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Each new class of insecticides that emerged during the development of plant protection gradually found the most suitable group of insect pests for application. At the same time, for each individual insecticide, a balance was sought between its effectiveness, on the one hand, and its safety for non-target organisms and the ecosystem as a whole, on the other hand. Neonicotinoids, diamides and pyrethroids, as effective control agents, dominate the insecticide market, but do not have outstanding performance in selectivity and biodegradation. The biodegradation of insecticides is one of the most important indicators, representing what will be said about the hidden costs for the resulting harvest paid by the environment and human health. Oligonucleotide insecticides (contact unmodified antisense DNA (CUAD) biotechnology, or 'genetic zipper' method) and RNA biocontrols (double-stranded RNA technology) as natural polymers and the next-generation classes of insecticides possess unique characteristics in fast biodegradation and high selectivity in action. While current chemical insecticides require days, months and even years for biodegradation by bacteria and fungi, oligonucleotide insecticides and RNA biocontrols are substantially biodegraded within hours in the presence of nucleases. Nucleic acid-based insecticides have the potential to complement the existing insecticide market and set an eco-precedent for crop protection products where the effectiveness of the insecticide will be determined by its safety for non-target organisms, and other factors being equal, the choice of a particular control agent will be determined by its biodegradability. It should be noted that not a single class of insecticides that once appeared has completely disappeared; rather, it has occupied its niche, gradually declining under the pressure of new classes of insecticides. At the same time, the common trend in plant protection is towards use of insecticides with higher biodegradability, which gives hope for a safer future of the planet. [ABSTRACT FROM AUTHOR]

Fluorescence-based aptasensors have been regarded as innovative analytical tools for the detection and quantification of analytes in many fields, including medicine and therapeutics. Using DNA aptamers as the biosensor recognition component, conventional molecular beacon aptasensor designs utilise target-induced structural switches of the DNA aptamers to generate a measurable fluorescent signal. However, not all DNA aptamers undergo sufficient target-specific conformational changes for significant fluorescence measurements. Here, the use of complementary 'antisense' strands is proposed to enable fluorescence measurement through strand displacement upon target binding. Using a published target-specific DNA aptamer against the receptor binding domain of SARS-CoV-2, we designed a streptavidin-aptamer bead complex as a fluorescence displacement assay for target detection. The developed assay demonstrates a linear range from 50 to 800 nanomolar (nM) with a limit of detection calculated at 67.5 nM and a limit of quantification calculated at 204.5 nM. This provides a 'fit-for-purpose' model assay for the detection and quantification of any target of interest by adapting and functionalising a suitable target-specific DNA aptamer and its complementary antisense strand. [ABSTRACT FROM AUTHOR]

Recently, sustainable and ecological approach in plant protection has produced many innovative ideas, including insecticides based on nucleic acids. Oligonucleotide insecticides (contact unmodified antisense DNA biotechnology, CUAD biotechnology) and RNA-based insecticides (double-stranded RNA technology) are pest control agents with different mechanisms of action, but they can complement each other's insecticidal potential in the control of insect pests from different orders, and may be present together in complex preparations. The insect physiology shows that the contact method of delivery of such controlling agents is very perspective enabling avoidance of meeting with nucleases in digestive tract. The future of CUAD biotechnology and double-stranded RNA technology looks alluring: two natural molecular genetic mechanisms for insect pest control in the service of plant protection with minimal harm to non-target organisms. As clean technologies, insecticides based on DNA and RNA can make a significant contribution to environmental health improvment. [ABSTRACT FROM AUTHOR]

Infectious laryngotracheitis (ILT) poses a significant threat to the poultry industry, and vaccines play an important role in protection. However, due to the increasing scale of poultry production, there is an urgent need to develop vaccines that are suitable for convenient immunization methods such as spraying. Previous studies have shown that Newcastle disease virus (NDV)-ILT vaccines administered via intranasal and intraocular routes to commercial chickens carrying maternally-derived antibodies (MDAs) are still protective against ILT. In this study, a recombinant NDV (rNDV) was generated to express infectious laryngotracheitis virus (ILTV) glycoprotein B (gB), named rLS-gB, based on a full-length cDNA clone of the LaSota strain. The protective effect of different doses of rLS-gB administered by spray vaccination to commercial chickens at 1 d of age (doa) was evaluated. The chickens were exposed to 160-μm aerosol particles for 10 min for spray vaccination, and no adverse reactions were observed after vaccination. Despite the presence of anti-NDV MDAs and anti-ILTV MDAs in chickens, the ILTV- and NDV-specific antibody titres were significantly greater in the vaccinated groups than in the unvaccinated group. After challenge with a virulent ILTV strain, no clinical signs were observed in the 107 EID50/ml group compared to the other groups. Furthermore, vaccination with 107 EID50/ml rLS-gB significantly reduced the ILTV viral load and ameliorated gross and microscopic lesions in the trachea of chickens. Overall, these results suggested that rLS-gB is a safe and efficient candidate spray vaccine for ILT and is especially suitable for scaled chicken farms. [ABSTRACT FROM AUTHOR]

We previously demonstrated that postruminal casein infusion and exogenous glucagon-like peptide 2 (GLP-2) administration independently stimulated growth and carbohydrase activity of the pancreas and jejunal mucosa in cattle. The objective of the current study was to profile the jejunal mucosal transcriptome of cattle using next-generation RNA sequencing in response to postruminal casein infusion and exogenous GLP-2. Twenty-four Holstein steers [250 ± 23.1 kg body weight (BW)] received a continuous abomasal infusion of 3.94 g raw corn starch/kg of BW combined with either 0 or 1.30 g casein/kg of BW for 7 d. Steers received subcutaneous injections at 0800 and 2000 h to provide either 0 or 100 μg GLP-2/kg of BW per day. At the end of the 7-d treatment period, steers were slaughtered for collection of the jejunal mucosa. Total RNA was extracted from jejunal mucosal tissue, strand-specific cDNA libraries were prepared, and RNA sequencing was conducted to generate 150-bp paired-end reads at a depth of 40 M reads per sample. Differentially expressed genes (DEG), KEGG pathway enrichment, and gene ontology enrichment were determined based on the FDR-corrected P-value (padj). Exogenous GLP-2 administration upregulated (padj < 0.05) 667 genes and downregulated 1,101 genes of the jejunal mucosa. Sphingolipid metabolism, bile secretion, adherens junction, and galactose metabolism were among the top KEGG pathways enriched with upregulated DEG (padj < 0.05) in response to exogenous GLP-2 administration. The top gene ontologies enriched with upregulated DEG (padj < 0.05) in response to exogenous GLP-2 administration included nutrient metabolic processes, brush border and bicellular tight junction assembly, and enzyme and transporter activities. Exogenous GLP-2 administration increased or tended to increase (padj < 0.10) brush border carbohydrase (MGAM, LCT, TREH), hexose transporter (SLC5A1, SLC2A2), and associated transcription factor (HNF1, GATA4, KAT2B) mRNA expression of the jejunal mucosa. Gene ontologies and KEGG pathways that were downregulated (padj < 0.05) in response to exogenous GLP-2 were related to genetic information processing. Postruminal casein infusion downregulated (padj < 0.05) 7 jejunal mucosal genes that collectively did not result in enriched KEGG pathways or gene ontologies. This study highlights some of the transcriptional mechanisms associated with increased growth, starch assimilation capacity, and barrier function of the jejunal mucosa in response to exogenous GLP-2 administration. [ABSTRACT FROM AUTHOR]

Total suspended solids (TSS) are a major contributor of anthropogenic impacts to aquatic systems. TSS exposure have been shown to affect the function of gills, but the mode of action is unclear. Zebrafish (Danio rerio) is emerging as an excellent model for mechanistic toxicology, and as there are no baseline studies on TSS effects in zebrafish gills, we tested the hypothesis that environmental concentrations of TSS damages gill structure and function in this species. Adult zebrafish were exposed to either 0, 10, 100, 500, 1000, or 2000 mg/L TSS for 4 days to assess the gill morphology. The minimal concentration that affected the gill structure was further tested for the distribution of key ion transporters, including Na+/K+- ATPase (NKA) and vacuolar-type H+-ATPase (VHA), using confocal microscopy. Our results reveal that TSS concentration as low as 100 mg/L alters the morphology of gills, including greater filament thickness, lamellae thickness, and epithelial lifting. This was also associated with a reduction in NKA immunoreactive (IR) cell count and intensity in the 100 mg/L TSS group, while there was neither a change in the VHA-IR cell count or expression nor the transcript abundance of atp6v1a and atp1a1a4 in the gills. Markers of stress response in these animals, including levels of cortisol, glucose, lactate, and glycogen were not altered after 4 days of TSS exposure. Overall, environmentally relevant concentrations of TSS can damage the gill structure and function in zebrafish and has the potential to enhance the toxicity of contaminants acting via the gills. [ABSTRACT FROM AUTHOR]

Background: Based on our previous research, a full-length cDNA sequence of HvANS gene was isolated from purple and white Qingke. The open reading frame (ORF) in the purple variety Nierumuzha was 1320 base pairs (bp), encoding 439 amino acids, while the ORF in the white variety Kunlun 10 was 1197 bp, encoding 398 amino acids. A nonsynonymous mutation was found at the position of 1195 bp (T/C) in the coding sequence (CDS) of the HvANS gene. We carried out a series of studies to further clarify the relationship between the HvANS gene and anthocyanin synthesis in Qingke. Results: The conservative structural domain prediction results showed that the encoded protein belonged to the PLN03178 superfamily. Multiple comparisons showed that this protein had the highest homology with Hordeum vulgare, at 88.61%. The approximately 2000 bp promoter sequence of the HvANS gene was identical in both varieties. The real-time fluorescence PCR (qRT-PCR) results revealed that HvANS expression was either absent or very low in the roots, stems, leaves, and awns of Nierumuzha. In contrast, the HvANS expression was high in the seed coats and seeds of Nierumuzha. Likewise, in Kunlun 10, HvANS expression was either absent or very low, indicating a tissue-specific and variety-specific pattern for HvANS expression. The subcellular localization results indicated that HvANS was in the cell membrane. Metabolomic results indicated that the HvANS gene is closely related to the synthesis of three anthocyanin substances (Idaein chloride, Kinetin 9-riboside, and Cyanidin O-syringic acid). Yeast single hybridization experiments showed that the HvANS promoter interacted with HvANT1, which is the key anthocyanin regulatory protein. In a yeast two-hybrid experiment, we obtained two significantly different proteins (ZWY2020 and POMGNT2-like) and verified the results by qRT-PCR. Conclusions: These results provide a basis for further studies on the regulatory mechanism of HvANS in the synthesis of anthocyanins in Qingke purple grains. [ABSTRACT FROM AUTHOR]

The chloride channel 7 gene (CLC7) of the Hong Kong oyster Crassostrea hongkongensis was cloned and named ChCLC7. The cDNA was 2 572 bp in length, with a 5′ non-coding region containing 25 bp, a 3′ non-coding region containing 327 bp, and an open reading frame of 2 298 bp. ChCLC7 has 96.8% and 92.1% homology with CLC7 of Crassostrea gigas and Crassostrea virginica, respectively, and it was clustered with CLC7 of C. gigas and C. virginica. QRT-PCR showed that ChCLC7 was expressed in all eight tissues, with the highest in adductor muscle and second in gill. The ChCLC7 expression pattern in gill was altered significantly under high salinity stress with an overall upward and then downward trend. After RNA interference, the expression of ChCLC7 and survival rate of oyster under high salinity stress was reduced significantly, and so did the concentration of hemolymph chloride ion in 48–96 h after RNA interference. We believed that ChCLC7 could play an important role in osmoregulation of C. hongkongensis by regulating Cl− transport. This study provided data for the analysis of molecular mechanism against oyster salinity stress. [ABSTRACT FROM AUTHOR]

Antisense oligonucleotides (ASOs) have been developed as potential therapeutic agents for genetic disorders and various life‐threatening conditions. This class of therapeutics facilitates the treatment of the currently undruggable targets. However, their low stability and poor activity must be overcome by developing new analogs with improved functions. Therefore, in this study, we synthesized 4′‐C‐(S)‐ and 4′‐C‐(R)‐2‐aminopropoxy−thymidine and investigated the stability of the modified oligonucleotides containing these analogs. Interestingly, both analogs responded differently to thermal denaturation, with the former exhibiting good stability closer to that of the unmodified oligonucleotide and the latter exhibiting slight thermal destabilization. Notably, DNA/RNA heteroduplexes modified with these analogs elicited RNase H activity by degrading the heteroduplexes upon treatment. In addition, the serum stability of the modified oligodeoxynucleotides was significantly enhanced by the introduction of these analogs. Overall, our findings highlight the potential application of these analogs as ASO therapeutic candidates. [ABSTRACT FROM AUTHOR]

This article discusses the use of contact unmodified antisense DNA (CUAD) biotechnology and oligonucleotide insecticides for pest control in agriculture. The development of this technology has allowed for the successful targeting of pest species using unmodified antisense DNA fragments. Oligonucleotide insecticides act through the DNA containment mechanism, which involves the interaction of antisense DNA oligonucleotides with target ribosomal RNA, leading to substantial insect pest mortality. These insecticides are highly selective in action, safe for non-target organisms, and have a low carbon footprint. The article provides an overview of the experiments conducted using oligonucleotide insecticides and highlights the potential of CUAD biotechnology in agronomy. The document discusses the use of oligonucleotide insecticides as a method of controlling various insect pests, targeting specific regions of the pests' RNA. The article suggests that more complex formulations of these insecticides could enhance their effectiveness against a wider range of insects, but further research and premarket environmental risk assessments are needed to ensure their safety and efficacy. The document also includes references to studies on the effectiveness of DNA insecticides against various insect pests and their potential benefits compared to traditional insecticides. [Extracted from the article]

In virology, the term reverse genetics refers to a set of methodologies in which changes are introduced into the viral genome and their effects on the generation of infectious viral progeny and their phenotypic features are assessed. Reverse genetics emerged thanks to advances in recombinant DNA technology, which made the isolation, cloning, and modification of genes through mutagenesis possible. Most virus reverse genetics studies depend on our capacity to rescue an infectious wild-type virus progeny from cell cultures transfected with an "infectious clone". This infectious clone generally consists of a circular DNA plasmid containing a functional copy of the full-length viral genome, under the control of an appropriate polymerase promoter. For most DNA viruses, reverse genetics systems are very straightforward since DNA virus genomes are relatively easy to handle and modify and are also (with few notable exceptions) infectious per se. This is not true for RNA viruses, whose genomes need to be reverse-transcribed into cDNA before any modification can be performed. Establishing reverse genetics systems for members of the Caliciviridae has proven exceptionally challenging due to the low number of members of this family that propagate in cell culture. Despite the early successful rescue of calicivirus from a genome-length cDNA more than two decades ago, reverse genetics methods are not routine procedures that can be easily extrapolated to other members of the family. Reports of calicivirus reverse genetics systems have been few and far between. In this review, we discuss the main pitfalls, failures, and delays behind the generation of several successful calicivirus infectious clones. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) can cause self-limiting acute and chronic hepatitis infections, particularly in immunocompromised individuals. In developing countries, HEV is mainly transmitted via drinking contaminated water, whereas zoonotic transmission dominates the route of infection in developed countries, including Japan. Pigs are an important reservoir for HEV infection. Wild boars, which share the same genus and species as domestic pigs, are also an HEV reservoir. During our nationwide study of HEV infection in wild boar populations in Japan, a genotype 6 (HEV-6) strain, wbJHG_23, was isolated in Hyogo Prefecture in 2023. The genomic length was 7244 nucleotides, excluding the poly(A) tract. The wbJHG_23 strain exhibited the highest nucleotide identity throughout its genome with two previously reported HEV-6 strains (80.3–80.9%). Conversely, it displayed lower similarity (73.3–78.1%) with the HEV-1–5, HEV-7, and HEV-8 strains, indicating that, although closely related, the wbJHG_23 strain differs significantly from the reported HEV-6 strains and might represent a novel subtype. The wbJHG_23 strain successfully infected the human-derived cancer cell lines, PLC/PRF/5 and A549 1-1H8 cells, suggesting that HEV-6 has the potential for zoonotic infection. An infectious cDNA clone was constructed using a reverse genetics system, and a cell culture system supporting the efficient propagation of the HEV-6 strain was established, providing important tools for further studies on this genotype. Using this cell culture system, we evaluated the sensitivity of the wbJHG_23 strain to ribavirin treatment. Its good response to this treatment suggested that it could be used to treat human infections caused by HEV-6. [ABSTRACT FROM AUTHOR]

Myostatin is a known negative regulator of muscle tissue growth. Thus, an inhibitor of myostatin may be therapeutically useful as an anabolic agent for the muscle tissue. A promising gene-silencing approach for gene therapy is DNA interference (DNAi), a sequence that is complementary to the promoter region of a target gene. To confer resistance to nuclease digestion, several modifications such as methylphosphonate or phosphorothioate have been proposed, wherein a nonbridging oxygen atom in the oligonucleotide phosphate backbone is replaced by sulphur. The aim of the present study was to assess the effectiveness of the DNAi molecule with phosphorothioate (PS) and without phosphorothioate (WPS) modification for inhibition of myostatin gene expression in mice. Eighteen four-week-old male BALB/c mice were randomly divided into three groups: DNAi-PS (n = 6), DNAi-WPS (n = 6) and control (n = 6). Intraperitoneal injections of DNAi (10 mg/kg) were given once a week, and mice body weights were measured weekly and sacrificed after three weeks. The expression of myostatin was assessed using real-time quantitative polymerace chain reaction. For histological evaluation, the skeletal muscle tissue was dissected from the biceps. The results were analysed by a t-test. Results demonstrated that administration of DNAi intraperitoneally with modification could suppress myostatin expression by up to 70%. Leg weight and histological analysis proved that chemically modified DNAi significantly suppressed the myostatin gene in mice. Overall, the results on DNA-induced gene silencing by antisense DNA oligonucleotides in animals can provide insight into the treatment of inherited diseases. [ABSTRACT FROM AUTHOR]

Small RNAS (sRNAs) participate in regulatory RNA interference (RNAi) mechanisms in a wide range of eukaryotic organisms, including fungi. The fungus Fusarium fujikuroi, a model for the study of secondary metabolism, contains a complete set of genes for RNAi pathways. We have analyzed by high-throughput sequencing the content of sRNAs in total RNA samples of F. fujikuroi grown in synthetic medium in the dark or after 1 h of illumination, using libraries below 150 nt, covering sRNAs and their precursors. For comparison, a parallel analysis with Fusarium oxysporum was carried out. The sRNA reads showed a higher proportion of 5′ uracil in the RNA samples of the expected sizes in both species, indicating the occurrence of genuine sRNAs, and putative miRNA-like sRNAs (milRNAS) were identified with prediction software. F. fujikuroi carries at least one transcriptionally expressed Ty1/copia-like retrotransposable element, in which sRNAs were found in both sense and antisense DNA strands, while in F. oxysporum skippy-like elements also show sRNA formation. The finding of sRNA in these mobile elements indicates an active sRNA-based RNAi pathway. Targeted deletion of dcl2, the only F. fujikuroi Dicer gene with significant expression under the conditions tested, did not produce appreciable phenotypic or transcriptomic alterations. [ABSTRACT FROM AUTHOR]

A signal-amplified platform was designed to construct a label-free electrochemical aptasensor for lead ions (Pb2+) assay. First, flower-like molybdenum disulfide-supported AuNPs (AuNPs@MoS2) nanocomposites were synthesized and used as substrates for modifying the electrode. The AuNPs@MoS2 material possessed large surface area and superior biocompatibility, which was beneficial to improve the loading amount of the complementary DNA (cDNA) and amplified the response signal. Importantly, the prepared core–shell Pt@Pd bimetallic nanoparticles (Pt@PdNPs) were used to conjugate with redox marker thionine (Thi) and aptamer (Apt) for further signal amplification; the obtained signal probes (Thi-Pt@PdNPs-Apt) were connected by the cDNA assembled on the electrode through DNA hybridization. Differential pulse voltammetry was performed to monitor the signal of Thi. After incubating of aptasensor with Pb2+, the specific recognition of Pb2+ and Apt resulted in the dissociation of aptamer-cDNA complex, thereby the Thi-Pt@PdNPs-Apt separated from the electrode surface and decreased current response was obtained. The prepared electrochemical sensor exhibited linear response to Pb2+ in the range 5.0 × 10−4–100 nM and a detection limit of 1.0 × 10−4 nM was achieved. The sensor was applied to the determination of Pb2+ in actual sample with high sensitivity and accuracy, demonstrating potential applications in heavy metal monitoring. [ABSTRACT FROM AUTHOR]

The present study accomplished the successful cloning and sequencing of the JfCXCL9_L and JfCXCL10_L genes found in the spleen cDNA of the Japanese flounder (Paralichthys olivaceus). Additionally, the tissue distribution of these two genes was examined at the zero-hour mark. Through qRT-PCR analysis, it was observed that the expression of JfCXCL10_L closely mirrored that of IL1-β, displaying an upregulation following the application of Poly I:C (Viral mimic) and formalin-killed Edwardsiella tarda (Gram-Negative mimic), while showing a downregulation after the application of formalin-killed Streptococcus iniae (Gram-Positive mimic) treatment. This finding strongly suggests a role for JfCXCL10_L in the immune response to viral and gram-negative bacterial stimuli. Regarding JfCXCL9_L, mRNA levels were found to be significantly downregulated after FKCET and FKCSI treatments, though to varying extents. Interestingly, at specific time points, JfCXCL9_L levels were even lower compared to Poly I:C treatment. These intriguing results shed valuable light on the immune response mechanisms and potential functions of both JfCXCL9_L and JfCXCL10_L in the context of the Japanese flounder's immune system. [ABSTRACT FROM AUTHOR]

Introduction: Porcine Reproductive and Respiratory Syndrome virus (PRRSV) causes high abortion rates in gestating sows and stillbirths, as well as high piglet mortality, seriously jeopardizing the pig industry in China and worldwide. Methods: In this study, an infectious clone containing the full-length genome of NADC34-like PRRSV was constructed for the first time using reverse genetic techniques. The gene was amplified segmentally onto a plasmid, transfected into BHK-21 cells, and the transfected supernatant was harvested and transfected into PAM cells, which showed classical cytopathic effects (CPE). Results: The virus rJS-KS/2021 was successfully rescued which could be demonstrated by Western Blot and indirect immunofluorescence assays. Its growth curve was similar to the original strain. Replace the 5'UTR and 3'UTR of rJS-KS/2021 with 5'UTR and 3'UTR of HP-PRRSV (strain SH1) also failed to propagate on MARC-145. Discussion: In this study, an infectious clone of NADC34-like was constructed by reverse genetics, replacing the UTR and changing the cellular tropism of the virus. These findings provide a solid foundation for studying the recombination of different PRRSVs and the adaption of PRRSVs on MARC-145 in the future. [ABSTRACT FROM AUTHOR]

【Objective】Pogostemon cablin (Blanco) Benth. is a frequently-used aromatic herb eliminating dampness in clinical settings, with its primary bioactive compound, patchouli alcohol, demonstrating potent antiviral and anti-inflammatory and other beneficial properties. Patchoulialcohol synthase (PcPTS) plays an essential role in the biosynthesis pathway of patchouli alcohol. However, the precise mechanisms by which PcPTS modulates patchouli alcohol production at the level of protein-protein interactions is still unclear. This study aims to identify interacting proteins of PcPTS in order to elucidate its regulatory mechanisms and functions in the biosynthesis pathway of patchouli alcohol.【Method】The yeast two-hybrid technique and GST pull-down combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were employed to identify potential interacting proteins of PcPTS. The annotation of these interacting proteins was conducted by the MASCOT software and BLAST analysis. Proteins known to be involved in protein translocation, modification, degradation and regulation of secondary metabolism were selected for further analysis. Their interactions with PcPTS proteins were initially confirmed through yeast two-hybrid technology, and subsequently analyzed by using bioinformatics tools. 【Result】The cDNA sequence of PcPTS was successfully cloned by PCR, resulting in an open reading frame (ORF) of 1 659 bp, encoding 552 amino acids. The primary cDNA library and the yeast hybrid cDNA library were constructed and obtained, with capacities of 7.6×106 and 8.6×106 colony-forming units (CFU), respectively. Both libraries achieved a 100% recombination rate, with an average length of the inserted fragment greater than 800 bp. The constructed bait vector pGBKT7-PcPTS exhibited no toxicity towards yeast strains and did not exhibit any self-activating activity in yeast cells. The positive clones identified through yeast two-hybrid screening were subjected to sequencing and BLAST analysis, resulting in the identification of 17 candidate proteins. Subsequently, the GST-PcPTS expression vector was effectively constructed, and the GST-PcPTS bait protein was obtained through induced expression purification. This bait protein was utilized to capture interacting proteins from the total protein of Pogostemon cablin, and 98 candidate interacting proteins were identified. Among these candidates, 14 proteins potentially interacting with PcPTS were selected and confirmed through point-to-point verification with yeast two-hybridization. The results indicated that PcC3H6, PcENO3, PcACR11 and PcHAD interacted with PcPTS. Bioinformatics analysis indicated that they belonged to the CCCH zinc finger protein family, enolase proteins, ACR subfamily and HAD-like superfamily, respectively. 【Conclusion】 Four proteins (PcC3H6, PcENO3, PcACR11, and PcHAD) were identified as interacting with PcPTS through initial screening and validation. This finding aids in elucidating the role of PcPTS in the biosynthesis pathway of patchouli alcohol and provides a robust basis for future investigations into the regulatory mechanisms of patchouli alcohol synthesis. [ABSTRACT FROM AUTHOR]

The combination of chemotherapy and gene therapy holds great promise for the treatment and eradication of tumors. However, due to significant differences in physicochemical properties between chemotherapeutic agents and functional nucleic acid drugs, direct integration into a single nano-agent is hindered, impeding the design and construction of an effective co-delivery nano-platform for synergistic anti-tumor treatments. In this study, we have developed an mRNA-responsive two-in-one nano-drug for effective anti-tumor therapy by the direct self-assembly of 2′-fluoro-substituted antisense DNA against P-glycoprotein (2′F-DNA) and chemo drug paclitaxel (PTX). The 2′-fluoro modification of DNA could significantly increase the interaction between the therapeutic nucleic acid and the chemotherapeutic drug, promoting the successful formation of 2′F-DNA/PTX nanospheres (2′F-DNA/PTX NSs). Due to the one-step self-assembly process without additional carrier materials, the prepared 2′F-DNA/PTX NSs exhibited considerable loading efficiency and bioavailability of PTX. In the presence of endogenous P-glycoprotein mRNA, the 2′F-DNA/PTX NSs were disassembled. The released 2′F-DNA could down-regulate the expression of P-glycoprotein, which decreased the multidrug resistance of tumor cells and enhanced the chemotherapy effect caused by PTX. In this way, the 2′F-DNA/PTX NSs could synergistically induce the apoptosis of tumor cells and realize the combined anti-tumor therapy. This strategy might provide a new tool to explore functional intracellular co-delivery nano-systems with high bioavailability and exhibit potential promising in the applications of accurate diagnosis and treatment of tumors. [Display omitted] [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]

Reverse transcriptase-Cas1 (RT-Cas1) fusion proteins found in some CRISPR systems enable spacer acquisition from both RNA and DNA, but the mechanism of RNA spacer acquisition has remained unclear. Here, we found that Marinomonas mediterranea RT-Cas1/Cas2 adds short 3'-DNA (dN) tails to RNA protospacers, enabling their direct integration into CRISPR arrays as 3'-dN-RNAs or 3'-dN-RNA/cDNA duplexes at rates comparable to similarly configured DNAs. Reverse transcription of RNA protospacers is initiated at 3' proximal sites by multiple mechanisms, including recently described de novo initiation, protein priming with any dNTP, and use of short exogenous or synthesized DNA oligomer primers, enabling synthesis of near full-length cDNAs of diverse RNAs without fixed sequence requirements. The integration of 3'-dN-RNAs or single-stranded DNAs (ssDNAs) is favored over duplexes at higher protospacer concentrations, potentially relevant to spacer acquisition from abundant pathogen RNAs or ssDNA fragments generated by phage defense nucleases. Our findings reveal mechanisms for site-specifically integrating RNA into DNA genomes with potential biotechnological applications. [ABSTRACT FROM AUTHOR]

A gene library is a collection of different DNA sequences from an organism, which has been cloned into vectors for ease of purification, storage, and analysis. There are two types of gene libraries based on the availability of a source of DNA used. The genomic library contains DNA fragments that represent the entire genome of an organism, whereas CDNA libraries will contain only the complementary DNA molecules synthesized from the mRNA molecules in a cell. For the construction of gene libraries, we have to know the size of the gene and insert size or capacity of vectors. Vectors used for gene library construction are plasmid, lambda phage, Cosmid, bacterial artificial chromosome, and yeast artificial chromosome. The most commonly used vector type is lambda, but most genomic libraries are constructed using BAC and YAC vectors. For the construction of gene libraries, the steps followed are the isolation of bacteria followed by cloning and sequencing DNA. The main use of genetic libraries is to increase the likelihood that a particular portion of a DNA source will be found within the collection. It is used to collect and store information as a set of DNA molecules. Gene libraries are stored inside bacterial cells. Bacterial colonies have the ability to store different genes, similar to how books are stored in a library. [ABSTRACT FROM AUTHOR]

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Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with Kd values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD. [ABSTRACT FROM AUTHOR]

The genus Paracoccidioides includes Paracoccidioides lutzii and the Paracoccidioides brasiliensis complex, which comprises four phylogenetic species. A key feature distinguishing planktonic growth from biofilm is the presence of a 3D extracellular matrix (ECM). Therefore, in this study, we analyzed biofilm formation in dierent species of Paracoccidioides yeast phase, characterized the structural elements of the matrix of P. brasiliensis (Pb18), P. lutzii (Pl01 and 8334) and P. restrepiensis (339 and 192) and evaluated the expression of glucan genes, according to the stage of biofilm evolution for P. brasiliensis. The strains were cultivated in planktonic and biofilm form for 24–144 h. The fungi biomass and metabolic activity were determined by crystal violet and tetrazolium salt reduction (XTT) tests and colony-forming unit (CFU) by plating. The biofilm structure was designed using scanning electron microscopy and confocal laser scanning microscopy techniques. The extracellular matrix of P. brasiliensis and P. lutzii biofilms was extracted by sonication, and polysaccharides, proteins, and extracellular DNA (eDNA) were quantified. The RNA was extracted with the Trizol® reagent and quantified; then, the cDNA was synthesized to analyze the enolase expression, 14-3-3, FKS1, AGS1, GEL3, and KRE6 genes by real-time PCR. All strains of Paracoccidioides studied form a biofilm with more significant metabolic activity and biomass values in 144 h. The extracellular matrix of P. brasiliensis and P. lutzii had a higher content of polysaccharides in their composition, followed by proteins and eDNA in smaller quantities. The P. brasiliensis biofilm kinetics of formation showed greater expression of genes related to glucan’s synthesis and its delivery to the external environment in addition adhesins during the biofilm’s adhesion, initiation, and maturation. The GEL3 and enolase genes increased in expression within 24 h and during the biofilm maturation period, there was an increase in 14-3-3, AGS1, and FKS1. Furthermore, at 144 h, there was a decrease in KRE6 expression and an increase in GEL3. This study highlights the potential for biofilm formation for three species of Paracoccidioides and the main components of the extracellular matrix that can contribute to a better understanding of biofilm organization. [ABSTRACT FROM AUTHOR]

Cornus iridoid glycosides (CIGs), including loganin and morroniside, are the main active components of Cornus officinalis. As one of the key enzymes in the biosynthesis of CIGs, geranyl pyrophosphate synthase (GPPS) catalyzes the formation of geranyl pyrophosphate, which is the direct precursor of CIGs. In this study, the C. officinalis geranyl pyrophosphate synthase (CoGPPS) sequence was cloned from C. officinalis and analyzed. The cDNA sequence of the CoGPPS gene was 915 bp (GenBank No. OR725699). Phylogenetic analysis showed that CoGPPS was closely related to the GPPS sequence of Actinidia chinensis and Camellia sinensis, but relatively distantly related to Paeonia lactiflora and Tripterygium wilfordii. Results from the quantitative real-time PCR showed the spatiotemporal expression pattern of CoGPPS; that is, CoGPPS was specifically expressed in the fruits. Subcellular localization assay proved that CoGPPS was specifically found in chloroplasts. Loganin and morroniside contents in the tissues were detected by high-performance liquid chromatography, and both compounds were found to be at higher levels in the fruits than in leaves. Thus, this study laid the foundation for further studies on the synthetic pathway of CIGs. [ABSTRACT FROM AUTHOR]

Objective: We previously elucidated that long non-coding RNA Promoter of CDKN1A Antisense DNA damage Activated RNA (PANDAR) as a p53-dependent oncogene to promote cisplatin resistance in ovarian cancer (OC). Intriguingly, high level of p53-independent PANDAR was found in cisplatin-resistant patients with p53 mutation. Here, our study probed the new roles and the underlying mechanisms of PANDAR in p53-mutant OC cisplatin-resistance. Methods: A2780 and A2780-DDP cells were served as OC cisplatin-sensitive and cisplatinresistant cells. HO-8910PM cells were subjected to construct chemotherapy-induced extracellular vesicles (Chemo-EVs). Transmission electron microscopy (TEM) and nanoparticle tracking analysis were employed to evaluate Chemo-EVs. Cell viability was assessed using cell counting kit-8 and colony formation assays. Cell apoptosis was assessed using Annexin V and propidium iodide staining. The relationships between PANDAR, serine and arginine-rich premRNA splicing factor 9 (SRSF9) were verified by RNA immunoprecipitation and fluorescence in situ hybridization. Tumor xenograft experiment was employed to evaluate the effects of PANDAR-Chemo-EVs on OC cisplatin-resistance in vivo. Immunofluorescent staining and immunohistochemistry were performed in tumor tissue. Results: PANDAR level increased in OC patients with p53-mutation. PANDAR efflux enacted via exosomes under cisplatin conditions. Additionally, exosomes from OC cell lines carried PANDAR, which significantly increased cell survival and chemoresistance in vitro and tumor progression and metastasis in vivo. During cisplatin-induced stress, SRSF9 was recruited to nuclear bodies by increased PANDAR and muted apoptosis in response to cisplatin. Besides, SRSF9 significantly increased the ratio of SIRT4/SIRT6 mRNA in OC. Conclusion: Cisplatin-induced exosomes transfer PANDAR and lead to a rapid adaptation of OC cell survival through accumulating SRSF9 following cisplatin stress exposure. [ABSTRACT FROM AUTHOR]

The capacity to identify small amounts of pathogens in real samples is extremely useful. Herein, we proposed a sensitive platform for detecting pathogens using cyclic DNA nanostructure@AuNP tags (CDNA) and a cascade primer exchange reaction (cPER). This platform employs wheat germ agglutinin-modified Fe3O4@Au magnetic nanoparticles (WMRs) to bind the E. coli O157:H7, and then triggers the cPER to generate branched DNA products for CDNA tag hybridization with high stability and amplified SERS signals. It can identify target pathogens as low as 1.91 CFU/mL and discriminate E. coli O157:H7 in complex samples such as water, milk, and serum, demonstrating comparable or greater sensitivity and accuracy than traditional qPCR. Moreover, the developed platform can detect low levels of E. coli O157:H7 in mouse serum, allowing the discrimination of mice with early-stage infection. Thus, this platform holds promise for food analysis and early infection diagnosis. [ABSTRACT FROM AUTHOR]

Resistance against aminoglycosides is widespread in bacteria. This study aimed to identify genes that are important for growth of E. coli during aminoglycoside exposure, since such genes may be targeted to re-sensitize resistant E. coli to treatment. We constructed three transposon mutant libraries each containing > 230.000 mutants in E. coli MG1655 strains harboring streptomycin (aph(3″)-Ib/aph(6)-Id), gentamicin (aac(3)-IV), or neomycin (aph(3″)-Ia) resistance gene(s). Transposon Directed Insertion-site Sequencing (TraDIS), a combination of transposon mutagenesis and high-throughput sequencing, identified 56 genes which were deemed important for growth during streptomycin, 39 during gentamicin and 32 during neomycin exposure. Most of these fitness-genes were membrane-located (n = 55) and involved in either cell division, ATP-synthesis or stress response in the streptomycin and gentamicin exposed libraries, and enterobacterial common antigen biosynthesis or magnesium sensing/transport in the neomycin exposed library. For validation, eight selected fitness-genes/gene-clusters were deleted (minCDE, hflCK, clsA and cpxR associated with streptomycin and gentamicin resistance, and phoPQ, wecA, lpp and pal associated with neomycin resistance), and all mutants were shown to be growth attenuated upon exposure to the corresponding antibiotics. In summary, we identified genes that are advantageous in aminoglycoside-resistant E. coli during antibiotic stress. In addition, we increased the understanding of how aminoglycoside-resistant E. coli respond to antibiotic exposure. [ABSTRACT FROM AUTHOR]

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]

Chitin deacetylase (CDA) can catalyze the deacetylation of chitin to produce chitosan. In this study, we identified and characterized a chitin deacetylase gene from Euphausia superba (EsCDA-9k), and a soluble recombinant protein chitin deacetylase from Euphausia superba of molecular weight 45 kDa was cloned, expressed, and purified. The full-length cDNA sequence of EsCDA-9k was 1068 bp long and encoded 355 amino acid residues that contained the typical domain structure of carbohydrate esterase family 4. The predicted three-dimensional structure of EsCDA-9k showed a 67.32% homology with Penaeus monodon. Recombinant chitin deacetylase had the highest activity at 40 °C and pH 8.0 in Tris-HCl buffer. The enzyme activity was enhanced by metal ions Co2+, Fe3+, Ca2+, and Na+, while it was inhibited by Zn2+, Ba2+, Mg2+, and EDTA. Molecular simulation of EsCDA-9k was conducted based on sequence alignment and homology modeling. The EsCDA-9k F18G mutant showed a 1.6-fold higher activity than the wild-type enzyme. In summary, this is the first report of the cloning and heterologous expression of the chitin deacetylase gene in Euphausia superba. The characterization and function study of EsCDA-9k will serve as an important reference point for future application. [ABSTRACT FROM AUTHOR]

MYB transcription factors (TFs) have been shown to play a key role in plant growth and development and are in response to various types of biotic and abiotic stress. Here, we clarified the structure, expression patterns, and function of a MYB TF, SlMYB86-like (Solyc06g071690) in tomato using an inbred tomato line exhibiting high resistance to bacterial wilt (Hm 2-2 (R)) and one susceptible line (BY 1-2 (S)). The full-length cDNA sequence of this gene was 1226 bp, and the open reading frame was 966 bp, which encoded 321 amino acids; its relative molecular weight was 37.05055 kDa; its theoretical isoelectric point was 7.22; it was a hydrophilic nonsecreted protein; and it had no transmembrane structures. The protein also contains a highly conserved MYB DNA-binding domain and was predicted to be localized to the nucleus. Phylogenetic analysis revealed that SlMYB86-like is closely related to SpMYB86-like in Solanum pennellii and clustered with other members of the family Solanaceae. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of the SlMYB86-like gene was tissue specific and could be induced by Ralstonia solanacearum, salicylic acid, and jasmonic acid. The results of virus-induced gene silencing (VIGS) revealed that SlMYB86-like silencing decreased the resistance of tomato plants to bacterial wilt, suggesting that it positively regulates the resistance of tomatoes to bacterial wilt. Overall, these findings indicate that SlMYB86-like plays a key role in regulating the resistance of tomatoes to bacterial wilt. [ABSTRACT FROM AUTHOR]

The aim of this research was to study the impact of squalene synthase gene silencing on the features of transgenic chrysanthemum plants, and on the synthesis of some sesquiterpenes—caryophyllene, germacrene and farnesene. We have cloned chrysanthemum squalene synthase gene cDNA (CmSQS) and obtained five lines stably transformed with vector for CmSQS silencing. Silencing of CmSQS gene transcription was detected in all five transgenic lines, the decrease in the squalene content was found in three of them, by 1.8–2.0 times depending on the line. The content of triterpenes α- and β-amirin, derivatives of squalene, clearly correlated with its content in plants. The β-caryophyllene, germacrene D and β-farnesene were identified in chrysanthemum using GC-MS analysis; the content of these compounds in transgenic plants did not change appreciably compared to control ones. Silencing of squalene synthase had no effect on plants morphology. In two transgenic lines, a decrease in growth rate relative to non-transgenic plants was observed; the growth rate of three other lines did not differ from the control. Thus, in our experiments no well-defined relationship between CmSQS gene transcription level, the content of squalene and studied sesquiterpenes was found. The obtained data provide additional information on mechanisms of plant secondary metabolism regulation including the relation between β-caryophyllene, germacrene D and β-farnesene content with squalene synthesis level. [ABSTRACT FROM AUTHOR]

Background: Intellectual disability (ID) is a con neurodevelopmental disorder in children. The genetic etiology of ID is complex, but more subtypes are defined due to the broad application of next‐generation sequencing. Methods: Whole‐exome sequencing (WES) and Sanger sequencing was applied in a family with ID. Results: We report a Chinese 7.5‐year‐old boy, born to non‐consanguineous parents. He showed severe intellectual disability, seizures and autistic features. Two previously unreported variants in MBOAT7, c.669C>G (p.(Tyr223*)) and c.1095C>G (p.(Ser365Arg)) were identified by trio‐WES. His mother is a heterozygous carrier of the c.1095C>G variant. The c.669C>G variant is a de novo variant which was undetected in his parents. By construction of the full‐length cDNA of the patient's MBOAT7, we verified that these two variants were trans‐compound heterozygous variants, which support the genetic etiology of this patient. Conclusion: This patient is the first Chinese case of intellectual developmental disorder (IDD), autosomal recessive 57 (OMIM:617188) with two unreported MBOAT7 variants. [ABSTRACT FROM AUTHOR]

This article discusses a study on the detection of Neoparamoeba perurans, a parasite that causes amoebic gill disease (AGD) in salmon. The study compares the use of DNA-based and RNA-based assays for detecting N. perurans in seawater samples. The researchers found that the RNA-based assay was more sensitive and reliable than the DNA-based assay, detecting N. perurans even at low concentrations. The study suggests that targeting intracellular RNA may provide a more accurate measure of amoeba abundance. Further research is needed to determine if RNA-based quantification of N. perurans can be used as an early indicator of AGD. [Extracted from the article]

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]

Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) (B. dorsalis) is an important agricultural, major invasive, and quarantine pest that can cause significant damage to the economic value of the fruit and vegetable industry. Male bait is one of the most effective methods of surveying, monitoring, and controlling B. dorsalis. In our study, we constructed cDNA libraries using total RNA extracted independently from the antennae, mouthparts, and thoracic legs of male and female adults and the ovipositors of female adults and screened out four aldehyde-oxidase-related genes (AOX-related), C58800, C66700, C67485, and C67698. Molecular docking predictions showed that eight compounds, including 3,4-dimethoxycinnamyl alcohol, 3,4-dimethoxy-cinnamaldehyde, deet, ethyl N-acetyl-N-butyl-β-alaninate, n-butyl butyrate, n-butyl butyrate, ethyl butyrate, methyl eugenol, and ethyl acetate, could combine with proteins encoded by the four B. dorsalis AOX-related genes. Furthermore, QPCR was performed to confirm that four compounds, including 3,4-dimethoxy cinnamic aldehyde, butyl levulinic acid ethyl ester (mosquito repellent), butyl butyrate, and methyl eugenol, induced significant changes in the AOX-related genes of B. dorsalis. These results provide useful information and guidance for the batch screening of potentially useful compounds and the search for effective attractants of B. dorsalis. [ABSTRACT FROM AUTHOR]

Rhododendron fortunei belongs to a scented Rhododendron species native to China, which produces fragrant flowers of great ornamental and environmental values for landscaping or indoor beautification. However, the scents in Rhododendron fortunei have not yet been investigated, let alone the mechanism of the formation of these fragrance in the flowers. In this study, we measured the scents in terms of its volatile components and contents (VOC) in Rhododendron fortunei at four different flowering stages and in different tissues of the plant by headspace solid-phase micro-extraction combined (HS-SPME) with gas chromatography-mass spectrometry (GC-MS). Then the characteristic aromatic values, which reflects the degree of scent perception by human, of each VOC in the plant was calculated according to its respective aromatic thresholds. Results showed that three main VOCs measured from highest to lowest are methyl benzoates, terpenes and fatty acid derivatives. Their content increased after the flower bud opening and reached the highest at half to full blossom. In a flower most VOC contents were measured in petals and only trace amount in other tissues such as stamen, pistil. A small amount of VOCs was determined in leaves as well.All aromatic values were almost corresponded to the contents of three main VOCs, indicating that the flower fragrance arises truly from these VOC components. S-adenosyl-L-methionine: benzoic acid carboxyl methyl transferase (BAMT) catalyzes the final step to form methyl benzoates. To understand the mechanism of the formation of this main type fragrance and its regulation, we firstly isolate a gene of RfBAMT from petal of Rhododendron fortunei by using homologous cloning and RACE technology. The full length of its cDNA was 1383 bp，with an open reading frame of 1104 bp, encoding a total of 368 amino acids. The phylogenetic tree analysis showed that RfBAMT was the closest to the BSMT of Camellia japonica, belonging to methyltransferases family. Then we measured the expression level of RfBAMT again at four flower developmental stages and in different flower tissues and leaves. The results showed that the expression level of this gene was highly positively correlated with the emitted content of methyl benzoates in the flowering, implying that RfBAMT plays a pivotal role in the formation and regulation of methyl benzoates in Rhododendron fortune.This researchshowed that the RfBAMT was cloned and identified in our study and its expression level was highly positively correlated with the emitted content of methyl benzoates in the flowers and leaves, which indicated this gene may play an important role on regulation of methyl benzoate synthesis in Rhododendron fortunei. [ABSTRACT FROM AUTHOR]

As a model organism, the cell culture method of Rana dybowskii (Rd) has not received widespread attention. We obtained kidney cells from Rana dybowskii by tissue block culture and enzyme digestion, and optimized the culture method by changing different culture conditions. We can more conveniently study the operation of the Rana dybowskii immune system through cell lines. One of the most important adaptor proteins in the Toll-like receptors (TLRs) signaling pathway, TIRdomain-containing adaptor inducing interferon-ß (TRIF), plays a role in the apoptosis pathway and participates in the ubiquitin-mediated protein hydrolysis pathway. This rationalizes the significance of TRIF in the immune cascade signaling pathway. To obtain and analyze the full-length sequence of cDNA, sequential structures of amino acids, as well as Rana dybowskii TRIF expression profiles, the present work carried out multiple-sequence alignment, rapid amplification of cDNA ends (RACE), the establishment of a phylogenetic tree, search for the conserved domain, and qRT-PCR. Results revealed 1733-bp full-length cDNA of RdTRIF with an open reading frame (ORF) of 1281 bp, which encoded one protein containing 426 amino acid residues. The amino acid sequence of RdTRIF confirmed the presence of one Toll/interleukin-1 receptor (TIR) domain. As a kind of hydrophilic protein, the pI and molecular weight of RdTRIF were 5.67 and 24.76 kDa, respectively. We predicted 51 candidate phosphorylation sites, including 32 serine residues, 16 threonine residues along with 3 tyrosine residues. The phylogenetic tree and multiple-sequence alignment substantiated the highest homology of RdTRIF protein to Xenopus tropicalis homologs. According to the qRT-PCR results, the expression of RdTRIF was noted under the stress of Ah in the kidney. [ABSTRACT FROM AUTHOR]

A poly(n-butyl acrylate)-gold-MXene quantum dots (PBA-Au-MXene QD) nanocomposite-based biosensor is presented that is modified by unique antisense single-stranded DNA (ssDNA) and uses the electrochemical detection methods of DPV, CV, and EIS to early detect miR-122 as a breast cancer biomarker in real clinical samples. This fabrication method is based on advanced nanotechnology, at which a poly(n-butyl acrylate) (PBA) as a non-conductive polymer transforms into a conductive composite by incorporating Au-MXene QD. This biosensor had a limit of detection (LOD) of 0.8 zM and a linear range from 0.001 aM to 1000 nM, making it capable of detecting the low concentrations of miR-122 in patient samples. Moreover, it allows approximately 100% sensitivity and 100% specificity for miR-122 without extraction. The synthesis and detection characteristics were evaluated by different complementary tests such as AFM, FTIR, TEM, and FESEM. This new biosensor can have a high potential in clinical applications to detect breast cancer early and hence improve patient outcomes. [ABSTRACT FROM AUTHOR]

Worldwide, genotype 3 is the second most prevalent major variant among patients with chronic hepatitis C virus (HCV) infection and the most difficult to treat with direct-acting antivirals (DAAs). Further, subtype 3b, which is highly prevalent in Southeast Asia with increasing transmission in high-risk populations, carries paired NS5A resistance-associated substitutions (RAS), NS5A-A30K+L31M, conferring resistance to DAA therapy and lowering cure rates with pan-genotypic regimens. However, no complete genomic sequence or infectious clone exists for HCV genotype 3b. We determined the entire genome sequences, including 5' and 3' termini, of HCV genotype 3b isolates from three treatment naïve chronic hepatitis C patients, and by clonal analysis of the entire coding sequence demonstrated heterogeneous genome population compositions all carrying RAS A30K+L31M in NS5A. We generated a full-length HCV genotype 3b cDNA clone (pODN) and transfected Huh7.5 and Huh-Lunet/SEC14L2 cells with derived RNA transcripts without detecting HCV antigens by immunofluorescence staining. In contrast, intrahepatic transfection with RNA transcripts from pODN, and subsequent virus passages, in human-liver chimeric mice resulted in robust infection with serum HCV RNA titers of up to 7.9 log10 genome equivalents/mL. Consensus HCV sequences of virus recovered from the transfected mouse contained no coding mutations exceeding 5% frequency, and sequences from the passage-infected mice likewise had no consensus changes. Thus, we developed the first HCV genotype 3b full-length cDNA clone which by its infectivity and genetic stability in human-liver chimeric mice proved functionality, and potential utility in future development of infectious cell culture systems needed for this DAA treatment-resistant subtype. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is a severe heavy metal pollutant globally. Zoysia japonica is an important perennial warm-season turf grass that potentially plays a role in phytoremediation in Cd-polluted soil areas; however, the molecular mechanisms underlying its Cd stress response are unknown. To further investigate the early gene response pattern in Z. japonica under Cd stress, plant leaves were harvested 0, 6, 12, and 24 h after Cd stress (400 μM CdCl2) treatment and used for a time-course RNA-sequencing analysis. Twelve cDNA libraries were constructed and sequenced, and high-quality data were obtained, whose mapped rates were all higher than 94%, and more than 601 million bp of sequence were generated. A total of 5321, 6526, and 4016 differentially expressed genes were identified 6, 12, and 24 h after Cd stress treatment, respectively. A total of 1660 genes were differentially expressed at the three time points, and their gene expression profiles over time were elucidated. Based on the analysis of these genes, the important mechanisms for the Cd stress response in Z. japonica were identified. Specific genes participating in glutathione metabolism, plant hormone signal and transduction, members of protein processing in the endoplasmic reticulum, transporter proteins, transcription factors, and carbohydrate metabolism pathways were further analyzed in detail. These genes may contribute to the improvement of Cd tolerance in Z. japonica. In addition, some candidate genes were highlighted for future studies on Cd stress resistance in Z. japonica and other plants. Our results illustrate the early gene expression response of Z. japonica leaves to Cd and provide some new understanding of the molecular mechanisms of Cd stress in Zosia and Gramineae species. [ABSTRACT FROM AUTHOR]

During the interaction between Brassica napus and Sclerotinia sclerotiorum, the expression of JA (jasmonic acid) synthesis-related genes increases, while JA signal transduction is inhibited. It is speculated that the effector proteins secreted by S. sclerotiorum may directly or indirectly interact with JAR1 protein, reducing its activity and thus inhibiting the defense system of B. napus dependent on JA pathway. The full-length sequence of the differentially expressed gene BnJarl was obtained by transcriptome sequencing in the early stage of this research. In order to further reveal the mechanism of BnJarl gene involved in the interaction between B. napus and S. sclerotiorum, this study analyzed and predicted the bioinformatics of BnJAR1 protein, constructed a yeast expression gene cDNA library of B. napus-S. sclerotiorum interaction, and used BnJar1 as bait protein to screen the library by yeast two-hybrid. Through the study, 5 interacting proteins from B. napus were obtained: BnAOC2, BnCOP9, BnDcoH, BnNIT2, and BnTSJT1; and 2 interacting proteins from S. sclerotiorum: SsMFS and SsRho3. The relevant results contributed to our research on the interaction between B. napus and S. sclerotiorum, exploration of pathogenic or resistance-related genes, and on further elucidation of their mechanisms. [ABSTRACT FROM AUTHOR]

Increasing evidence shows the African lineage Zika virus (ZIKV) displays a more severe neurovirulence compared to the Asian ZIKV. However, viral determinants and the underlying mechanisms of enhanced virulence phenotype remain largely unknown. Herein, we identify a panel of amino acid substitutions that are unique to the African lineage of ZIKVs compared to the Asian lineage by phylogenetic analysis and sequence alignment. We then utilize reverse genetic technology to generate recombinant ZIKVs incorporating these lineage-specific substitutions based on an infectious cDNA clone of Asian ZIKV. Through in vitro characterization, we discover a mutant virus with a lysine to arginine substitution at position 101 of capsid (C) protein (termed K101R) displays a larger plaque phenotype, and replicates more efficiently in various cell lines. Moreover, K101R replicates more efficiently in mouse brains and induces stronger inflammatory responses than the wild type (WT) virus in neonatal mice. Finally, a combined analysis reveals the K101R substitution promotes the production of mature C protein without affecting its binding to viral RNA. Our study identifies the role of K101R substitution in the C protein in contributing to the enhanced virulent phenotype of the African lineage ZIKV, which expands our understanding of the complexity of ZIKV proteins. The neurovirulence determinants of Zika virus remain not fully established. Song et al identified a single K101R substitution in the capsid protein that contributes to the lineage-specific virulence phenotypes. [ABSTRACT FROM AUTHOR]

The prevailing concept is that gestational alloimmune liver disease (GALD) is caused by maternal antibodies targeting a currently unknown antigen on the liver of the fetus. This leads to deposition of complement on the fetal hepatocytes and death of the fetal hepatocytes and extensive liver injury. In many cases, the newborn dies. In subsequent pregnancies early treatment of the woman with intravenous immunoglobulin can be instituted, and the prognosis for the fetus will be excellent. Without treatment the prognosis can be severe. Crucial improvements of diagnosis require identification of the target antigen. For this identification, this work was based on two hypotheses: 1. The GALD antigen is exclusively expressed in the fetal liver during normal fetal life in all pregnancies; 2. The GALD antigen is an alloantigen expressed in the fetal liver with the woman being homozygous for the minor allele and the father being, most frequently, homozygous for the major allele. We used three different experimental approaches to identify the liver target antigen of maternal antibodies from women who had given birth to a baby with the clinical GALD diagnosis: 1. Immunoprecipitation of antigens from either a human liver cell line or human fetal livers by immunoprecipitation with maternal antibodies followed by mass spectrometry analysis of captured antigens; 2. Construction of a cDNA expression library from human fetal liver mRNA and screening about 1.3 million recombinants in Escherichia coli using antibodies from mothers of babies diagnosed with GALD; 3. Exome/genome sequencing of DNA from 26 presumably unrelated women who had previously given birth to a child with GALD with husband controls and supplementary HLA typing. In conclusion, using the three experimental approaches we did not identify the GALD target antigen and the exome/genome sequencing results did not support the hypothesis that the GALD antigen is an alloantigen, but the results do not yield basis for excluding that the antigen is exclusively expressed during fetal life., which is the hypothesis we favor. [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]

Single-cell RNA sequencing (scRNA-seq) provides a powerful tool to evaluate the transcriptomic landscape and heterogeneity of thousands of cells in parallel. However, complex study designs or the unavailability of in-house instruments require the temporal disconnection between sample preparation and library construction, raising the need for efficient sample preservation methods which are compatible with scRNA-seq downstream analysis. Several studies evaluated the effect of methanol fixation as preservation method, yet none of them deeply assessed its effect on adult primary dissociated brain tissue. Here, we evaluated its effect on murine dentate gyrus (DG) single cell suspensions and on subsequent scRNA-seq downstream analysis by performing SOrting and Robot-assisted Transcriptome SEQuencing (SORT-seq), a partially robotized version of the CELseq2 protocol. Our results show that MeOH fixation preserves RNA integrity and has no apparent effects on cDNA library construction. They also suggest that fixation protects from sorting-induced cell stress and increases the proportion of high-quality cells. Despite evidence of mRNA leakage in fixed cells, their relative gene expression levels correlate well with those of fresh cells and fixation does not significantly affect the variance of the dataset. Moreover, it allows the identification of all major DG cell populations, including neural precursors, granule neurons and different glial cell types, with a tendency to preserve more neurons that are underrepresented in fresh samples. Overall, our data show that MeOH fixation is suitable for preserving primary neural cells for subsequent single-cell RNA profiling, helping to overcome challenges arising from complex workflows, improve experimental flexibility and facilitate scientific collaboration. [ABSTRACT FROM AUTHOR]

Tuberous sclerosis complex (TSC) is a relatively common autosomal dominant disorder characterized by multiple dysplastic organ lesions and neuropsychiatric symptoms caused by loss-of-function mutation of either TSC1 or TSC2. The genetic diagnosis of inherited diseases, including TSC, in the clinical field is widespread using next-generation sequencing. The mutations in proteincoding exon tend to be verified because mutations directly cause abnormal protein. However, it is relatively difficult to verify mutations in the intron region because it is required to investigate whether the intron mutations affect the abnormal splicing of transcripts. In this study, we developed a target-capture full-length double-stranded cDNA sequencing method using Nanopore longread sequencer (Nanopore long-read target sequencing). This method revealed the occurrence of intron mutation in the TSC2 gene and found that the intron mutation produces novel intron retention splicing transcripts that generate truncated proteins. The protein-coding transcripts were decreased due to the expression of the novel intron retention transcripts, which caused TSC in patients with the intron mutation. Our results indicate that Nanopore long-read target sequencing is useful for the detection of mutations and confers information on the full-length alternative splicing of transcripts for genetic diagnosis. [ABSTRACT FROM AUTHOR]

Bovine herpesvirus type 1 (BoHV-1) is an important agricultural pathogen that infects cattle and other ruminants worldwide. Though it was first sequenced and annotated over twenty years ago, the Cooper strain, used in this study, was sequenced as recently as 2012 and is currently said to encode 72 unique proteins. However, tandem mass spectrometry has identified several peptides produced during active infection that align with the BoHV-1 genome in unannotated regions. One of these abundant peptides, "ORF M", aligned antisense to the DNA helicase/primase protein UL5. This study characterizes the novel transcript and its protein product and provides evidence to support the existence of homolog protein-coding genes in other Herpesviruses. [ABSTRACT FROM AUTHOR]