene decreased the crude protein content in seeds. Quantitative real-time polymerase chain reaction analysis revealed that lncRNA43234 influenced the expression of XM_014775786.1 associated with phosphatidylinositol metabolism by acting as a decoy for miRNA10420, thereby affecting the content of soybean oil. Our results provide insights into how lncRNA-mediated competing endogenous RNA regulatory networks are involved in soybean oil synthesis.

The inhibitory activity of a new scorpion venom-derived antimicrobial peptide Hp1470 against Gram-positive bacteria

Antimicrobial peptides (AMPs) are a new type of antibiotic and target a variety of microbes, including antibiotic-resistant strains; thus, AMPs have attracted widespread interest. Scorpion venoms contain many bioactive peptides, including AMPs, and have become an important natural resource of peptide-based drugs. Here, the antibacterial peptide gene Hp1470 from the venom of the scorpion Heterometrus petersii was characterized, and its antibacterial activity was determined. The cDNA sequence of Hp1470 is 300 nt in length and contains an open reading frame (ORF) of 207 nt. The ORF was shown to encode 68 amino acid residues, including a signal peptide (23 aa), a mature peptide (13 aa), a C-terminal posttranslational processing signal (3 aa), and a propeptide (29 aa). Multiple sequence alignment results indicated that Hp1470 is an antibacterial peptide. The mature peptide Hp1470, which has a molecular mass of 1564.09 Da, was further chemically synthesized with a purity of greater than 95%. Antimicrobial assays showed that the synthesized Hp1470 exerted an inhibitory effect on Gram-positive bacteria and clinical drug-resistant strains, including PRSA and MRSA, but not Gram-negative bacteria. Hp1470 was further found to protect mice from MRSA infection, suggesting its potential application as an in vivo antimicrobial agent. Interestingly, Hp1470 only inhibited bacterial growth but did not kill bacteria, which was consistent with scanning electron microscopy results showing that Hp1470 did not lyse the cell membrane of Staphylococcus aureus. Our work provides a new direction for developing antibacterial agents with different modes of action from natural scorpion venoms.

New chemistries for DNA sequencing

Next-generation sequencing (NGS) has made analyzing DNA faster and cheaper, with effects felt in the lab, clinic, and beyond. A set of newly launched technologies is offering a twist on the chemistry behind the process. Experts say these efforts may signal the first big change to NGS in years. NGS relies on sequencing by synthesis: the sequence of a template DNA strand is determined by synthesizing a complementary strand from fluorescently labeled bases. After each base is incorporated by a polymerase and is imaged, its fluorescent tag is removed and another base can be added. Now, companies are introducing sequencing platforms that separate fluorescent labeling from the extension of the complementary DNA strand, touting improvements in accuracy that result from optimizing each step. Late in May, Element Biosciences published on the chemistry behind its platform for avidity sequencing ( Nat. Biotechnol. 2023, doi: 10.1038/s41587-023-01750-7 ). Biochemists define avidity as the

Detoxification and antioxidant functions and regulatory mechanisms of two Delta-class GSTs in paddy crayfish (Procambarus clarkii) after imidacloprid stress

Glutathione S-transferases (GSTs) are phase II metabolic detoxification enzymes, which are widely found in organisms, and play an important role in helping organisms to resist toxic compounds. In this study, the two Delta-class GSTs cDNA sequences were cloned from Procambarus clarkii (designated as PcGSTD1 and PcGSTD2). Tissue specific expression profile showed that PcGST1,2 were expressed in all 6 tissues, with the highest expression in hepatopancreas. Subcellular localization assay showed that PcGSTD1, 2 were mainly expressed in the cytoplasm of HEK-293 T cells. Recombinant PcGSTD1, 2 showed the highest catalytic activity to the GST model substrate 1-chloro-2,4-dinitrobenzene (CDNB) at 20 and 30 °C, pH 8 and 7, respectively. The mRNA expression of PcGSTD1, 2 and the GSTs activity varied with the time of imidacloprid challenge. The BL21(DE3) expressing PcGSTD1, 2 proteins could more resistant to H2O2. The dsRNA experiments showed that PcKeap1b, PcNrf1, and PcMafK affected the transcription levels of PcGSTD1, 2. The GST-Pulldown results revealed that PcbZIP and PcMafK recombinant proteins could bind to each other in vitro. The gel mobility shift assay demonstrated that PcMafK recombinant protein had affinity with the promoter of PcGSTD2. The Dual luciferase assays analyzed the activity of the promoters after different truncations, the core region of PcGSTD1 promoter was at −440 bp to +54 bp, and that of PcGSTD2 promoter was between −1609∼−1125 bp. These results suggested that PcGSTD1, 2 respond positively to imidacloprid stress in P. clarkii, and the transcriptional expressions of PcGSTD1, 2 were influenced by the factors of PcKeap1b/PcNrf1/PcMafK.

A novel heterozygous PKD1 variant causing alternative splicing in a Chinese family with autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by pathogenic variants of PKD1 and PKD2. Compared to PKD2-related patients, patients with PKD1 pathogenic variants have more severe symptoms, present a gradual decline in renal function, and finally progress to end-stage kidney disease with an earlier mean onset age. In this study, trio exome sequencing (ES) was performed to reveal the genetic etiology in a Chinese family clinically diagnosed with polycystic kidney, followed by validation through Sanger sequencing on both genomic DNA and cDNA levels. Subsequently, targeted preimplantation genetic testing was provided for the family. A novel heterozygous PKD1 variant (c.1717_1722+11del) was detected in the proband and other clinically-affected relatives. Interestingly, cDNA sequencing demonstrated that the variant, despite being annotated as non-frameshift within exon 8, impacted the splicing of PKD1. Two abnormal transcription products were formed: one induced frameshift, while the other caused 133 amino acids to be inserted between exon 8 and exon 9. Our study revealed a novel PKD1 variant using ES as the cause of ADPKD in a Chinese family with multiple affected members. The variant at the exon-intron boundary would induce alternative splicing, which should not be excluded from genetic analysis. Validated on the cDNA level could provide more comprehensive genetic information for disease stratification. And the novel variant expands the spectrum of PKD1 variants in ADPKD. The recurrent risk could be blocked accordingly for the families' offspring.

Programming DNA Circuits for Controlled Immunostimulation through CpG Oligodeoxynucleotide Delivery.

Herein, we present a DNA circuit programmed for the delivery of CpG oligodeoxynucleotides (CpG ODNs) with the pharmacological immunostimulation function. The circuit employs a complementary DNA (cDNA) strand to deactivate the biological function of CpG ODNs via hybridization, while T7 exonuclease mediates the activation by hydrolyzing the cDNA and releasing the CpG ODN as an active moiety. We investigated the influence of several factors on the kinetic profile and temporal behavior of the circuit. These include the design of the cDNA strand, the concentration of the DNA duplex, and the concentration of T7 exonuclease. The DNA circuit's in vitro activation resulted in toll-like receptor 9 stimulation in the HEK-engineered cell line, as well as tumor necrosis factor-alpha release by J774A.1 macrophages. By programming the DNA circuit to control the release of the CpG ODN, we achieved an altered pharmacological profile with acute and potent immunostimulation, in comparison to a system without controlled CpG ODN release, which exhibited a slow and delayed response. Our findings demonstrate the potential of DNA circuits in controlling the pharmacological activity of DNA strands for controlled drug delivery.

Effects of NUCB2/nesfatin-1 on lipid metabolism in the hepatopancreas of largemouth bass (Micropterus salmoides)

Although nucleobindin-2 (NUCB2)/nesfatin-1 has multiple biological functions, there is a scarcity of information regarding the regulatory effect of NUCB2/nesfatin-1 on lipid metabolism in mammals and fish. In order to investigate the mechanism underlying the effect of nesfatin-1 on lipid metabolism in largemouth bass (Micropterus salmoides), the sequence of NUCB2 was cloned and nesfatin-1 was expressed in prokaryotic system. Intraperitoneal injection of 5, 50 or 500 ng/g nesfatin-1 protein to determine differentially expressed genes (DEGs) in largemouth bass by transcriptomic profiling. The triglyceride and total cholesterol levels in the hepatopancreas and plasma were subsequently determined. The results showed that the NUCB2 cDNA sequence was 2015 bp in length and analyzed that NUCB2 mRNA was abundantly expressed in the hepatopancreas. RNA-seq analysis revealed that 385, 1647, and 750 genes were differentially expressed in the three groups, respectively, of which 33, 69, and 61 DEGs, respectively, were involved in lipid metabolism. GO and KEGG analysis showed that the DEGs were enriched in 24 signaling pathways related to lipid metabolism. The PPAR signaling pathway was significantly enriched following treatment with nesfatin-1. The fatty acid transport genes (ACSL, FABP3, FATP, ACBP) were significantly downregulated in the pathway (p < 0.05). Additionally, the TG (p < 0.01) and T-CHO (p < 0.05) levels in the hepatopancreas were significantly down-regulated at 50 ng/g nesfatin-1 protein injection dose, while TG and T-CHO levels in plasma were not significantly altered (p > 0.05). The present study demonstrated that at an optimal concentration, nesfatin-1 affected lipid metabolism in the hepatopancreas of largemouth bass primarily via the PPAR signaling pathway, to down-regulate the expression of fatty acid transport genes, which in turn decreased lipid synthesis in the hepatopancreas. The findings provide useful insights for further analysis of the mechanism underlying the effect of nesfatin-1, and for elucidating the regulation of lipid metabolism homeostasis in the aquaculture production of largemouth bass.

First report of meadow saffron breaking virus on wild Colchicum autumnale from a stricly protected Natura2000 site at a Hungarian National Park.

In mid-April of 2018 light green to greenish yellow linear stripes (Fig. S1.) were observed on the foliage of meadow saffron (Colchicum autumnale) plants - which are native to Hungary - at a strictly protected Natura2000 site maintained by the Duna-Ipoly National Park (DNPI). By autumn, during the flowering season, flower breaking symptoms (Fig. S2.) were noticed, which indicated possible viral infection. With the permit of the Government Office of Pest County and the DNPI, 200 mg leaf sample was collected from one symptomatic plant in spring 2021 and stored at -70 °C until further processing. At the time of the sampling about 2.5 % of the ~ 5000 meadow saffron were symptomatic. Multiplex RT-PCR testing of the sample and an asymptomatic C. autumnale plant for cucumber mosaic virus, tomato spotted wilt virus (Nemes and Salánki 2020) and Nepovirus subgroup-A (Digiaro et al. 2007) gave negative results. The asymptomatic plant also tested negative for potyviruses (Salamon and Palkovics 2005). The asymptomatic (healthy) C. autumnale plant was inoculated with leaf sap of the sample (0.02M Sörensen's phosphate buffer pH 7.2 + 2 % PVP-40 (m/v)) resulting in symptoms of flower breaking in autumn of 2021, and linear stripes on the foliage in spring 2022, identical to symptoms on the originally infected plant. ELISA tests were carried out on the source plants in duplicate using potyvirus-specific MAb PTY1 antibodies (Jordan and Hammond 1991) (Agdia, Elkhart, IN, USA). Absorbance values were 1.519 and 1.530, while the negative controls were 0.003 and 0.007, respectively indicating potyvirus infection of the sample. Molecular tests were carried out on the source and inoculated plant samples in 2022. Total nucleic acid was extracted with the modified CTAB protocol of Xu et al. (2004), and reverse transcription was carried out with Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) with poly T2 (5'-CGGGGATCCTCGAGAAGCTTTTTTTTTTTTTTTTT-3') primer (Salamon and Palkovics 2005). PCR amplification was carried out with poty7941 (5'-GGAATTCCCGCGGNAAYAAYAGYGGNCARCC-3') and poly T2 primers as described earlier (Salamon and Palkovics 2005). A PCR product of ~ 1.6 kb was obtained in each case (Fig. S3.), cloned into pGEM®-T Easy vector (Promega, Madison, WI, USA) and transformed into E. coli DH5α strain. The obtained 1642 nucleotide (nt) sequence encompassing the complete coat protein (CP) was determined (Accession No: OP057214). The virus sequence present in the source and inoculated plants shared 100% nt identity. EcoRV digestion of the PCR products yielded two restriction fragments (369/1273 bp), indicating the presence of a single potyvirus in the infected plant tissue (Fig. S3.). BLASTN analysis of the CP cistron revealed highest nt identity (93.91 %) to meadow saffron breaking virus (MSBV) isolate FR GenBank Acc. No.: AY388995. MSBV was first reported in the Alsace region of France at an INRA research station in cultivated meadow saffron plants showing similar symptoms and the disease reached 100% incidence within a year (Poutaraud et al. 2004). Potyviruses are transmitted mechanically and by aphids (Inoue-Nagata et al. 2022). The spread of MSBV could lead to the infection and decline of the population of Colchicum in protected ecosystems. To our knowledge, this is the first report of MSBV on wild meadow saffron plant from a strictly protected Natura2000 site at a Hungarian National Park.

Synthesis and Characterization of Abasic β-Diol-C-Nucleosides

Modified nucleobases are potentially useful building blocks when containing catalytically active functionalities and could be introduced in chiral tridimensional molecules such as nucleic acids, which creates the premises for the development of novel catalytic species. Herein, we describe the synthesis of a novel β-C-nucleoside bearing a diol group at anomeric position, amenable as a metal ligand or organocatalyst. An abasic ligand was successfully prepared and inserted into a complementary DNA strand.

In Vitro FVIII Encoding Transgenic Mesenchymal Stem Cells Maintain A Successful Coagulation in FVIII- Deficient Plasma Mimicking Hemophilia A.

Hemophilia A is an X-linked recessive bleeding disorder caused by deficiency of plasma coagulation factor VIII (FVIII), and accounts for about 80-85% of patients with hemophilia. Plasma-derived therapies or recombinant factor VIII concentrates and also FVIII mimicking antibodies are used to prevent and treat the bleeding symptoms. Recently, EMA granted a conditional marketing approval for the first gene therapy of Hemophilia A. Aim of this study was to determine the effectiveness of coagulation in correcting FVIII deficiency with FVIII secreting transgenic mesenchymal stem cells (MSC). A lentiviral vector encoding B-domain-deleted Factor VIII cDNA sequence with CD45 R0 truncated (CD45R0t) surface marker was designed to develop a transgenic FVIII expressing primary cell line by transducing MSC. Efficacy and functionality of secreted FVIII from MSCs was assessed with anti- FVIII ELISA, CD45R0t flow cytometry, FVIII Western Blot and Mixing Test analysis in vitro. Results of this study showed that the transgenic MSCs maintain a persistent FVIII secretion. There was no significant difference in FVIII secretion over time, suggesting stable FVIII expression from the MSCs. Functionality of FVIII protein secreted in MSC supernatant was demonstrated by using Mixing Test in coagulation analysis. In the Mixing Test analysis, FVIIIdeficient human plasma products were mixed either with saline control or FVIII-secreted MSC supernatant. Mean FVIII levels of saline control group were 0.41±0.03 IU/dL whereas mean FVIII levels were 25.41±33.38 IU/dL (p<0.01) in FVIII-secreting MSC supernatant mixed group. Mean activated partial thromboplastin time (aPTT) of saline control group was 92.69±11.38 seconds whereas in FVIII-secreting MSC supernatant mixed group, mean aPTT levels were decreased to 38.60±13.38 seconds (p<0.001). Findings of this in vitro study shows a promising new method applicable to treat Hemophilia A. Accordingly, a FVIII secreting transgenic mesenchymal stem cell study will be initiated in FVIII knock-out animal model.

In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A

Hemophilia A is an X-linked recessive bleeding disorder caused by a deficiency of plasma coagulation factor VIII (FVIII), and it accounts for about 80%-85% of all cases of hemophilia. Plasma-derived therapies or recombinant FVIII concentrates are used to prevent and treat the bleeding symptoms along with FVIII-mimicking antibodies. Recently, the European Medicines Agency granted conditional marketing approval for the first gene therapy for hemophilia A. The aim of this study was to determine the effectiveness of coagulation in correcting FVIII deficiency with FVIII-secreting transgenic mesenchymal stem cells (MSCs). A lentiviral vector encoding a B domain-deleted FVIII cDNA sequence with CD45R0 truncated (CD45R0t) surface marker was designed to develop a transgenic FVIII-expressing primary cell line by transducing MSCs. The efficacy and functionality of the FVIII secreted from the MSCs was assessed with anti-FVIII ELISA, CD45R0t flow cytometry, FVIII western blot, and mixing test analysis in vitro. The findings of this study showed that the transgenic MSCs maintained persistent FVIII secretion. There was no significant difference in FVIII secretion over time, suggesting stable FVIII expression from the MSCs. The functionality of the FVIII protein secreted in the MSC supernatant was demonstrated by applying a mixing test in coagulation analysis. In the mixing test analysis, FVIII-deficient human plasma products were mixed with either a saline control or FVIII-secreted MSC supernatant. The mean FVIII level of the saline control group was 0.41±0.03 IU/dL, whereas the mean level was 25.41±33.38 IU/dL in the FVIII-secreting MSC supernatant mixed group (p<0.01). The mean activated partial thromboplastin time (aPTT) of the saline control group was 92.69±11.38 s, while in the FVIII-secreting MSC supernatant mixed group, the mean aPTT level decreased to 38.60±13.38 s (p<0.001). The findings of this in vitro study suggest that the new method presented here is promising as a possible treatment for hemophilia A. Accordingly, a study of FVIII-secreting transgenic MSCs will next be initiated in a FVIII-knockout animal model.

Molecular Characterization of Immunoglobulin M (IgM) and Polymeric Immunoglobulin Receptor (pIgR) and Expression Response to Vibrio harveyi Challenge in Leopard Coral Grouper (Plectropomus leopardus)

Immunoglobulin M (IgM) is the primary antibody in fish, which is transported from epithelial cells into the external secretion system by polymeric immunoglobulin receptor (pIgR). In this study, the full length of IgM and pIgR complementary DNA sequences from leopard coral grouper (Plectropomus leopardus) was characterized, and their expression levels under Vibrio harveyi infection were studied. cDNA sequence analysis showed that the pl-IgM heavy chain cDNA contained an open reading frame (ORF) of 1797 bp encoding a polypeptide of 597 amino acids consisting of a signal peptide, a variable region, and 4 constant regions. pl-pIgR (1041 bp) encodes 346 amino acids with 2 Ig-like domains (ILDs). The expression levels of pl-IgM and pl-pIgR in healthy fish were mainly in the intestine and spleen of fish, which indicates the relationship between the mucosal-associated lymphoid tissue and system lymphoid tissue. Messenger RNA (mRNA) expression profiles of pl-IgM and pl-pIgR were significantly increased in the intestine, head kidney, spleen, and liver after V. harveyi infection. The cDNA sequences of two ILDs were cloned and expressed in Escherichia coli BL21 (DE3). The recombinant pl-pIgR protein had antibacterial function against V. harveyi and Vibrio alginolyticus at 200 ng/μl. Our results indicate that pl-IgM and pl-pIgR are involved in the mucosal-associated immune and systemic immune response in the antibacterial immunity of leopard coral grouper.

Molecular Characterization and Expression Pattern of leptin in Yellow Cheek Carp (Elopichthys bambusa) and Its Transcriptional Changes in Response to Fasting and Refeeding.

Leptin, a secretory protein encoded by obese genes, plays an important role in regulating feeding and energy metabolism in fish. To study the structure and function of the Leptin gene in yellow cheek carp (Elopichthys bambusa), the full-length cDNA sequence of leptin was cloned, named EbLep. The full-length cDNA of Eblep was 1140 bp, and the length of the open reading frame (ORF), which can encode a protein of 174 amino acids, was 525 bp. The signal peptide was predicted to contain 33 amino acids. Sequence alignment showed that the amino acid sequence of Leptin was conserved in cyprinid fish. Despite large differences between primary structures, the tertiary structure of the EbLep protein was similar to that of the human protein and had four α-helices. The EbLep mRNA transcript was detected in all tested tissues, with the highest expression in the liver and lowest expression in the spleen. In this study, short-term fasting significantly increased the mRNA expression of EbLep in the liver, which returned to a normal level after 6 days of refeeding and was significantly lower than the normal level after 28 days of refeeding. In the brain, the mRNA expression of EbLep significantly decreased during short-term fasting and significantly increased to a higher value than the control group after 1 h of refeeding. It then rapidly decreased to a lower value than the control group after 6 h of refeeding, returning to the normal level after 1 day of refeeding, and significantly decreasing to a lower value than the control group after 28 days of refeeding. To sum up, the change in the mRNA expression of EbLep in the brain and liver may be an adaptive strategy for different energy levels.

Recombinant glutathione transferases from flufenacet-resistant black-grass (Alopecurus myosuroides Huds.) form different flufenacet metabolites and differ in their interaction with pre- and post-emergence herbicides.

Black-grass (Alopecurus myosuroides Huds.) has become a problematic weed in cereals in Europe. Besides resistance to post-emergent herbicides becoming increasingly widespread, enhanced metabolism of inhibitors of the synthesis of very-long-chain fatty acids (VLCFAs), such as flufenacet, is evolving. Yet, cross-resistance patterns and evolution of this resistance remains poorly understood. The cDNA sequences of five glutathione transferases (GSTs) upregulated in flufenacet resistant black-grass were identified and used for recombinant protein expression. Moderate to slow detoxification of flufenacet was verified for all candidate GSTs expressed in E. coli, and the most active protein produced flufenacet-alcohol instead of a glutathione conjugate, in the presence of reduced glutathione (GSH). Moreover, cross-resistance to other VLCFA-inhibitors e.g., acetochlor and pyroxasulfone and the ACCase inhibitor fenoxaprop was verified in vitro. Various other herbicides of different modes of action including VLCFA-inhibitors were not detoxified by the candidate GSTs. As several in planta upregulated GSTs detoxified flufenacet in vitro, the shift in sensitivity observed in black-grass populations, is likely a result of an additive effect. The polygenic character and the relatively low turnover rate of the individual GSTs may explain the slow evolution of flufenacet resistance. In addition, flufenacet resistance was accompanied by cross-resistance with some, but not all, herbicides of the same mode of action, and furthermore to the ACCase inhibitor fenoxaprop-ethyl. Hence, not only the rotation of herbicide modes of action, but also of individual active ingredients is important for resistance management. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Visible-light driven and efficient photoelectrochemical aptasensor constructed with N-doped carbon quantum dots-decorated TiO2 nanorods for determination of di-2-ethylhexyl phthalate

Here, a visible-light driven and efficient photoelectrochemical (PEC) aptasensor for highly sensitive and specific detection of di-2-ethylhexyl phthalate (DEHP) was constructed based on N-doped carbon quantum dots-decorated TiO2 nanorods (N-CQDs-decorated TiO2 NRs) subtly integrated with biocatalytic precipitation (BCP). The N-CQDs-decorated TiO2 NRs synthesized through a facile hydrothermal method possess enhanced visible-light absorption capability and excellent PEC performance. The synthesized composites were used as the sensing material for developing a PEC aptasensor. Amino-functionalized complementary strand (cDNA) was first covalently immobilized on the N-CQDs-decorated TiO2 NRs, and then horseradish peroxidase (HRP)-labeled aptamer molecules were introduced onto the surface of composites by hybridization with cDNA. HRP-induced biocatalytic precipitation (BCP) caused the decrease of PEC detection signal, playing a role of signal amplification. The proposed PEC aptasensor shows good detection performance for DEHP with high sensitivity, selectivity, stability and reproducibility, and the detection limit is as low as 0.017 ng/L. Moreover, satisfying results were achieved for the DEHP assays in real environmental samples using the proposed PEC aptasensor. Thus, this present method would contribute a fresh idea to monitor DEHP or other pollutants in the environment.

Characterization and tissue expression of twelve selenoproteins in yellow catfish Pelteobagrus fulvidraco fed diets varying in oxidized fish oil and selenium levels

BackgroundSelenium (Se) functions through selenoproteins and is essential to growth and metabolism of vertebrates. The present study was conducted to identify twelve selenoproteins genes (selenoe, selenof, selenoh, selneoi, selenom, selenok, selneon, selenoo, selenot, selenos, selenou and msrb1) from yellow catfish. Their mRNA expression patterns, as well as their response to dietary oxidized fish oils and Se addition were explored. MethodsWe use 3′and 5′ RACE PCR to clone full-length cDNA sequence of twelve selenoprotein genes from yellow catfish. Their mRNA expression patterns were assessed via quantitative real-time PCR. Yellow catfish were fed diet adequate Se+ fresh fish oil, adequate Se+ oxidized fish oil, high Se+ fresh fish oil and high Se+ oxidized fish oil, respectively, for 10 weeks. Their kidney, heart, brain and testis were used to assess the mRNA expression of twelve selenoprotein. ResultsTwelve selenoprotein genes had similar domains with mammals and the other fish. Their mRNAs were expressed widely in eleven tissues but varied with the tissues. Dietary oxidized fish oils and Se addition influenced their mRNA abundances of twelve selenoproteins in a tissue-dependent manner. ConclusionOur study demonstrated the characterization and expression of twelve selenoproteins, and elucidated their responses in yellow catfish fed diets varying in oxidized fish oils and Se addition, which increased our knowledge into the biological function and regulatory mechanism of Se and selenoproteins in fish.

Nanopore-based RNA sequencing deciphers the formation, processing, and modification steps of rRNA intermediates in archaea.

Ribosomal RNA (rRNA) maturation in archaea is a complex multistep process that requires well-defined endo- and exoribonuclease activities to generate fully mature linear rRNAs. However, technical challenges prevented detailed mapping of rRNA processing steps and a systematic analysis of rRNA maturation pathways across the tree of life. In this study, we used long-read (PCR)-cDNA and direct RNA nanopore-based sequencing to study rRNA maturation in three archaeal model organisms, namely the Euryarchaea Haloferax volcanii and Pyrococcus furiosus and the Crenarchaeon Sulfolobus acidocaldarius Compared to standard short-read protocols, nanopore sequencing facilitates simultaneous readout of 5'- and 3'-positions, which is required for the classification of rRNA processing intermediates. More specifically, we (i) accurately detect and describe rRNA maturation stages by analysis of terminal read positions of cDNA reads and thereupon (ii) explore the stage-dependent installation of the KsgA-mediated dimethylations in H. volcanii using base-calling and signal characteristics of direct RNA reads. Due to the single-molecule sequencing capacity of nanopore sequencing, we could detect hitherto unknown intermediates with high confidence, revealing details about the maturation of archaea-specific circular rRNA intermediates. Taken together, our study delineates common principles and unique features of rRNA processing in euryarchaeal and crenarchaeal representatives, thereby significantly expanding our understanding of rRNA maturation pathways in archaea.

Characterization of novel CACNA1A splice variants by RNA-sequencing in patients with episodic or congenital ataxia.

Loss of function variants in CACNA1A cause a broad spectrum of neurological disorders, including episodic ataxia, congenital or progressive ataxias, epileptic manifestations or developmental delay. Variants located on the AG/GT consensus splice sites are usually considered as responsible of splicing defects, but exonic or intronic variants located outside of the consensus splice site can also lead to abnormal splicing. We investigated the putative consequences on splicing of 11 CACNA1A variants of unknown significance (VUS) identified in patients with episodic ataxia or congenital ataxia. In silico splice predictions were performed and RNA obtained from fibroblasts was analyzed by Sanger sequencing. The presence of abnormal transcripts was confirmed in 10/11 patients, nine of them were considered as deleterious and one remained of unknown significance. Targeted next-generation RNA sequencing was done in a second step to compare the two methods. This method was successful to obtain the full cDNA sequence of CACNA1A. Despite the presence of several isoforms in the fibroblastic cells, it detected most of the abnormally spliced transcripts. In conclusion, RNA sequencing was efficient to confirm the pathogenicity of nine novel CACNA1A variants. Sanger or Next generation methods can be used depending on the facilities and organization of the laboratories.

Shape-induced crystallization of binary DNA-functionalized nanocubes.

Leveraging the anisotropic shape of DNA-functionalized nanoparticles holds potential for shape-directed crystallization of a wide collection of superlattice structures. Using coarse-grained molecular dynamics simulations, we study the self-assembly of a binary mixture of cubic gold nanoparticles, which are functionalized by complementary DNA strands. We observe the spontaneous self-assembly of simple cubic (SC), plastic body-centered tetragonal (pBCT), and compositionally disordered plastic body-centered tetragonal (d-pBCT) phases due to hybridization of the DNA strands. We systematically investigate the effect of length, grafting density, as well as rigidity of the DNA strands on the self-assembly behavior of cubic nanoparticles. We measure the potential of mean force between DNA-functionalized nanocubes for varying rigidity of the DNA strands and DNA lengths. Using free-energy calculations, we find that longer and flexible DNA strands can lead to a phase transformation from SC to the pBCT phase due to a gain in entropy arising from the orientational degrees of freedom of the nanocubes in the pBCT phase. Our results may serve as a guide for self-assembly experiments on DNA-functionalized cubic nanoparticles.