SMA20/PMIS2 Is a Rapidly Evolving Sperm Membrane Alloantigen with Possible Species-Divergent Function in Fertilization.

Immunodominant alloantigens in pig sperm membranes include 15 known gene products and a previously undiscovered Mr 20,000 sperm membrane-specific protein (SMA20). Here we characterize SMA20 and identify it as the unannotated pig ortholog of PMIS2. A composite SMA20 cDNA encoded a 126 amino acid polypeptide comprising two predicted transmembrane segments and an N-terminal alanine- and proline (AP)-rich region with no apparent signal peptide. The Northern blots showed that the composite SMA20 cDNA was derived from a 1.1 kb testis-specific transcript. A BLASTp search retrieved no SMA20 match from the pig genome, but it did retrieve a 99% match to the Pmis2 gene product in warthog. Sequence identity to predicted PMIS2 orthologs from other placental mammals ranged from no more than 80% overall in Cetartiodactyla to less than 60% in Primates, with the AP-rich region showing the highest divergence, including, in the extreme, its absence in most rodents, including the mouse. SMA20 immunoreactivity localized to the acrosome/apical head of methanol-fixed boar spermatozoa but not live, motile cells. Ultrastructurally, the SMA20 AP-rich domain immunolocalized to the inner leaflet of the plasma membrane, the outer acrosomal membrane, and the acrosomal contents of ejaculated spermatozoa. Gene name search failed to retrieve annotated Pmis2 from most mammalian genomes. Nevertheless, individual pairwise interrogation of loci spanning Atp4a-Haus5 identified Pmis2 in all placental mammals, but not in marsupials or monotremes. We conclude that the gene encoding sperm-specific SMA20/PMIS2 arose de novo in Eutheria after divergence from Metatheria, whereupon rapid molecular evolution likely drove the acquisition of a species-divergent function unique to fertilization in placental mammals.

[Research progress on chemical constituents and pharmacological activities of small molecule compounds in scorpions].

Scorpions, a group of oldest animals with wide distribution in the world, have a long history of medicinal use. Scorpio, the dried body of Buthus martensii, is a rare animal medicine mainly used for the treatment of liver diseases, spasm, and convulsions in children in China. The venom has been considered as the active substance of scorpions. However, little is known about the small molecules in the venom of scorpions. According to the articles published in recent years, scorpions contain amino acids, fatty acids, steroids, and alkaloids, which endow scorpions with antimicrobial, anticoagulant, metabolism-regulating, and antitumor activities. This paper summarizes the small molecule chemical components and pharmacological activities of scorpions, with a view to providing valuable information for the discovery of new active molecules and the clinical use of scorpions.

Photoperiodism of Diapause Induction in the Silkworm, Bombyx mori.

The silkworm Bombyx mori exhibits a photoperiodic response (PR) for embryonic diapause induction. This article provides a comprehensive review of literature on the silkworm PR, starting from early works on population to recent studies uncovering the molecular mechanism. Makita Kogure (1933) conducted extensive research on the PR, presenting a pioneering paper on insect photoperiodism. In the 1970s and 80s, artificial diets were developed, and the influence of nutrition on PR was well documented. The photoperiodic photoreceptor has been investigated from organ to molecular level in the silkworm. Culture experiments demonstrated that the photoperiodic induction can be programmed in an isolated brain (Br)-subesophageal ganglion (SG) complex with corpora cardiaca (CC)-corpora allata (CA). The requirement of dietary vitamin A for PR suggests the involvement of opsin pigment in the photoperiodic reception, and a cDNA encoding an opsin (Boceropsin) was cloned from the brain. The effector system concerning the production and secretion of diapause hormone (DH) has also been extensively investigated in the silkworm. DH is produced in a pair of posterior cells of SG, transported to CC by nervi corporis cardiaci, and ultimately released into the hemolymph. Possible involvement of GABAergic and corazonin (Crz) signal pathways was suggested in the control of DH secretion. Knockout (KO) experiments of GABA transporter (GAT) and circadian clock genes demonstrated that GAT plays a crucial role in PR through circadian control. A model outlining the PR mechanism, from maternal photoperiodic light reception to DH secretion, has been proposed.

Expression of mRNAs Encoding Hypothalamic Small Proteins, Neurosecretory Protein GL and Neurosecretory Protein GM, in the Japanese Quail, Coturnix japonica.

Neurosecretory protein GL (NPGL) and neurosecretory protein GM (NPGM) are novel neuropeptides that have been discovered in the hypothalamic infundibulum of chickens. NPGL and NPGM play important roles in lipid metabolism in juvenile chickens. The physiological functions of NPGL and NPGM in sexually mature birds remain unknown. The Japanese quail (Coturnix japonica) seems to be an appropriate model for analyzing NPGL and NPGM during sexual maturity. However, studies on NPGL or NPGM have yet to be reported in the Japanese quail. In the present study, we identified cDNAs encoding precursor proteins of NPGL and NPGM in the quail hypothalamus. In situ hybridization revealed that NPGL mRNA-expressing cells in the hypothalamus were localized in the infundibular nucleus and median eminence, and NPGM mRNA-expressing cells were only found in the mammillary nucleus. Immunohistochemistry revealed that NPGM-like immunoreactive cells were distributed in the mammillary nucleus, whereas NPGL-like immunoreactive cells were not detected in the hypothalamus. Real-time PCR analysis indicated that the expression of NPGL mRNA was higher in the hypothalamus of females than in that of males, and NPGM mRNA expression showed no sex differences. NPGL and NPGM mRNA expression in males was upregulated after 24 h of food deprivation. In females, only NPGM mRNA expression was increased by fasting. These results suggest that the physiological functions of NPGL and NPGM are different in quail, and these factors are involved in sex differences in energy metabolism.

Mechanisms used for cDNA synthesis and site-specific integration of RNA into DNA genomes by a reverse transcriptase-Cas1 fusion protein.

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.

RT-qPCR based quantitative analysis of ARO and ADH genes in Saccharomyces cerevisiae and Metschnikowia pulcherrima strains growth white grape juice.

BACKGROUND: Yeast biosynthesizes fusel alcohols in fermentation through amino acid catabolism via the Ehrlich pathway. ARO8 and ARO9 genes are involved in the first step of the Ehrlich pathway, while ADH2 and ADH5 genes are involved in the last step. In this study, we describe RT-qPCR methods to determine the gene expression level of genes (ARO8, ARO9, ADH2, ADH5) found in Saccharomyces cerevisiae (Sc) and Metschnikowia pulcherrima (Mp) strains growth pasteurized white grape juice. METHODS AND RESULTS: We used RNA extraction and cDNA synthesis protocols. The RT-qPCR efficiency of primer pairs was evaluated by generating a standard curve through serial dilution of yeast-derived cDNA. Method performance criteria were determined for each RT-qPCR assay. Then, we evaluated the gene expression levels of the four genes in all samples. RNA extraction and cDNA synthesis from yeast samples demonstrated the method's capability to generate high-yield, high-purity nucleic acids, supporting further RT-qPCR analysis. The highest normalized gene expression levels of ARO8 and ARO9 were observed in SC1, SC4, and SC5 samples. No significant difference in ADH2 gene expression among Mp strains was observed during the examination of ADH2 and ADH5 genes (p < 0.05). We observed no expression of the ADH5 gene in Mp strains except MP6 strain. The expression of ADH2 and ADH5 genes was higher in Sc strains compared to Mp strains. CONCLUSIONS: The results suggest that the proposed RT-qPCR methods can measure gene expression of ARO8, ARO9, ADH2, and ADH5 in Sc and Mp strains growing in pasteurized white grape juice.

Updated mutational spectrum and genotype-phenotype correlations in ichthyosis patients with ABCA12 pathogenic variants.

Autosomal recessive congenital ichthyoses (ARCI) is a genetically heterogeneous condition that can be caused by pathogenic variants in at least 12 genes, including ABCA12. ARCI mainly consists of congenital ichthyosiform erythroderma (CIE), lamellar ichthyosis (LI) and harlequin ichthyosis (HI). The objective was to determine previously unreported pathogenic variants in ABCA12 and to update genotype-phenotype correlations for patients with pathogenic ABCA12 variants. Pathogenic variants in ABCA12 were detected using Sanger sequencing or a combination of Sanger sequencing and whole-exome sequencing. To verify the pathogenicity of a previously unreported large deletion and intron variant, cDNA analysis was performed using total RNA extracted from hair roots. Genetic analyses were performed on the patients with CIE, LI, HI and non-congenital ichthyosis with unusual phenotypes (NIUP), and 11 previously unreported ABCA12 variants were identified. Sequencing of cDNA confirmed the aberrant splicing of the variant ABCA12 in the patients with the previously unreported large deletion and intron variant. Our findings expand the phenotype spectrum of ichthyosis patients with ABCA12 pathogenic variants. The present missense variants in ABCA12 are considered to be heterogenous in pathogenicity, and they lead to varying disease severities in patients with ARCI and non-congenital ichthyosis with unusual phenotypes (NIUP).

Expression of PTGS2 along with genes regulating VEGF signalling pathway and association with high-risk factors in locally advanced oral squamous cell carcinoma.

BACKGROUND: PTGS2 encodes cyclooxygenase-2 (COX-2), which catalyses the committed step in prostaglandin synthesis. Various in vivo and in vitro data suggest that COX-2 mediates the VEGF signalling pathway. In silico analysis performed in TCGA, PanCancer Atlas for head and neck cancers, demonstrated significant expression and co-expression of PTGS2 and genes that regulate VEGF signalling. This study was designed to elucidate the expression pattern of PTGS2 and genes regulating VEGF signalling in patients with locally advanced oral squamous cell carcinoma (OSCC). METHODOLOGY: Tumour and normal tissue samples were collected from patients with locally advanced OSCC. RNA was isolated from tissue samples, followed by cDNA synthesis. The cDNA was used for gene expression analysis (RT-PCR) using target-specific primers. The results obtained were compared with the in silico gene expression of the target genes in the TCGA datasets. Co-expression analysis was performed to establish an association between PTGS2 and VEGF signalling genes. RESULTS: Tumour and normal tissue samples were collected from 24 OSCC patients. Significant upregulation of PTGS2 expression was observed. Furthermore, VEGFA, KDR, CXCR1 and CXCR2 were significantly upregulated in tumour samples compared with paired normal samples, except for VEGFB, whose expression was not statistically significant. A similar expression pattern was observed in silico, except for CXCR2 which was highly expressed in the normal samples. Co-expression analysis showed a significant positive correlation between PTGS2 and VEGF signalling genes, except for VEGFB which showed a negative correlation. CONCLUSION: PTGS2 and VEGF signalling genes are upregulated in OSCC, which has a profound impact on clinical outcomes.

A single amino acid at position 31 in the N-terminus of the coat protein of cucumber mosaic virus determines its avirulence function for RCY1-conferred virus resistance.

The coat protein (CP) of the cucumber mosaic virus (CMV) yellow strain [CMV(Y)], but not the CMV B2 strain [CMV(B2)], serves as an avirulence determinant against the NB-LRR class RCY1 of Arabidopsis thaliana. To investigate the avirulence function, a series of binary vectors were constructed by partially exchanging the CP coding sequence between CMV(Y) and CMV(B2) or introducing nucleotide substitutions. These vectors were transiently expressed in Nicotiana benthamiana leaves transformed with modified RCY1 cDNA. Analysis of hypersensitive resistance-cell death (HCD), CP accumulation, and defense gene expression at leaf sites infiltrated with Agrobacterium indicated that a single amino acid at position 31 of the CP seems to determine the avirulence function.

Unraveling the chicken T cell repertoire with enhanced genome annotation.

T cell receptor (TCR) repertoire sequencing has emerged as a powerful tool for understanding the diversity and functionality of T cells within the host immune system. Yet, the chicken TCR repertoire remains poorly understood due to incomplete genome annotation of the TCR loci, despite the importance of chickens in agriculture and as an immunological model. Here, we addressed this critical issue by employing 5' rapid amplification of complementary DNA ends (5'RACE) TCR repertoire sequencing with molecular barcoding of complementary DNA (cDNA) molecules. Simultaneously, we enhanced the genome annotation of TCR Variable (V), Diversity (D, only present in ß and δ loci) and Joining (J) genes in the chicken genome. To enhance the efficiency of TCR annotations, we developed VJ-gene-finder, an algorithm designed to extract VJ gene candidates from deoxyribonucleic acid (DNA) sequences. Using this tool, we achieved a comprehensive annotation of all known chicken TCR loci, including the α/δ locus on chromosome 27. Evolutionary analysis revealed that each locus evolved separately by duplication of long homology units. To define the baseline TCR diversity in healthy chickens and to demonstrate the feasibility of the approach, we characterized the splenic α/ß/γ/δ TCR repertoire. Analysis of the repertoires revealed preferential usage of specific V and J combinations in all chains, while the overall features were characteristic of unbiased repertoires. We observed moderate levels of shared complementarity-determining region 3 (CDR3) clonotypes among individual birds within the α and γ chain repertoires, including the most frequently occurring clonotypes. However, the ß and δ repertoires were predominantly unique to each bird. Taken together, our TCR repertoire analysis allowed us to decipher the composition, diversity, and functionality of T cells in chickens. This work not only represents a significant step towards understanding avian T cell biology, but will also shed light on host-pathogen interactions, vaccine development, and the evolutionary history of avian immunology.

Identification of novel pathogenic variants of CUBN in patients with isolated proteinuria.

BACKGROUND: Although proteinuria is long recognized as an independent risk factor for progressive chronic kidney diseases, not all forms of proteinuria are detrimental to kidney function, one of which is isolated proteinuria caused by cubilin (CUBN)-specific mutations. CUBN encodes an endocytic receptor, initially found to be responsible for the Imerslund-Gräsbeck syndrome (IGS; OMIM #261100) characterized by a combined phenotype of megaloblastic anemia and proteinuria. METHODS: After analyzing their clinical and pathological characterizations, next-generation sequencing for renal disease genes or whole-exome sequencing (WES) was performed on four patients with non-progressive isolated proteinuria. CUBN biallelic pathogenic variants were identified and further analyzed by cDNA-PCR sequencing, immunohistochemistry, minigene assay, and multiple in silico prediction tools, including 3D protein modeling. RESULTS: Here, we present four patients with isolated proteinuria caused by CUBN C-terminal biallelic pathogenic variants, all of which showed no typical IGS symptoms, such as anemia and vitamin B12 deficiency. Their urine protein levels fluctuated between +~++ and estimated glomerular filtration rate (eGFR) were normal or slightly higher. Mild mesangial hypercellularity was found in three children's renal biopsies. A homozygous splice-site variant of CUBN (c.6821+3 (IVS44) A>G) was proven to result in the exon 44 skipping and premature translation termination by cDNA sequencing and immunohistochemistry. Compound heterozygous mutations were identified among the other three children, including another novel splice-site variant (c.10764+1 (IVS66) G>A) causing the retention of first 4 nucleotides in intron 66 by minigene assay, two unreported missense mutations (c.4907G>A (p.R1636Q); c. 9095 A>G (p.Y3032C)), and two reported missense mutations in China (c.8938G>A (p.D2980N); c. 9287T>C (p.L3096P)), locating behind the vitamin B12-binding domain, affecting CUB11, CUB16, CUB22, CUB23, and CUB27 domains, respectively. CONCLUSION: These results demonstrate that above CUBN mutations may cause non-progressive and isolated proteinuria, expanding the variant spectrum of CUBN and benefiting our understanding of proteinuria and renal function.

Colorimetric and fluorescent independent dual "signal on" biosensor for accurate detection of ochratoxin A based on aptamer-triggered biocatalytic reactions.

Ochratoxin A (OTA) is a hazardous food contaminant with significant health risks. Dual-channel OTA detection is noted for its cross-reference capability and high accuracy. Still, challenges in addressing in-system corrections and "signal off" related false positives and limited signal gains remain. Herein, we developed a dual-channel "signal on" aptasensor with one recognition process and two independent signal outputs for OTA analysis. The OTA aptamer binds to magnetic beads (MBs) and partially hybridizes with a complementary-trigger (cDNA-Trigger) sequence. Adding OTA disrupts the duplex sequence, leading to G-quadruplex (G4) formation and enrichment on the MBs, which then interacts with hemin to catalyze a color signal. Concurrently, the freed cDNA-Trigger catalyzes an enzyme-free DNA circuit, producing a fluorescence signal. The magnetic enrichment and signal amplification strategies make the proposed assay demonstrate excellent sensitivity toward OTA, with limits of detection (LOD) of 0.017 pM in the fluorescence channel and 48.1 pM in the colorimetric channel. Both channels have effectively detected OTA in grape juice and baijiu, demonstrating their applicability and reliability. Moreover, given the widespread use of smartphones globally, a mini-program with a self-correction function was designed to facilitate on-site colorimetric channel monitoring, making OTA detection more accessible and user-friendly.

A Flexible Mouse Model of Autoimmune Thyroiditis Induced by Immunization with an Adenovirus Containing Full-Length Thyroglobulin cDNA.

The main challenge in the "post-GWAS" era is to determine the functional meaning of genetic variants and their contribution to disease pathogenesis. Development of suitable mouse models is critical because disease susceptibility is triggered by complex interactions between genetic, epigenetic, and environmental factors that cannot be modeled by in vitro models. Thyroglobulin (TG) is a key gene for autoimmune thyroid disease (AITD) and several single nucleotide polymorphisms (SNPs) in the TG coding region have been associated with AITD. The classical model of experimental autoimmune thyroiditis (EAT), based on immunization of genetically susceptible mouse strains with purified TG protein in adjuvant, does not allow testing the impact of TG sequence variants on the development of autoimmune thyroiditis. Here we describe a protocol for the induction of EAT by immunization of mice susceptible to thyroiditis with an adenovirus vector carrying full-length human TG cDNA (Ad-TG EAT). We also provide support protocols for evaluation of autoimmune thyroiditis including serological assessment of TG antibodies, in vitro splenocyte proliferation assay and cytokines secretion, thyroid histology, and evaluation of thyroid lymphocytic infiltration by immunostaining. This protocol for EAT induction allows manipulation of the TG cDNA to introduce variants associated with AITD, enabling the testing of the functional effects of susceptible variants and their haplotypes on the immunogenicity of TG. Furthermore, the Ad-TG EAT mouse model is a valuable model for studying the interactions of the TG variants with non-genetic factors influencing AITD development (e.g., cytokines, iodine exposure) or with variants of other susceptible genes (e.g., HLA-DRß1). © 2024 Wiley Periodicals LLC. Basic Protocol: Development of a mouse model of autoimmune thyroiditis induced by immunization with adenovirus containing full-length thyroglobulin cDNA Support Protocol 1: Splenocytes isolation Support Protocol 2: T cell stimulation and carboxyfluorescein diacetate succinimidyl ester (CFSE) based cell proliferation assay Support Protocol 3: Cytokine assays: measuring levels of interferon gamma (IFNγ) and interleukins IL-2, IL-4, and IL-10 in splenocyte supernatants Support Protocol 4: Evaluating thyroid histology and infiltration with immune cells: hematoxylin-eosin staining of mice thyroid glands Support Protocol 5: Immunohistochemistry of thyroid tissues: Immunofluorescence protocol of paraffin-embedded thyroid sections Support Protocol 6: Anti-thyroglobulin antibody measurement in mice sera by enzyme-linked immunosorbent assay (ELISA).

Mg2+-promoted high-efficiency DNA conjugation on polydopamine surfaces for aptamer-based ochratoxin A detection.

BACKGROUND: Surface immobilization of DNA is the foundation of a broad range of applications in biosensing and specific DNA extraction. Polydopamine (PDA) coatings can serve as intermediate layers to immobilize amino- or thiol-labelled molecules, including DNA, onto various materials through Michael addition and/or Schiff base reactions. However, the conjugation efficiency is limited by electrostatic repulsion between negatively charged DNA and PDA. Recently, it has been reported that polyvalent metal ions (such as Mg2+ and Ca2+) can mediate the adsorption of DNA on PDA surfaces. Inspired by this, in this work we aimed to exploit polyvalent metal ions to facilitate the conjugation of DNA on PDA. RESULTS: Mg2+ was used to promote the conjugation of amino-terminated DNA complementary to ochratoxin A (OTA) aptamer (cDNA-NH2) on PDA-coated magnetic nanoparticles (Fe3O4@PDA). After the reaction, the unlinked cDNA-NH2 adsorbed on Fe3O4@PDA mediated by Mg2+ was removed with EDTA. In the presence of 20 mM Mg2+, the amount of covalently linked cDNA-NH2 increased approximately 11-fold compared to that in the absence of Mg2+. The resulting Fe3O4@PDA@cDNA conjugates exhibited superior hybridization capacity towards OTA aptamers, minimal nonspecific adsorption, and excellent chemical stability. The conjugates combined with fluorophore-labelled aptamers were employed for OTA detection, achieving a limit of detection (LOD) of 2.77 ng mL-1. To demonstrate versatility, this conjugation method was extended to Ca2+-promoted conjugation of cDNA-NH2 on Fe3O4@PDA nanoparticles and Mg2+-promoted conjugation of cDNA-NH2 on PDA-coated 96-well plates. SIGNIFICANCE: The conjugation efficiency of DNA on PDA was significantly improved with the assistance of polyvalent metal ions (Mg2+ and Ca2+), providing a facile and efficient method for DNA immobilization. Due to the substrate-independent adhesion property of PDA, this method demonstrates versatility in DNA surface modification and holds great potential for applications in target extraction, biosensing, and other fields.

[LncRNA SNHG11 promotes malignant progression of colorectal cancer cells through the PI3K/Akt/mTOR signaling pathway].

Objective: To investigate the effects of lncRNA SNHG11 on proliferation, migration, invasion and apoptosis of colorectal cancer cancer cells and possible mechanisms. Methods: qRT-PCR was performed to detect the expression level of lncRNA SNHG11 in colorectal cancer tissues and its related cell lines. The correlation between SNHG11 expression and clinical prognosis of patients was assessed by bioinformatics techniques. Cultured CRC cell lines were transfected with shCtrl (shCtrl group), shSNHG11#1 (shSNHG11#1 group), shSNHG11#2 (shSNHG11#2 group), Control cDNA (Control cDNA group), and SNHG11 cDNA (SNHG11 cDNA), respectively. Thiazolyl blue (MTT), clone formation assay, Transwell assay, cell scratch assay, and flow cytometry were used to detect the proliferation, migration, invasion, and apoptosis of CRC cells in each group. Western protein blotting was used to detect the expression of relevant proteins in each group, and the effect of lncRNA SNHG11 knockdown on the growth of tumour cells in vivo was analysed by nude mice tumouring assay. Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signalling pathway inhibitor LY294002 was used for rescue experiments. Results: The expression of lncRNA SNHG11 was significantly higher in colorectal cancer cells and tissues than in normal tissues (P<0.05). Survival analysis showed that the expression level of SNHG11 was not statistically associated with CRC survival (P>0.05). shSNHG11#2 group compared with shCtrl group. MTT OD490/570 values decreased, the number of CRC cell clones decreased, the number of Transwell cells decreased, the area of cell scratch decreased, and the apoptosis rate increased (P<0.05). The mesenchymal markers matrix metalloproteinase (MMP9), N-cadherin and vimentin were significantly reduced, and the expression of the epithelial marker E-cadherin was upregulated. The expression of anti-apoptotic proteins Bcl-2 and Bcl-xl was decreased, and the expression of pro-apoptotic protein Bax was increased (P<0.05).In vivo experiments showed that lncRNA SNHG11 knockdown inhibited the growth of colorectal cancer cells, and the expression of Ki67 was reduced in tumours (P<0.05). LncRNA SNHG11 knockdown inhibited the expression of p-PI3K, p-Akt and p-mTOR.The PI3K/Akt/mTOR signaling pathway inhibitor LY294002 was able to restore the malignant cytological progression of colorectal cancer cells induced by the overexpression of lncRNA SNHG11. Conclusions: LncRNA SNHG11 is highly expressed in colorectal cancer. lncRNA SNHG11 can promote the malignant progression of colorectal cancer cells by regulating the PI3K/Akt/mTOR signaling pathway, and this finding provides a new theoretical basis for targeted therapy of colorectal cancer.

A PAM-Free One-Step Asymmetric RPA and CRISPR/Cas12b Combined Assay (OAR-CRISPR) for Rapid and Ultrasensitive DNA Detection.

Current research endeavors have focused on the combination of various isothermal nucleic acid amplification methods with CRISPR/Cas systems, aiming to establish a more sensitive and reliable molecular diagnostic approach. Nevertheless, most assays adopt a two-step procedure, complicating manual operations and heightening the risk of contamination. Efforts to amalgamate both assays into a single-step procedure have faced challenges due to their inherent incompatibility. Furthermore, the presence of the protospacer adjacent motif (PAM) motif (e.g., TTN or TTTN) in the target double-strand DNA (dsDNA) is an essential prerequisite for the activation of the Cas12-based method. This requirement imposes constraints on crRNA selection. To overcome such limitations, we have developed a novel PAM-free one-step asymmetric recombinase polymerase amplification (RPA) coupled with a CRISPR/Cas12b assay (OAR-CRISPR). This method innovatively merges asymmetric RPA, generating single-stranded DNA (ssDNA) amenable to CRISPR RNA binding without the limitations of the PAM site. Importantly, the single-strand cleavage by PAM-free crRNA does not interfere with the RPA amplification process, significantly reducing the overall detection times. The OAR-CRISPR assay demonstrates sensitivity comparable to that of qPCR but achieves results in a quarter of the time required by the latter method. Additionally, our OAR-CRISPR assay allows the naked-eye detection of as few as 60 copies/µL DNA within 8 min. This innovation marks the first integration of an asymmetric RPA into one-step CRISPR-based assays. These advancements not only support the progression of one-step CRISPR/Cas12-based detection but also open new avenues for the development of detection methods capable of targeting a wide range of DNA targets.

Reverse transcription of plasma-derived HIV-1 RNA generates multiple artifacts through tRNA(Lys-3)-priming.

In vitro reverse transcription of full-length HIV-1 RNA extracted from the blood plasma of people living with HIV-1 remains challenging. Here, we describe the initiation of reverse transcription of plasma-derived viral RNA in the absence of an exogenous primer. Real-time PCR and Sanger sequencing were applied to identify the source and to monitor the outcome of this reaction. Results demonstrated that during purification of viral RNA from plasma, tRNA(Lys-3) is co-extracted in a complex with the viral RNA. In the presence of a reverse transcription enzyme, this tRNA(Lys-3) can induce reverse transcription, a reaction that is not confined to transcription of the 5' end of the viral RNA. A range of cDNA products is generated, most of them indicative for the occurrence of in vitro strand transfer events that involve translocation of cDNA from the 5' end to random positions on the viral RNA. This process results in the formation of cDNAs with large internal deletions. However, near full-length cDNA and cDNA with sequence patterns resembling multiple spliced HIV-1 RNA were also detected. Despite its potential to introduce significant bias in the interpretation of results across various applications, tRNA(Lys-3)-driven reverse transcription has been overlooked thus far. A more in-depth study of this tRNA-driven in vitro reaction may provide new insight into the complex process of in vivo HIV-1 replication.IMPORTANCEThe use of silica-based extraction methods for purifying HIV-1 RNA from viral particles is a common practice, but it involves co-extraction of human tRNA(Lys-3) due to the strong interactions between these molecules. This co-extraction becomes particularly significant when the extracted RNA is used in reverse transcription reactions, as the tRNA(Lys-3) then serves as a primer. Reverse transcription from tRNA(Lys-3) is not confined to cDNA synthesis of the 5' end of the viral RNA but extends across various regions of the viral genome through in vitro strand transfer events. Co-extraction of tRNA(Lys-3) has been overlooked thus far, despite its potential to introduce bias in downstream, reverse transcription-related applications. The observed events in the tRNA(Lys-3)-induced in vitro reverse transcription resemble in vivo replication processes. Therefore, these reactions may offer a unique model to better understand the replication dynamics of HIV-1.

A novel ß-cyclodextrin-assisted enhancement strategy for portable and sensitive detection of miR-21 in human serum.

Benefiting from our discovery that ß-cyclodextrin (ß-CD) could enhance the catalytic activity of invertase through hydrogen bonding to improve detection sensitivity, a highly sensitive and convenient biosensor for the detection of miR-21 was proposed, which is based on the simplicity of reading signals from a personal glucose meter (PGM), combined with self-assembled signal amplification probes and the performance of ß-CD as an enhancer. In the presence of miR-21, magnetic nanoparticle coupled capture DNA (MNPs-cDNA) could capture it and then connect assist DNA/H1-invertase (aDNA/H1) and self-assembled signal amplification probes (H1/H2) in turn. As a result, a "super sandwich" structure was formed. The invertase on MNPs-cDNA could catalyze the hydrolysis of sucrose to glucose and this catalytic process could be enhanced by ß-CD. The PGM signal exhibited a linear correlation with miR-21 concentration within the range of 25 pmol L-1 to 3 nmol L-1, and the detection limit was as low as 5 pmol L-1 with high specificity. Moreover, the recoveries were 103.82-124.65% and RSD was 2.59-6.43%. Furthermore, the biosensor was validated for the detection of miR-21 in serum, and the results showed that miR-21 levels in serum samples from patients with Diffuse Large B-Cell Lymphoma (DLBCL) (n = 12) were significantly higher than those from healthy controls (n = 12) (P < 0.001). Therefore, the ingenious combination of PGM-based signal reading, self-assembled signal amplification probes and ß-CD as an enhancer successfully constructed a convenient, sensitive and specific biosensing method, which is expected to be applied to clinical diagnosis.

Engineering of stable infectious cDNA constructs of a fluorescently tagged tomato chlorosis virus.

Tomato chlorosis virus (ToCV) is an emerging pathogen that cause severe yellow leaf disorder syndrome in tomato plants. In this study, we aimed to generate a recombinant ToCV tagged with green fluorescent protein (GFP) to enable real-time monitoring of viral infection in living plants. Transformation of the full-length cDNA construct of ToCV RNA1 into Escherichia coli resulted in instability issues, which were successfully overcome by inserting a plant intron into RNA1. Subsequently, a GFP tag was engineered into a cDNA construct of ToCV RNA2. The resulting recombinant ToCV-GFP could systemically infect Nicotiana benthamiana plants, and GFP expression was observed along the major veins. Utilizing ToCV-GFP, we also showed that ToCV engages in antagonistic relationships with two different tomato-infecting viruses in mixed infections in N. benthamiana. This study demonstrates the potential of ToCV-GFP as a valuable tool for the visual tracking of infection and movement of criniviruses in living plants.

Development of full-length infectious cDNA clones and host range identification of an echinacea strain of tobacco streak virus.

Tobacco streak virus induces severe diseases on a wide range of plants and becomes an emerging threat to crop yields. However, the infectious clones of TSV remain to be developed for reverse genetics studies. Here, we obtained the full genome sequence of a TSV-CNB isolate and analyzed the phylogenetic characteristics. Subsequently, we developed the full-length infectious cDNA clones of TSV-CNB driven by 35 S promoter using yeast homologous recombination. Furthermore, the host range of TSV-CNB isolate was determined by Agrobacterium infiltration and mechanical inoculation. The results reveal that TSV-CNB can infect 10 plant species in 5 families including Glycine max, Vigna radiate, Lactuca sativa var. Ramosa, Dahlia pinnate, E. purpurea, Calendula officinalis, Helianthus annuus, Nicotiana. Benthamiana, Nicotiana tabacum and Chenopodium quinoa. Taken together, the TSV infectious clones will be a useful tool for future studies on viral pathogenesis and host-virus interactions.